WO2018037416A1 - Procédés et compositions pour le traitement de maladies auto-immunes - Google Patents

Procédés et compositions pour le traitement de maladies auto-immunes Download PDF

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WO2018037416A1
WO2018037416A1 PCT/IL2017/050945 IL2017050945W WO2018037416A1 WO 2018037416 A1 WO2018037416 A1 WO 2018037416A1 IL 2017050945 W IL2017050945 W IL 2017050945W WO 2018037416 A1 WO2018037416 A1 WO 2018037416A1
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peptide
cell
seq
amino acid
acid sequence
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PCT/IL2017/050945
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English (en)
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Yechiel Shai
Etai ROTEM
Yoel KLUG
Roland SCHWARZER
Omri FAINGOLD
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Yeda Research And Development Co. Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • the present invention in some embodiments thereof, relates to isolated peptides, pharmaceutical compositions and methods of inhibiting T cell proliferation and, more particularly, but not exclusively, to methods of treating autoimmune diseases.
  • HTLV-1 human T-lymphotropic virus type 1
  • ATL adult T cell leukemia
  • HAM HTLV-1 -associated myelopathy
  • TSP tropical spastic paraparesis
  • HTLV-1 While only a small percentage of HTLV-1 patients eventually develop ATL, it is a rapid and fatal lymphoproliferative disease.
  • the leukemic cells in ATL are mainly CD4 T cells, in which 90-99 % of HTLV-1 DNA segregates in the peripheral blood of ATL patients [1].
  • the immunopathogenesis of this retrovirus is unique, since its lifelong persistence in CD4 + lymphocytes determines a prolonged interaction between the virus and the immune system, which may result in the broad spectrum of diseases associated with HTLV-1.
  • HIV-1 Human immunodeficiency virus type 1
  • HTLV-1 Human immunodeficiency virus type 1
  • HTLV-1 have a close common ancestor and are proposed to share the same infection mechanism [2].
  • the HTLV envelope is comprised of a surface glycoprotein termed gp62 which is cleaved into two subunits, the transmembrane protein gp21 and the envelope protein gp46. These proteins are proposed "homologs" to the HIV's gpl60, gp41 and gpl20 respectively [3]. While the infection mechanism of HIV and HTLV is proposed to be similar, their receptors are known to be different.
  • the receptor for HIV infection is the CD4, which is adjacent to the TCR when activated.
  • HTLV receptor however is still under debate, with glucose transporter 1 (Glut-1), neuropilin-1 (NRP-1) and heparan sulfate proteoglycans (HSPG) all found to be involved in HTLV-1 binding and entry [3-10], yet no evidence exists that HTLV binds to the CD4 on the T cell. Similar to HIV, HTLV also possesses proteins that modulate the immune response and its transmembrane protein was shown to be one of them [11-16].
  • Glut-1 glucose transporter 1
  • NBP-1 neuropilin-1
  • HSPG heparan sulfate proteoglycans
  • the CKS-17 peptide (SEQ ID NO: 11), which is derived from the gp21 of the virus, was shown to be immunosuppressive by inhibiting the production of interferon ⁇ (IFN- ⁇ ), interleukin (IL)- 2, and tumor necrosis factor a (TNF-a) in vitro and in vivo [17]. Additionally, patients infected with HTLV-1 were shown to be more prone to opportunistic infections such as strongyloides stercoralis. HTLV-1 carriers infected with this parasite were shown to have a very low serum antibody levels of IgG to S. stercoralis larvae which eventually became undetectable despite continued infection with the parasite. Moreover, patients with HTLV- 1-associated disease were found to have very low total serum IgE levels.
  • Enveloped viruses such as HIV and HTLV require fusion of the viral membrane with their host cell membrane in order to initiate a successful infection.
  • the fusion process of the HIV membrane and the T cell membrane is catalyzed by its fusion peptide (FP) which is located on the N terminus of the gp41 [18, 19].
  • FP fusion peptide
  • the T cell receptor (TCR) together with the CD3 coreceptors are adjacent to the fusion site as they are located closely to CD4 [20, 21]. HIV exploits this proximity by utilizing its FP to specifically bind the TCR and was shown to suppress T cell activation in vitro and in vivo [22] .
  • a method of inhibiting proliferation of a T cell comprising contacting the T cell with a peptide comprising an amino acid sequence which comprises a consensus sequence motif selected from the group consisting of SEQ ID NOs: 12-17 and 85, wherein the peptide is capable of inhibiting T cell proliferation, thereby inhibiting the proliferation of the T cell.
  • a method of treating a T cell mediated autoimmune disease comprising administering to a subject in need thereof a therapeutically effective amount of a peptide comprising an amino acid sequence which comprises a consensus sequence motif selected from the group consisting of SEQ ID NOs: 12-17 and 85, wherein the peptide is capable of inhibiting T cell proliferation, thereby treating the T cell mediated autoimmune disease.
  • a method of treating a T cell mediated autoimmune disease comprising administering to a subject in need thereof a therapeutically effective amount of a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 2, thereby treating the T cell mediated autoimmune disease.
  • a therapeutically effective amount of a peptide comprising an amino acid sequence which comprises a consensus sequence motif selected from the group consisting of SEQ ID NOs: 12-17 and 85, wherein the peptide is capable of inhibiting T cell proliferation, for use in treating a T cell mediated autoimmune disease.
  • a pharmaceutical composition comprising a peptide comprising an amino acid sequence which comprises a consensus sequence motif selected from the group consisting of SEQ ID NOs: 12-17 and 85, wherein the peptide is capable of inhibiting T cell proliferation, and a pharmaceutically acceptable carrier.
  • a peptide comprising an amino acid sequence which comprises a consensus sequence motif selected from the group consisting of SEQ ID NOs: 12-17 and 85, wherein the peptide is capable of inhibiting T cell proliferation.
  • the peptide is not a native peptide.
  • the peptide is capable of inhibiting T cell proliferation.
  • the method is effected in vitro. According to some embodiments of the invention, the method is effected in vivo. According to some embodiments of the invention, the T cell is activated.
  • the amino acid sequence does not exceed 50 amino acids in length.
  • the peptide specifically inhibits proliferation of a T cell mediating the autoimmune disease.
  • the peptide is comprised in a pharmaceutical composition with a pharmaceutically acceptable carrier.
  • the amino acid sequence does not exceed 50 amino acids in length.
  • the consensus sequence motif is set forth by SEQ ID NO: 12, then the peptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 23-40, 86-87.
  • the consensus sequence motif is set forth by SEQ ID NO: 13, then the peptide comprises the amino acid sequence is selected from the group consisting of SEQ ID NOs: 20, 41-59.
  • the consensus sequence motif is set forth by SEQ ID NO: 14, then the peptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 60-65.
  • the consensus sequence motif is set forth by SEQ ID NO: 15, then the peptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 66-71.
  • the consensus sequence motif is set forth by SEQ ID NO: 16, then the peptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 72-80.
  • the consensus sequence motif is set forth by SEQ ID NO: 17, then the peptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 81-83.
  • the consensus sequence motif is set forth by SEQ ID NO: 85, then the peptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 6. According to some embodiments of the invention, the consensus sequence motif is set forth by SEQ ID NO: 85, then the peptide comprises the amino acid sequence set forth in SEQ ID NO: 2.
  • At least one amino acid of the peptide is a dexter optical isomer (D-enantiomer) amino acid.
  • an activation of the T cell is performed by contacting the T cell with an antigen presenting cell (APC) and an autoimmune antigen.
  • APC antigen presenting cell
  • an activation of the T cell is performed by contacting the T cell with CD3 and CD28 antibodies.
  • an activation of the T cell is performed by contacting the T cell with phorbol 12-myristate 13-acetate (PMA) and ionomycin.
  • PMA phorbol 12-myristate 13-acetate
  • the activated T cell is a T helper cell.
  • the peptide is capable of modulating an expression or secretion of a factor produced by the T cell.
  • the factor comprises a cytokine.
  • the factor comprises a transcription factor.
  • the peptide is capable of altering a balance between T helper 1 (Thl) and T helper 2 (Th2) cell response.
  • the autoimmune disease is selected from the group consisting of: arthritis, diabetes mellitus type 1, multiple sclerosis, psoriasis, celiac, Hashimoto's thyroiditis, Polymyositis, Allergic contact dermatitis, and Transfusion-associated graft versus host disease.
  • the autoimmune disease is multiple sclerosis.
  • FIGs. 1A-D depict detection of active FPs in T cell's retroviruses.
  • Figure 1A is a schematic illustration depicting the sequences of the fusion peptides (FP) of the HIV, HTLV, Measles, BLV and JDV viruses (SEQ ID NOs: 1-5) taken from the UniProtKB database.
  • Figure IB is a histogram depicting the percentage of inhibition by the viral FPs (SEQ ID NOs: 1-5 as shown in Figure 1A) of T cell proliferation.
  • T cells were activated with the MOG 35 -55 peptide (SEQ ID NO: 8) and irradiated antigen presenting cells (APCs) in the presence of 10 ⁇ of the viruses' FP peptides.
  • MOG 35 -55 peptide SEQ ID NO: 8
  • APCs irradiated antigen presenting cells
  • the proliferative responses were assessed by H -thymidine incorporation and normalized to the untreated proliferated T cells (i.e., activated T cells in the absence of the virus FP peptides.
  • the data is presented as mean of percentage inhibition of three independent assays, each made with six repetitions.
  • the results show that similar to the HIVl-33 FP, the HTLVl-33 FP is a potent inhibitor of T cell proliferation.
  • the JDV virus is a retrovirus, however, is not known as capable of attacking T cells.
  • Figure 1C is qPCR relative gene expression following HTLVl-33 FP treatment on activated T-cells.
  • Figure ID is cytokine secretion detected via ELISA following HTLVl-33 FP treatment on activated T-cells.
  • FIGs. 2A-D depict detection of the Minimal Active Segment of the HTLV FPi_ 33 peptide.
  • Figure 2A depicts candidate active segments derived from the HTLV's FP. Shown are the designations and sequences (SEQ ID NOs: 2, 6 and 7) of the candidate peptides synthesized post secondary structure consensus prediction.
  • Figure 2B - Circular dichroism (CD) spectroscopy structure analysis of HTLV FPi_ 33 (SEQ ID NO: 2), FPs_i 3 (SEQ ID NO: 6) and FP 9 _ 22 (SEQ ID NO: 7).
  • CD Circular dichroism
  • T cells were activated with the MOG 35-55 peptide (SEQ ID NO: 8) and APC in the presence of HTLV FPi_33 or HTLV FPs_i3 at a concentration of 10 ⁇ .
  • the proliferative responses were assessed by H -thymidine incorporation and normalized to the untreated proliferated T cells.
  • the data is presented as mean inhibitions of two or more independent assays, each made with six repetitions. * means that P ⁇ 0.05.
  • FIGs. 3A-F depict specificity and activity of the HTLV FPs_i3 peptide.
  • Figure 3A T cell proliferation inhibition by HIV FP and HTLV FP at 5 ⁇ (Black) and 10 ⁇ (Grey).
  • T cells were activated with the MOG 35-55 peptide (SEQ ID NO: 8) and APC in the presence of HIV FP and HTLV FP peptides at different concentrations.
  • the proliferative responses were assessed by H -thymidine incorporation and normalized to the untreated proliferated T cells. The data is presented as mean inhibitions of three independent assays, each made with six repetitions.
  • FIG. 3B T cell proliferation inhibition after activation with APC (black), CD3 and CD28 antibodies (bright gray) or PMA and Ionomycin (dark gray).
  • T cells were activated in the presence of different HIV and HTLV derived peptides at a concentration of 10 ⁇ .
  • the proliferative responses were assessed by H -thymidine incorporation and normalized to the untreated proliferated T cells. The data is presented as mean inhibitions of three independent assays, each made with six repetitions.
  • HIV FPi_33 and HIV FP 5-1 3 the inhibition of proliferation by the HIV FP peptides (HIV FPi_33 and HIV FP 5-1 3) is depended on the mode of activation of the T cells, i.e., highest inhibition of T cells proliferation is achieved when the cells are activated with APC and the MOG 35 -55 peptide
  • inhibition of T cell proliferation by the HTLV FP peptide is independent of the mode of T cell activation.
  • the HTLV FPi_33 and HTLV FPs_i3 peptides are efficient in inhibiting T cells proliferation.
  • FIGS 3C-E HTLV FP peptide preferentially binds T cells over B cells. Splenocytes derived from C57BL/6J mice were incubated with rhodamine-labeled HTLV FP1-33 (SEQ ID NO: 2) or the LL37 (SEQ ID NO: 10) peptides.
  • Figure 3F TNF-a secretion from activated macrophages following HTLV1-33 FP treatment.
  • FIG. 3C defining a lymphocyte gate using forward (FS) and side (SS) scatter analysis on splenocytes.
  • Figure 3D defining a rhodamine-labeled lymphocyte gate.
  • Figure 3E the percentage of T cells and B cells in the rhodamine-labeled cells was analyzed using anti- CD3 and anti-B220 antibody, respectively.
  • the HTLV FPi_3 3 peptide preferentially binds to T cells (73.2%, Figure 3E, lower left panel) as compared to B cells (3.2%, Figure 3E, lower right panel).
  • FIGs. 4A-E demonstrate that the HTLV FPs_i 3 is localized to the membrane.
  • mMOG 3 5-55 T cells were incubated with rho-labeled peptides, either LL37 (a control peptide known to bind to T cells membrane) or HTLV FPs_i 3 , then loaded with the membrane fluorescent dye DiD and finally labeled FITC-CD3 antibodies. The cells were analyzed via ImageStreamX (Amnis Corp.) imaging flow cytometer.
  • Figure 4A Representative cells demonstrating LL37 cellular distribution. Note the presence of the LL37 peptide in the T cells membrane.
  • Figure 4B Representative cells demonstrating HTLV FP 5 _i 3 cellular distribution.
  • HTLV FPs_i 3 peptide is also presence in the T cell membrane.
  • Figure 4C Localization of the LL37 peptide as compared to the membrane DiD staining.
  • LL37 as expected is localized to membrane, demonstrated by its calculated similarity to the DiD distribution.
  • Its max contour position relative to CD3 was also calculated for an additional localization indicator.
  • the max contour position feature calculates the location of the highest intensity concentration of the staining.
  • the similarity feature is the log transformed Pearson' s Correlation Coefficient and is a measure of the degree to which two images are linearly correlated within a masked region.
  • Figure 4D Localization of the HTLV FPs_i 3 peptide as compared to the membrane DiD staining.
  • HTLV FP 5 _i 3 median values of similarity to DiD and max contour position relative to CD3, are not significantly different for the median values of LL37.
  • Figure 4E Inhibition of T cell proliferation by HTLV FP 5 _i (the "L" enantiomer) and HTLV FP 5 _i D (the "D" enantiomer) at a final concentration of 10 ⁇ .
  • T cells were activated in the presence of HTLV FP derived peptides and the proliferative responses were assessed by H -thymidine incorporation and normalized to the untreated proliferated T cells. The data is presented as mean inhibitions of three independent assays, each made with six repetitions. Note that both enantiomers are efficient in inhibiting T cell proliferations, and the D enantiomer, which is not recognized by the human body's enzymes is even more efficient in inhibition of T cells proliferation.
  • FIGs. 5A-D demonstrate that the HIV and HTLV FPs differ in structure, alignment and regional phylogenetic distance.
  • Figure 5 A Shown are the active segments 5-13 derived from the HTLV's and HIV's FP peptides along with the peptide sequences, molecular weight (MW) and grand average of hydropathicity (GRAVY). MW and GRAVY were calculated using ProtParam [23] .
  • Figure 5B CD spectroscopy structure analysis of HTLV FP 5 _i 3 (SEQ ID NO: 6) and HIV FP 5 _i 3 (SEQ ID NO: 9).
  • FIG. 6 depicts the HTLV FPi_ 33 Consensus secondary structure prediction. Secondary structure is predicted by three methods: DSC (King and Stenberg, 1996), MLRC (Guermeur ei al., 1998) and PHD (Rost et ah, 1994). A consensus secondary structure is then being predicted [26] .
  • FIG. 7 depicts the HTLV FP 5 _i 3 and HTLV FPi_ 33 toxicity assay.
  • mMOG (35-55)- specific line T cells were plated onto round 96-well plates over-night (O.N.). Peptides were added at different concentrations and were incubated for additional 24 hours. XTT assay was performed and measured after 3 hours. Results were normalized to the untreated T cells. The data is presented as mean % viability of three repetitions. The results show that both of these peptides are not toxic.
  • FIG. 8 is a histogram depicting the percentage of inhibition by the MUMPS FP peptide (SEQ ID NO: 18) and the HTLV TMD peptide (SEQ ID NO: 22) of T cell proliferation.
  • MOG 35 _55-antigen specific T cells were activated by irradiated MOG 35 -55 presenting APCs in the presence of HTLV TMD and MUMPS FP derived peptides at a final concentration of 10 ⁇ .
  • the results show that both the MUMPS FP peptide (SEQ ID NO: 18) and the HTLV TMD peptide (SEQ ID NO: 22) are potent inhibitor of T cell proliferation.
  • FIGs. 9A-D are graphs depicting monitoring of T-bet expression in activated and non-activated T-cells.
  • Figure 9A Gating on lymphocytes.
  • Figure 9B Gating on T-bet positively stained cells.
  • Figure 9C T-bet expression in non-activated and activated T- cells.
  • Figure 9D T-bet expression in non-activated and activated T-cells over the course of 72 hours.
  • FIGs. lOA-C are graphs depicting reduction in T-bet expression induced by the HTLV FP. (Figure 10A) 24, ( Figure 10B) 48 and ( Figure IOC) 72 hours following activation. Each time point is represented as a cell count vs. APC fluorescence histogram and percent of activated cells chosen as described in result section.
  • FIGs. 11A-D are graphs depicting monitoring of Gata3 expression in activated and non-activated T-cells.
  • Figure 11A Gating on lymphocytes.
  • Figure 11B Gating on Gata3 positively stained cells.
  • Figure 11C An example of an increase in Gata3 expression.
  • Figure 1 ID Gata3 expression in non-activated and activated T-cells over the course of 72 hours.
  • FIGs. 12A-C are graphs depicting elevation in Gata3 expression induced by the HTLV FP. ( Figure 12A) 24, ( Figure 12B) 48 and ( Figure 12C) 72 hours following activation. Each time point is represented as a count vs. APC fluorescence histogram and percent of activated cells chosen as described in result section.
  • FIGs. 13A-F are graphs depicting reduced progression and severity of experimental autoimmune encephalomyelitis (EAE) in mice treated with HTLV FPi_ 33 (as set forth in SEQ ID NO: 2) as compared to mice not treated with the HTLV FPi_ 33 peptide (i.e. saline group).
  • EAE was induced in C57BL/6 mice.
  • Two groups of 10 mice each were examined where one group received the HTLV1-33 fusion peptide (FP) together with EAE induction.
  • Clinical scoring ( Figure 13A) and weight measurements (Figure 13B) of the mice were carried out at the time points presented in the graphs.
  • a comparison of the cumulative EAE score (Figure 13C), maximal EAE score (Figure 13D), cumulative initial weight (Figure 13E), and IFNy secretion (Figure 13F) is also presented.
  • FIGs. 14A-B are graphs depicting the ability of the peptides listed in Table 10 (herein below) to inhibit T-cell proliferation ( Figure 14A) without affecting their viability ( Figure 14B).
  • the present invention in some embodiments thereof, relates to isolated peptides, pharmaceutical compositions and methods of inhibiting T cell proliferation and, more particularly, but not exclusively, to methods of treating autoimmune diseases.
  • the present inventors have generated a bioinformatic screen in which they identified several conserved motifs within the fusion proteins of many different viruses, these are set forth in SEQ ID Nos: 12-17 and 85). Based on this screen, the present inventors have synthesized various peptides (see e.g. Tables 3-8 below) in order to determine their suppressive effect on T cells. The results of the present study indicate, that although exerting the same specificity and suppressive effect on T cells as HIV derived peptides, some of these peptides operate through different suppression mechanisms.
  • the present inventors uncovered that the viral envelope derived peptides, including the fusion peptide (FP) domains and transmembrane domains (TMDs) of several retroviruses can effectively inhibit T cell proliferation and accordingly can be used to treat various T cell mediated autoimmune diseases.
  • FP fusion peptide
  • TMDs transmembrane domains
  • the present inventors tested if T cell viruses can utilize their fusion peptide (FP) to modulate the immune response.
  • FP fusion peptide
  • the present inventors synthesized and examined the FPs and TMDs of several viruses, among them Mumps, Measles, Ebola, human T-lymphotropic virus (HTLV), Hepatitis C virus (HCV) and Human parainfluenza virus (hPIV), and examined their ability to inhibit T cell proliferation in vitro.
  • the FP of HTLV is one of the most potent FPs that specifically inhibits T cell activation (see Examples 2-3 of the Examples section which follows) and modulates the balance between T helper 1 and T helper 2 cell response (see Example 5 of the Examples section which follows).
  • HIV and HTLV share a common ancestor, their FPs were found to be different in structure, alignment, and mode of action (see Example 6 of the Examples section which follows).
  • the present inventors have further illustrated that viral FP and TMD peptides (e.g. HTLV- 1 FP, HTLV-1 TMD, Mumps FP, Measles FP and hPIV FP) can be used to inhibit proliferation of T cells without affecting T cell viability (see Example 8 of the Examples section which follows).
  • the present inventors have further illustrated that the peptides of the invention can be used for the prevention and treatment of autoimmune diseases, such as, multiple sclerosis (see Examples 9-10 of the Examples section which follows). Taken together, the peptides of the invention can be used as therapeutics in any indication in which downregulation of T cell activity or proliferation is warranted.
  • a method of inhibiting proliferation of a T cell comprising contacting the T cell with a peptide comprising an amino acid sequence which comprises a consensus sequence motif selected from the group consisting of SEQ ID NOs: 12-17 and 85, wherein the peptide is capable of inhibiting T cell proliferation, thereby inhibiting the proliferation of the T cell.
  • T cell refers to a T lymphocyte which is characterized by CD4 and/or CD8 phenotypes.
  • the T cell is a helper T cell, a regulatory T cells (e.g. suppressor T cells) and/or an effector T cell.
  • a regulatory T cells e.g. suppressor T cells
  • an effector T cell can be a cytotoxic T cell.
  • the T cell can include naive T cells or memory T cells.
  • the T cells used by specific embodiments of the invention can be primary T cells or a T cell line.
  • the T cells can be obtained from a biological sample of a subject such as a blood sample, a spleen sample and a lymph node sample. T cells can be isolated and purified according to methods known in the art [e.g., as described in Mor and Cohen, 1995, J. Immunol. 155:3693-3699 which is fully incorporated herein by reference].
  • the T cell is an activated T cell.
  • Methods of activating T cells are known in the art and include antigen-specific activation and non-antigen specific polyclonal activation.
  • the T cell is activated ex-vivo.
  • the non-antigen specific polyclonal activated T cells are obtained by exposure of T cells to various molecules such as protein(s) (e.g., various antibodies, cytokines, chemokines), Toll-like receptor (TLR) ligand(s), lectin(s) [e.g., concavalin A, phytohaemagglutinin] and ionomycin, which bind and activate the T cell receptor.
  • protein(s) e.g., various antibodies, cytokines, chemokines
  • TLR Toll-like receptor
  • lectin(s) e.g., concavalin A, phytohaemagglutinin
  • ionomycin bind and activate the T cell receptor.
  • activation of the T cell is performed by contacting the T cell with an antigen presenting cell (APC) and an autoimmune antigen (i.e., the antigen involved in the pathogenesis of the autoimmune disease).
  • APC antigen presenting cell
  • an autoimmune antigen i.e., the antigen involved in the pathogenesis of the autoimmune disease
  • activation of the non-antigen specific polyclonal T cell is performed by incubation of a T cell with an anti CD3 antibody and an anti CD28 antibody or by Phorbol 12-myristate 13-acetate (PMA) and Ionomycin.
  • PMA Phorbol 12-myristate 13-acetate
  • activation of the T cell is performed by contacting the T cell with antibodies which specifically bind CD3 and/or CD28.
  • activation of T cells can be achieved by interacting the T cells with a combination of anti-CD3 and anti-CD28 antibodies, which stimulate both the primary and co- stimulatory signals that are required for activation and expansion of T cells.
  • the antibody can be attached to a surface such as beads, e.g., magnetic beads (e.g., Dynabeads® coupled to the antibodies, available from Invitrogen, Carlsbad, CA, USA).
  • T cells Purified T cells (e.g., 1-1.5 x 10 6 cells) are placed in a tissue culture vessel (e.g., a 24-well or 96-well tissue culture plate) in the presence of a medium.
  • the culture vessel can include anti-CD28 and anti-CD3 antibodies attached thereto.
  • the culture vessel does not include the anti-CD28 and anti-CD3 antibodies attached thereto, yet, these antibodies are attached to beads which are added to the medium within the culture vessel.
  • the T cells can be incubated in the Biotarget culture medium (Catalogue number 05-080-1, Biological Industries, Beit- Haemek, Israel) supplemented with 1 % Pen/Strep/Nystatin, 40 mM L-glutamine.
  • Dynabeads® Human T-Activator CD3/CD28 for cell expansion and activation are added to the culture medium at a bead-to-cell ratio of 1: 1 followed by the addition of 30 U/ml recombinant interleukin-2 (rIL-2).
  • the T cells are then incubated in a humidified C0 2 incubator at 37°C, and can be examined daily, noting cell size and shape. Cell shrinking and reduced proliferation rate is typically observed in exhausted cell cultures. When the cell density exceeds 2.5 x 10 6 cells/ml or when the medium turns yellow the cultures are split to a density of 0.5-lxlO 6 cells/ml in Biotarget culture medium containing 30 U/ml rIL-2. Typically activation of the T cells occurs within 2 days in the described culture conditions.
  • activation of the T cell is performed by contacting the T cell with phorbol 12-myristate 13-acetate (PMA) and ionomycin.
  • PMA phorbol 12-myristate 13-acetate
  • isolated refers to at least partially separated from the natural environment e.g., the human body.
  • the peptide is an isolated peptide, e.g., not forming part of a cell or cell components, and/or not forming part of an organism (e.g., a virus).
  • T cell proliferation can be performed within a subject (i.e., in vivo), within cells derived from a subject (i.e., ex vivo or in vitro).
  • the method of inhibiting T cell proliferation is effected in vitro. According to some embodiments of the invention, the method of inhibiting T cell proliferation is effected in vivo (e.g., for treating an autoimmune disease).
  • inhibiting proliferation of a T cell refers to preventing, reducing, downregulating and/or completely abolishing the proliferation of the T cell as compared to the proliferation of the T cell which is detected in the absence of the peptide.
  • T cell proliferation assay which can be used to qualify the ability of the peptide of some embodiments of the invention to inhibit T cell proliferation.
  • T cell proliferation assay Primary T cells specific to an autoantigenic peptide (e.g., MOG P35-55, SEQ ID NO: 8) are plated onto round 96-well plates in medium containing RPMI-1640 supplemented with 2.5% fetal calf serum (FCS), 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 50 ⁇ ⁇ -mercaptoethanol, and 2 mM L-glutamine.
  • FCS fetal calf serum
  • Each of the 96 wells has a final volume of 200 ⁇ and contains 20x10 3 T cells, 5x105 irradiated (25 gray) spleen cells [which include the antigen presenting cells (APCs)], and 5 ⁇ g/ml of the autoantigenic peptide (e.g., MOG P35-55).
  • the peptide is added to the reaction mixture in the wells, following which T cell proliferation is measured.
  • the autoantigenic peptide e.g., MOG P35-55
  • the APCs is added to the APCs in a first test tube, and in a second test tube the peptide of some embodiments of the invention is added to the T cells. After about 1 hour, the APCs (with the autoantigenic peptide) are mixed with the T cells (with the peptide of some embodiments of the invention) and are further incubated for about 48 hours in a 96 well round bottom plate.
  • the T cells are pulsed with 1 ⁇ (H ) thymidine, with a specific activity of 5.0 Ci/mmol, for 24 hours, and (H ) thymidine incorporation is measured using a 96-well plate beta- counter. Each read is made with minimum of five repeats. The mean cpm + Standard Deviation (SD) is calculated for each quadruplicate or more.
  • the results of the T cell proliferation experiments can be presented as the percentage of T cell proliferation triggered by the autoantigenic peptide (e.g., the MOG P35-55 peptide) in the presence of the peptide of some embodiments of the invention as compared to the proliferation of T cell in the absence of the peptide of some embodiments of the invention.
  • the T cells which are used in the above described proliferation assay can be activated by any T cell activation method known in the art and/or described hereinabove.
  • the T cells used in the proliferation assay can be activated in a non-antigen specific mode of activation, such as with pre-coated CD3 and CD28 antibodies (LEAFTM purified anti mouse clones 145-2-Cl l and 37.51, respectively from Biolegend) at final concentration of 2 ⁇ g/ml.
  • the T cells can be activated with 50 ng/mL of PMA (phorbol 12-myristate 13-acetate) together with 1 ⁇ of ionomycin (Sigma Chemical Co, Israel).
  • the T cell proliferation assay is performed with or without the peptide of some embodiments of the invention and the ratio between the measured T cell proliferation in the presence of the peptide of some embodiments of the invention and the measured T cell proliferation in the absence of the peptide of some embodiments of the invention is determined.
  • Non-limiting examples of the results of inhibition of T cell proliferation assays are shown in Figures IB, 2D, 3A-B, and 4E herein.
  • a very efficient inhibition e.g., 100% inhibition reflects no measured T cell proliferation at all in the presence of the peptide of some embodiments of the invention.
  • 0% inhibition reflects that the addition of the peptide of some embodiments of the invention had no effect on the proliferation of T cells as compared to the proliferation measured in the absence of the same peptide.
  • the proliferation of the T cell in the presence of the peptide of some embodiments of the invention is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100% as compared to the proliferation of the T cell in the absence of the same peptide.
  • the peptide is capable of modulating expression or secretion of a factor produced by a T cell.
  • modulating refers to increasing or decreasing the expression or secretion of a factor by the T cell.
  • Exemplary factors produced by T cells include, but are not limited to, cytokines including lymphokines, interleukins, and chemokines such as, but not limited to, IL-2, IL- 3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-10, IL-13, IL-15, IL-18, IL-20, IL-21, IL- 22, IL-23, IL-28, GM-CSF, Leukemia inhibitory factor (LIF), IFN- ⁇ , transforming growth factor beta (TGF- ⁇ ), tumor necrosis factor (TNF) family members (including, but not limited to, TNFa, TNF- ⁇ and Lymphotoxin-alpha (LT-alpha)), chemokines including C-C chemokines (e.g.
  • RANTES RANTES, MCP-1, MIP-la, and MIP-1B
  • C-X-C chemokines e.g. IL- 8
  • C chemokines e.g. Lymphotactin
  • CXXXC chemokines e.g. Fractalkine
  • transcription factors including, but not limited to, STAT4, STAT6, STAT1, STAT3, IRFs, T-bet and GATA3.
  • the peptide is capable of altering a balance between T helper 1 (Thl) response and T helper 2 (Th2) response.
  • the peptide of the invention is capable of increasing the activity of a type of T helper cello response while decreasing the activity of another type of T helper cell response, thereby altering their effect on the immune system (e.g. on T cytotoxic cells, on B cells, etc. e.g., for treating an autoimmune disease).
  • the peptide is capable of increasing the Th2 response while decreasing the Thl response.
  • increasing an expression or secretion of a factor by the T cell or in an activity of a T cell is by about 5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 99 % or by 100 % as compared to a T cell not treated with the peptide of some embodiments of the invention.
  • decreasing an expression or secretion of a factor by the T cell or in an activity of a T cell is by about 5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 99 % or by 100 % as compared to a T cell not treated with the peptide of some embodiments of the invention.
  • Measuring an activity of a T cell can be carried out using any method known in the art, as discussed above.
  • Thl response can be identified by expression or secretion of IFN- ⁇ , LT-a and expression of the T-bet or STAT4 transcription factors.
  • Th2 response can be identified, for example, by expression and secretion of IL-4, IL-10 and expression of the Gata3 transcription factor.
  • Consensus sequence motif refers to an amino acid sequence which is shared by a number of peptide sequences.
  • G/A refers to presence of either a “G” (i.e., Glycine) or an “A” (i.e., Alanine) amino acid.
  • the term "peptide” refers to natural, non-natural and/or chemically modified amino acid residues connected one to the other by peptide or non-peptide bonds.
  • the peptide of some embodiments of the invention encompasses native peptides (e.g., either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells.
  • Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, backbone modifications, and residue modification.
  • Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided herein under.
  • a “chemical derivative” as used herein refers to peptides containing additional chemical moieties not normally a part of the peptide molecule such as esters and amides of free carboxy groups, acyl and alkyl derivatives of free amino groups, esters and ethers of free hydroxy groups. Such modifications may be introduced into the peptide by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • the present invention also encompasses salts of the peptides, fragments, analogs, and chemical derivatives of the invention.
  • salts of carboxyl groups can be formed by means known in the art and include inorganic salts, for example aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc, and the like.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, ⁇ , ⁇ '- dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.
  • basic ion exchange resins such as
  • Acid addition salts include, for example, salts with mineral acids such as, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like.
  • mineral acids such as, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, ni
  • Natural aromatic amino acids, Trp, Tyr and Phe may be substituted by non-natural aromatic amino acids such as l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine, ring-methylated derivatives of Phe, halogenated derivatives of Phe or O- methyl-Tyr.
  • the peptides of some embodiments of the invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
  • modified amino acids e.g. fatty acids, complex carbohydrates etc.
  • amino acid residues are represented throughout the specification and claims by either one or three-letter codes, as is commonly known in the art.
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phospho threonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid includes both D- and L-amino acids.
  • Tables 1 and 2 below list naturally occurring amino acids (Table 1), and non- conventional or modified amino acids (e.g., synthetic, Table 2) which can be used with some embodiments of the invention.
  • Non-conventional amino aci Code Non-conventional amino acid Code
  • peptides of some embodiments of the invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.
  • the salts, analogs and the chemical derivatives of the peptides are preferably used to modify the pharmaceutical properties of the peptides insofar as stability, solubility, etc. are concerned.
  • the present peptides are preferably utilized in therapeutics or diagnostics which require the peptides to be in soluble form
  • the peptides of some embodiments of the invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.
  • the peptides of some embodiments of the invention may be synthesized by any techniques that are known to those skilled in the art of peptide synthesis.
  • solid phase peptide synthesis a summary of the many techniques may be found in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, W. H. Freeman Co. (San Francisco), 1963 and J. Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46, Academic Press (New York), 1973.
  • For classical solution synthesis see G. Schroder and K. Lupke, The Peptides, vol. 1, Academic Press (New York), 1965.
  • these methods comprise the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain.
  • amino acids or suitably protected amino acids Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group.
  • the protected or derivatized amino acid can then either be attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected, under conditions suitable for forming the amide linkage.
  • the protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support) are removed sequentially or concurrently, to afford the final peptide compound.
  • a preferred method of preparing the peptide compounds of some embodiments of the invention involves solid phase peptide synthesis.
  • the peptide is purified.
  • the peptide of some embodiments of the invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
  • standard protein purification techniques such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
  • the peptide is provided in a purified pharmaceutical grade, which is suitable for use for treating a subject (i.e., for administration into a subject).
  • the peptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 23-40, 86-87.
  • the peptide comprises the amino acid sequence as set forth in SEQ ID NO: 18.
  • the peptide comprises the amino acid sequence as set forth in SEQ ID NO: 86.
  • the peptide comprises the amino acid sequence as set forth in SEQ ID NO: 87.
  • the peptide comprises the amino acid sequence is selected from the group consisting of SEQ ID NOs: 20, 41-59.
  • the peptide comprises the amino acid sequence as set forth in SEQ ID NO: 20.
  • the peptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 60-65.
  • the peptide comprises the amino acid sequence as set forth in SEQ ID NO: 19.
  • the peptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 66-71.
  • the peptide comprises the amino acid sequence as set forth in SEQ ID NO: 21.
  • the peptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 72-80.
  • the peptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 81-83.
  • the peptide comprises the amino acid sequence as set forth in SEQ ID NO: 22.
  • the peptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 6.
  • the peptide comprises the amino acid sequence as set forth in SEQ ID NO: 2.
  • At least one amino acid of the peptide is a dexter optical isomer (D-enantiomer) amino acid.
  • D-enantiomer dextrorotatory optical isomer
  • the amino acid sequence of the peptide of some embodiments of the invention does not exceed 50 amino acids in length.
  • the amino acid sequence of the peptide of some embodiments of the invention does not exceed about 50, about 45, about 40, about 35, about 30, about 25, about 24, about 23, about 22, about 21, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 21, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, or about 5 amino acids in length.
  • the amino acid sequence of the peptide of some embodiments of the invention does not exceed 15 amino acids in length.
  • the amino acid sequence of the peptide of some embodiments of the invention does not exceed 25 amino acids in length.
  • the amino acid sequence of the peptide of some embodiments of the invention does not exceed 35 amino acids in length.
  • the amino acid sequence of the peptide of some embodiments of the invention is between 5-50, between 5-40, between 5-30, between 5- 20, between 5-15, between 5-10, between 5-9, between 5-7, between 7-50, between 7-40, between 7-30, between 7-20, between 7-15, between 7-10, between 7-9, between 9-50, between 9-40, between 9-30, between 9-20, between 9-15, between 9-12, between 9-11, between 15-50, between 15-40, between 15-35, between 15-30, between 15-20, between 25-50, between 25-40, between 25-35, between 25-30, between 35-50, between 35-40, or between 40-50 amino acids in length.
  • a method of treating a T cell mediated autoimmune disease comprising administering to a subject in need thereof a therapeutically effective amount of a peptide comprising an amino acid sequence which comprises a consensus sequence motif selected from the group consisting of SEQ ID NOs: 12-17 and 85, wherein the amino acid sequence is capable of inhibiting T cell proliferation, thereby treating the T cell mediated autoimmune disease.
  • a method of treating a T cell mediated autoimmune disease comprising administering to a subject in need thereof a therapeutically effective amount of a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 2, thereby treating the T cell mediated autoimmune disease.
  • a therapeutically effective amount of a peptide comprising an amino acid sequence which comprises a consensus sequence motif selected from the group consisting of SEQ ID NOs: 12-17 and 85, wherein the peptide is capable of inhibiting T cell proliferation, for use in treating a T cell mediated autoimmune disease.
  • treating refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology.
  • pathology disease, disorder or condition
  • Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
  • the term "subject” includes mammals, preferably human beings at any age which suffer from the pathology.
  • autoimmune disease refers to a disease which onset or progression is mediated by the immune system of the subject in response to autoantigenic peptides.
  • autoantigenic peptide refers to an antigen derived from an endogenous (i.e., self-protein) or a consumed protein (e.g., by food) against which an inflammatory response is elicited as part of an autoimmune inflammatory response.
  • presentation of an autoantigenic peptide on antigen presenting cells can result in recognition of the MHC-autoantigenic peptides by specific T cells, and consequently generation of an inflammatory response that can activate and recruit T cell and B cell responses against the APCs cells.
  • the autoimmune disease which is treated by the method of an aspect of the invention is mediated by T cells.
  • the autoimmune disease is selected from the group consisting of: rheumatoid arthritis, diabetes mellitus type 1, multiple sclerosis, psoriasis, celiac, Hashimoto's thyroiditis, Polymyositis, Allergic contact dermatitis, and Transfusion-associated graft versus host disease.
  • the autoimmune disease is multiple sclerosis.
  • the peptide specifically inhibits proliferation of a T cell mediating the autoimmune disease.
  • autoimmune diseases which can be treated according to the method of some embodiments of the invention.
  • the autoimmune disease(s) include, but is (are) not limited to, cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, cutaneous diseases, hepatic diseases, neurological diseases, muscular diseases, nephric diseases, diseases related to reproduction, connective tissue diseases and systemic diseases.
  • autoimmune cardiovascular diseases include, but are not limited to atherosclerosis (Matsuura E. et al, Lupus. 1998;7 Suppl 2:S 135), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S 132), thrombosis (Tincani A. et al, Lupus 1998;7 Suppl 2:S 107-9), Wegener's granulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S. et al, Wien Klin Klin Klin Klin Klinschr 2000 Aug 25;112 (15-16):660), anti-factor VIII autoimmune disease (Lacroix-Desmazes S.
  • autoimmune rheumatoid diseases include, but are not limited to rheumatoid arthritis (Krenn V. et al, Histol Histopathol 2000 Jul;15 (3):791; Tisch R, McDevitt HO. Proc Natl Acad Sci units S A 1994 Jan 18;91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al, Arthritis Res 2001; 3 (3): 189).
  • autoimmune glandular diseases include, but are not limited to, pancreatic disease, Type I diabetes, thyroid disease, Graves' disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitis and Type I autoimmune polyglandular syndrome, diseases include, but are not limited to autoimmune diseases of the pancreas, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S 125), autoimmune thyroid diseases, Graves' disease (Orgiazzi J.
  • autoimmune gastrointestinal diseases include, but are not limited to, chronic inflammatory intestinal diseases (Garcia Herola A. et al, Gastroenterol Hepatol. 2000 Jan;23 (1): 16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16; 138 (2): 122), colitis, ileitis and Crohn's disease.
  • autoimmune cutaneous diseases include, but are not limited to, autoimmune bullous skin diseases, such as, but are not limited to, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.
  • autoimmune hepatic diseases include, but are not limited to, hepatitis, autoimmune chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol 1990 Mar;54 (3):382), primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov;91 (5):551; Strassburg CP. et al, Eur J Gastroenterol Hepatol. 1999 Jun;l l (6):595) and autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug;33 (2):326).
  • autoimmune neurological diseases include, but are not limited to, multiple sclerosis (Cross AH. et al., J Neuroimmunol 2001 Jan 1 ; 112 (1-2): 1), Alzheimer's disease (Oron L. et al, J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999;18 (l-2):83; Oshima M. et al, Eur J Immunol 1990 Dec;20 (12):2563), neuropathies, motor neuropathies (Kornberg AJ. J Clin Neurosci.
  • autoimmune muscular diseases include, but are not limited to, myositis, autoimmune myositis and primary Sjogren's syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 Sep; 123 (1):92) and smooth muscle autoimmune disease (Zauli D. et al, Biomed Pharmacother 1999 Jun;53 (5-6):234).
  • autoimmune nephric diseases include, but are not limited to, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug;l (2): 140).
  • autoimmune diseases related to reproduction include, but are not limited to, repeated fetal loss (Tincani A. et ah, Lupus 1998;7 Suppl 2:S 107-9).
  • autoimmune connective tissue diseases include, but are not limited to, ear diseases, autoimmune ear diseases (Yoo TJ. et ah, Cell Immunol 1994 Aug; 157 (1):249) and autoimmune diseases of the inner ear (Gloddek B. et ah, Ann N Y Acad Sci 1997 Dec 29;830:266).
  • autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et ah, Immunol Res 1998; 17 (l-2):49) and systemic sclerosis (Renaudineau Y. et ah, Clin Diagn Lab Immunol. 1999 Mar;6 (2): 156); Chan OT. et al, Immunol Rev 1999 Jun;169: 107).
  • the peptide when the disease is multiple sclerosis, the peptide comprises the amino acid sequence as set forth in SEQ ID NO: 2.
  • the peptide of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the peptide of some embodiments of the invention accountable for the biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • neurosurgical strategies e.g., intracerebral injection or intracerebroventricular infusion
  • molecular manipulation of the active agent e.g., production of a chimeric fusion protein that comprises a transport peptide in combination with an agent that is itself incapable of crossing the blood brain barrier (BBB)
  • BBB blood brain barrier
  • pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers)
  • the transitory disruption of the integrity of the BBB by hyperosmotic disruption resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).
  • each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
  • tissue refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.
  • Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • compositions of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (the peptide of some embodiments of the invention) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., the autoimmune disease) or prolong the survival of the subject being treated.
  • a therapeutically effective amount means an amount of active ingredients (the peptide of some embodiments of the invention) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., the autoimmune disease) or prolong the survival of the subject being treated.
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
  • Dosage amount and interval may be adjusted individually to provide levels of the active ingredient that are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC).
  • MEC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • the peptide of some embodiments of the invention can be administered to a subject (e.g. human subject) before the onset of a disease (e.g., as a preventive measure e.g. years, months, days or hours before onset of a disease) especially in subjects who are at risk of developing the disease, e.g., as a results of a genetic background, environmental factors, life style, etc., or any time after onset of a disease (e.g. hours, days, months or years after diagnosis of a disease). Determination of disease onset can be determined by any one of skill in the art according to standard tests (e.g. blood tests, physical examination, MRI, CT, ultrasound, etc.)
  • the present invention further envisions administering to the subject an additional therapy such as immunotherapy (e.g. antibody immunotherapy), immunosuppressive therapy, anti-inflammatory therapy, steroids, cytokines/interferons (e.g. beta interferons) or combinations thereof.
  • immunotherapy e.g. antibody immunotherapy
  • immunosuppressive therapy e.g. anti-inflammatory therapy
  • steroids e.g. IL-12
  • cytokines/interferons e.g. beta interferons
  • compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
  • the kit may further comprise another active ingredient to improve therapeutic efficacy.
  • the peptides of the present invention may be administered in combination with immunotherapy (e.g. antibody immunotherapy), immunosuppressive agent, anti-inflammatory agent, steroids, cytokines/interferons (e.g. beta interferons).
  • immunotherapy e.g. antibody immunotherapy
  • immunosuppressive agent e.g. anti-inflammatory agent
  • steroids e.g. beta interferons
  • cytokines/interferons e.g. beta interferons
  • the peptides of some embodiments of the invention can be packaged in one container while the immunosuppressive agent may be packaged in a second container both for therapeutic treatment.
  • a peptide comprising an amino acid sequence which comprises a consensus sequence motif selected from the group consisting of SEQ ID NOs: 12-17 and 85, wherein the peptide is capable of inhibiting T cell proliferation.
  • a pharmaceutical composition comprising a peptide comprising an amino acid sequence which comprises a consensus sequence motif selected from the group consisting of SEQ ID NOs: 12-17 and 85, wherein the peptide is capable of inhibiting T cell proliferation, and a pharmaceutically acceptable carrier.
  • the peptide is not a native peptide.
  • the peptide is capable of inhibiting T cell proliferation.
  • the peptide comprises an amino acid sequence as set forth in SEQ ID NOs: 2 and 6.
  • the peptide comprises an amino acid sequence as set forth in SEQ ID NO: 2 (also referred to herein as HTLV FPi_3 3 peptide).
  • the peptide comprises an amino acid sequence as set forth in SEQ ID NO: 2
  • the peptide is not a naturally occurring peptide (e.g. is a non-native peptide).
  • the peptide comprises at least one non-conventional amino acid, e.g., as described in Table 2 hereinabove.
  • the peptide comprises at least two, three, four, five, six, seven, eight, nine or ten non-conventional amino acids.
  • the amino acid sequence of the peptide is different in at least one amino acid from the amino acid sequence of a corresponding naturally occurring peptide.
  • the amino acid sequence of the peptide is different in at least two, three, four, five, six, seven, eight, nine or ten amino acids from the amino acid sequence of a corresponding naturally occurring peptide.
  • the peptide is not the naturally occurring peptide selected from the group consisting of SEQ ID NOs: 23-40.
  • the non-native peptide comprises an amino acid sequence which does not exceed 50 amino acids in length.
  • the amino acid sequence of the non-native peptide of some embodiments of the invention does not exceed about 50, about 45, about 40, about 35, about 30, about 25, about 24, about 23, about 22, about 21, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, or about 5 amino acids in length.
  • the amino acid sequence of the non-native peptide of some embodiments of the invention does not exceed 15 amino acids in length.
  • the amino acid sequence of the non-native peptide of some embodiments of the invention does not exceed 25 amino acids in length.
  • the amino acid sequence of the non-native peptide of some embodiments of the invention does not exceed 35 amino acids in length.
  • the amino acid sequence of the non-native peptide of some embodiments of the invention is between 5-50, between 5-40, between 5-30, between 5-20, between 5-15, between 5-10, between 5-9, between 5-7, between 7-50, between 7-40, between 7-30, between 7-20, between 7-15, between 7-10, between 7-9, between 9-50, between 9-40, between 9-30, between 9-20, between 9-15, between 9-12, between 9-11, between 15-50, between 15-40, between 15-35, between 15-30, between 15-20, between 25-50, between 25-40, between 25-35, between 25-30, between 35-50, between 35-40, or between 40-50 amino acids in length.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in
  • mice - C57B1/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). All mice were 2-3 month-old when used in the experiments.
  • the IACUC of the Weizmann Institute has approved the experiments, permit number: 03530710-3, which was performed in accordance to its relevant guidelines and regulations.
  • Cell lines - Antigen- specific T cell lines were selected in vitro [27] from primed lymph node cells derived from C57B1/6J mice that had been immunized 9 days before with antigen (100 ⁇ g myelin peptide, MOG35-55) emulsified in complete Freund's adjuvant (CFA) containing 150 ⁇ g Mycobacterium tuberculosis (Mt) H37Ra (Difco Laboratories, Detroit, MI). All T cell lines were maintained in vitro in medium containing interleukin IL-2, with alternate stimulation with the antigen every 10 to 14 days.
  • CFA complete Freund's adjuvant
  • the human T cell-line, Jurkat E6-1 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health [28].
  • RAW264.7 macrophages were obtained from ATCC (ATCC TIB-71).
  • Peptide Synthesis and Fluorescent Labeling - Peptides were synthesized using the Fmoc solid phase method on Rink amide resin (0.68 meq/gm), as previously described [29].
  • the synthetic peptides were purified (greater than 98 % homogeneity) by reverse phase high performance liquid chromatography (RP-HPLC) on a C4 or C18 column using a linear gradient of 30 - 70 % acetonitrile in 0.1 % trifluoroacetic acid (TFA) for 40 minutes.
  • RP-HPLC reverse phase high performance liquid chromatography
  • TSA trifluoroacetic acid
  • the resins were washed thoroughly with DMF and then with methylene chloride, dried under nitrogen flow, and then cleaved for 3 hours with TFA 95 %, H 2 0 2.5 %, and triethylsilane 2.5 %.
  • the Fmoc protecting group was removed from the N-terminus of the resin-bound peptides by incubation with piperidine for 12 minutes, whereas all the other reactive amine groups of the attached peptides were kept protected.
  • the resin-bound peptides were washed twice with DMF, and then treated with rhodamine-N-hydroxysuccinimide (2-fold excess), in anhydrous DMF containing 2 % DIEA, leading to the formation of a resin-bound N- rhodamine peptide. After 24 hours, the resin was washed thoroughly with DMF and then with methylene chloride, dried under nitrogen flow, and then cleaved for 3 hours with TFA 95 %, H20 2.5 %, and triethylsilane 2.5 %.
  • the labeled peptides were purified on a RP-HPLC C4 or C18 column as described above. Unless stated otherwise, stock solutions of concentrated peptides were maintained in DMSO to avoid aggregation of the peptides prior to use.
  • 96-well plates were plated onto round 96-well plates in medium containing RPMI-1640 supplemented with 2.5 % fetal calf serum (FCS), 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 50 ⁇ ⁇ - mercaptoethanol, and 2 mM L-glutamine.
  • FCS fetal calf serum
  • Each of the 96 wells had a final volume of 200 ⁇ and contained 20 x 10 3 T cells, 5 x 105 irradiated (25 gray) spleen cells (APC), and 5 ⁇ g ml of MOG p 3 5_55.
  • APC x 105 irradiated (25 gray) spleen cells
  • MOG p 3 5_55 MOG p 3 5_55
  • the present inventors initially added the MOG p 3 5_55 antigen to the APCs in a test tube, and in a second test tube added the examined FP peptides to the T cells. After 1 hour, the APCs were mixed with the T cells and further incubated for 48 hours in a 96 well round bottom plate. The T cells were pulsed with 1 ⁇ (H 3 ) thymidine, with a specific activity of 5.0 Ci/mmol, for 24 hours, and (H 3 ) thymidine incorporation was measured using a 96-well plate beta-counter.
  • the mean cpm + Standard Deviation (SD) was calculated for each quadruplicate or more.
  • the results of T cell proliferation experiments are shown as the percentage of T cell proliferation inhibition triggered by the antigen (MOG p 3 5_55 antigen) as compared to the proliferation of T cell in the absence of the FP peptide.
  • MOG p 3 5_55 antigen the antigen
  • 100 % proliferation was achieved in the presence of APC and MOG peptide and in the absence of the FP peptide.
  • 0 % proliferation was achieved when the T cells were incubated with the APC without the MOG peptide.
  • CD4 T cells were activated with pre-coated CD3 and CD28 antibodies (LEAFTM purified anti-mouse clones 145-2-Cl l and 37.51, respectively from Biolegend) at final concentration of 2 ⁇ g/ml, and in other experiments the T cells were activated with 50 ng/mL of PMA (phorbol 12-myristate 13 -acetate) together with 1 ⁇ of ionomycin (Sigma Chemical Co, Israel). Of note, PMA and ionomycin activate the T cells by cytosolic activation.
  • PMA phorbol 12-myristate 13 -acetate
  • Cytokine secretion measurements - Antigen- specific T-cells were plated onto round 96-well plates in medium containing RPMI-1640 supplemented with 2.5 % fetal calf serum (FCS), 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 50 ⁇ ⁇ - mercaptoethanol, and 2 mM L-glutamine. Each of the 96 wells had a final volume of 200 ⁇ and contained 104 T-cells, 5 x 10 5 irradiated (25 gray) spleen cells, as APC, and 5 ⁇ g/ml of MOG p35-55. In addition, the relevant peptide was added. Each treatment was made with triplicates. Analysis of IFN- ⁇ , IL-4 and TNFa secretion was performed by ELISA 24 hours after cell activation according to standard protocols from R&D systems .
  • FCS fetal calf serum
  • RAW264.7 cells were cultured overnight in DMEM supplemented with 10 % FBS, L-glutamine, sodium pyruvate, non-essential amino acids, and antibiotics in a 96-well plate (10 5 cells/ well). The following day, media were replaced by fresh DMEM, including all supplements. Cells were incubated with the peptide for 2 hours, washed and incubated with fresh media containing Pam3CSK4 for 5 hours, and then analyzed for TNFa secretion according to standard protocols from R&D systems.
  • RNA isolation and quantitative real time PCR - Antigen- specific T- cells were plated onto round 12-well plates (10 6 cells/ well) and activated with 5 x 10 5 irradiated (25 gray) APC and 5 ⁇ g/ml of MOG p35-55 in the presence or absence of relevant peptides.
  • Total RNA from cells was isolated 24 hours following activation using the NucleoSpin RNA II kit (Macherey-Nagel, Duren, Germany). 2 ⁇ g aliquot of the total RNA was reverse transcribed into cDNA using Bio-RT (Bio-Lab, Jerusalem, Israel), dNTPs and random hexamer primers.
  • qRT-PCR was performed on Step One Plus, ABI instrument (Applied Biosystems, Grand Island, NY, USA) using SYBR Green PCR Master Mix (Quanta Biosciences, Gaithersburg, MD, USA). The values for the specific genes were normalized to Rpll3a (mouse) as housekeeping controls and the data are described in arbitrary units. PCR reactions were performed in duplicate. The specific primers used for qRT-PCR are available from Sigma (U.S.A).
  • T-bet and Gata3 expression detected by FACS - Antigen- specific T-cells were plated onto round 12-well plates (10 6 cells/ well) and activated with 5 x 10 5 irradiated (25 gray) APC and 5 ⁇ g/ml of MOG p35-55 in the presence or absence of relevant peptides. Cells were washed with PBS, blocked (5 % Donkey serum, 2 % BSA and 0.1 % Triton in PBS) and fixed with 4 % Paraformaldehyde (PFA) 24 hour following activation.
  • PFA Paraformaldehyde
  • EAE Experimental Autoimmune Encephalomyelitis
  • mice were injected subcutaneously above the lumbar spinal cord with 100 ⁇ of emulsion containing 200 ⁇ g/mouse of the encephalitogenic peptide in complete Freund's adjuvant (BD-Difco) enriched with 250 ⁇ g/mouse of heat- inactivated Mycobacterium tuberculosis (BD-Difco) at 0 days post- induction (DPI).
  • BD-Difco complete Freund's adjuvant
  • the HTLV FP was dissolved in PBS and added to the emulsion (1 mg/kg).
  • Pertussis toxin (Enzo Life Sciences) at a dose of 300 ng per mouse was injected intraperitoneally immediately after the encephalitogenic injection, as well as at 0 DPI.
  • EAE disease was scored using a five-point grading with 0, for no clinical disease; 1, tail weakness; 2, paraparesis (incomplete paralysis of one or two hind limbs); 3, paraplegia (complete paralysis of one or two hind limbs); 4, paraplegia with forelimb weakness or paralysis; 5, moribund or dead animals. The mice were examined daily.
  • Image Stream X analysis - mMOG35-55 T cells were incubated with rho-labeled peptides, either LL37 or HTLV FPs_i3, and then loaded with the membrane fluorescent dye DiD and finally labeled FITC-CD3 antibodies.
  • the cells were fixed in 3 % paraformaldehyde in order to preserve fragile fusion events. Images were compensated for fluorescent dye overlap by using single-stain controls. Cells were gated for single cells or doublets using the area and aspect ratio features, and for focused cells using the Gradient RMS feature.
  • Co-localization of the fully infectious virus and each peptide was determined using the similarity feature, which calculates the log-transformed Pearson's correlation coefficient between the two stainings, on a pixel by pixel basis. Localization of the peptide during fusion events was measured using the max contour position feature, which calculates the location of the highest intensity concentration of the staining relative to the entire cell mask (this was done on a threshold mask which takes the top 80 % intensity pixels, to eliminate staining background noise). Values closer to 1 represent the periphery of the doublet and closer to 0 the fusion interface.
  • Cytotoxicity Assay (XTT Proliferation) - Aliquots of HEK cells were distributed onto a 96-well plate (Falcon). Once reaching 70 % confluence, various peptide concentrations were added and incubated for additional 24 hours. Wells with medium only served as blank and wells with cells and medium served as 100 % survival controls. After incubation, the XTT reaction solution (sodium 3'-l-(phenyl-aminocarbonyl)-3,4- tetrazolium-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate and N-methyl dibenzopyrazine methyl sulfate, mixed in a proportion of 50: 1) was added for 6 additional hours. Optical density was read at a 450 nm wavelength in an enzyme-linked immunoabsorbent assay plate reader. All assays were performed in triplicates.
  • CD Circular Dichroism
  • Bioinformatics analysis A database was created from all the universal protein resource (UniProt) entries a transmembrane protein of the envelope of HIV and SIV. These protein sequences were then run in a motif-based sequence analysis tool (MEME) which analyzes sequences for similarities among them and produce a description (motif) for each pattern it discovers. A motif which is short and unique in the protein was sought; therefore the search was limited for a motif up to 10 residues long that does not repeat more than one time in the transmembrane protein. The motifs found were automatically arranged by their E-value, which is an estimate of the expected number of motifs with the given log likelihood ratio that one would find in a similarly sized set of random sequences.
  • E-value is an estimate of the expected number of motifs with the given log likelihood ratio that one would find in a similarly sized set of random sequences.
  • the log likelihood ratio is the logarithm of the ratio of the probability of the occurrences of the motif given the motif model versus their probability given the background model.
  • the background model here is a 0-order Markov model using the background letter frequencies.
  • the ⁇ ' axis is in BITS, which is equal to the relative entropy of the motif relative to a uniform background frequency model.
  • the relative entropy of the motif, computed in bits and relative to the background letter frequencies, is equal to the log- likelihood ratio (llr) divided by the number of contributing sites of the motif times l/ln(2).
  • the MEME results plus the flanking regions where ran through WebLogo which is designed to generate sequence logos, as a method for graphical representation of amino or nucleic acid sequence alignment.
  • FPs active fusion peptides
  • the present inventors synthesized the entire FP peptides of viruses from different species and genera ( Figure 1A, the HIV, HTLV, Measles, BLV (Bovine leukemia virus) and JDV (Jembrana disease virus) FPs). The ability of these FPs to inhibit T cell proliferation was measured using T cell proliferation assays.
  • Figure IB reveal that the most potent FP peptide in inhibiting T cell proliferation was that of the Human immunodeficiency virus (HIV).
  • HAV Human immunodeficiency virus
  • the present inventors focused on the human T-lymphotropic virus (HTLV) due to its FP activity, similarity, and phylogenetic proximity to HIV.
  • HTLV human T-lymphotropic virus
  • HTLV FP Thl-specific cytokines Interferon- ⁇ (IFN- ⁇ ), Lymphotoxin-a (LT-a) and the Thl key mediator Stat4 [33, 34] ( Figure 1C).
  • IFN- ⁇ Interferon- ⁇
  • LT-a Lymphotoxin-a
  • Stat4 Thl key mediator
  • the HTLV FP elevated the expression of the Th2-specific cytokines IL-4 and IL-10 ( Figure 1C). Yet, Tumor necrosis factor a (TNF-a), that is expressed by both subsets [35], was not affected ( Figure 1C).
  • TNF-a Tumor necrosis factor a
  • HTLV's FP has the ability to inhibit T cell proliferation
  • the present inventors investigated this peptide in order to obtain the minimal active segment.
  • new peptides were synthesized and tested for their ability to inhibit T cell proliferation.
  • candidate peptides for the active segment were selected by utilizing the NPS (Network protein sequence analysis) secondary consensus prediction method [26] on the HTLV's FPi_3 3 sequence ( Figure 6). Taking the secondary consensus prediction into consideration, two new peptides were synthesized and are shown in Figure 2A.
  • the FPs_i 3 peptide consists of only the helical predicted section of the HTLV FP. This peptide is located on the same region, previously found to be the active segment of the HIV FP, both at the 5-13 amino acid (aa) section
  • the other peptide, the FP 9 _ 2 2 was synthesized to overlap only part of the predicted helical section and consists of two consecutive repeats of the known GxxxG-like dimerization motif [36, 37]. These peptides together comprise most of the native HTLV FP sequence.
  • the actual secondary structures of the peptides were determined using circular dichroism (CD) ( Figure 2B).
  • FIG. 2B and 2C shows the structure of the newly found active HTLV FPi_ 33 , and the structure of FPs_i 3 and FP 9 _ 22. It is apparent that both the active FPi_ 33 and FPs_i 3 are mainly a-helical while FP 9 _ 22 is practically a random coil. The ability of FPi_ 33 and FPs_i 3 to suppress T cell activation was assessed via a T cell proliferation assay. Both FPi_ 33 and FPs_i 3 exhibit high levels of inhibition (Figure 2D).
  • FPi_ 33 and FPs_i 3 are highly similar and that the active motif of the HTLV's FP is concealed within the FPs_i 3 region, as in the case of the HIV's FP.
  • HTLV FP 5 Specificity and Activity of the HTLV FP 5 . 13 - Initially, both HTLV s and HIV's FP were examined in a dose dependent manner for their effect in T cell proliferation. The results show that these FPs are able to inhibit T cell proliferation with no statistical significant difference between them at different concentrations (Figure 3A). This indicates that HTLV's FP has a similar potential to that of HIV in inhibiting T cell proliferation. To verify that the results of T cell inhibition by the native HTLV FPs_i 3 were not due to peptide toxicity, a XTT proliferation assay was performed.
  • HTLV FUSION PEPTIDE EXHIBITS HIGHER AFFINITY TO T CELLS HTLV FP5.13 peptide exhibits higher affinity to T cells than to B cells or macrophages -
  • affinity of the peptides for lymphocyte populations was examined. Different Rho- labeled peptides were introduced to cells isolated from a naive mouse C57BL/6J spleen. The HTLV FP 5 _i 3 peptide was compared with the control peptide LL37 (SEQ ID NO: 10), which is an unrelated antimicrobial peptide [38].
  • the present inventors gated only on the lymphocytes that were labeled with rhodamine ( Figures 3C and 3D); then the percentage of the labeled cells that were T cells or B cells was examined utilizing antibodies for the CD3 and B220 markers, respectively ( Figure 3E).
  • the HTLV FP 5 _i 3 demonstrated much higher affinity for T cells than B cells (22-fold).
  • the LL37 control peptide demonstrated similar affinity for both lymphocyte populations. This result shows that the HTLV FP has the ability to specifically interact with particular cell types, i.e., T cells rather than B cells.
  • HTLV FPi_ 33 specificity was then tested by assessing its inhibitory activity on activated macrophages.
  • RAW264.7 cells were stimulated using the TLR1/2 ligand Pam3CSK4 and the effect of HTLV FP treatment on TNF-a secretion was measured by ELISA.
  • the HTLV FP had no effect on TNF-a secretion from cultured macrophages ( Figure 3F), demonstrating that the peptide selectively inhibits T cell activation.
  • the HTLV FP5.13 is also localized to the membrane - In order to establish the
  • HTLV FP mode of action the present inventors conducted T cell proliferation assays while activating the T cells in different steps of their signaling cascade.
  • the data demonstrates that HTLV's FP inhibits T cell proliferation induced by TCR, CD3 and CD28 antibodies and even by phorbol 12-myristate 13-acetate (PMA) and ionomycin (IONO) activation (Figure 3B).
  • the HIV's FP potency however diminished significantly when the activation was downstream to the TCR (note the less efficient inhibition of T cells proliferation by HIV FP peptides when the T cells were activated with CD3/CD28 or PMA and IONO as compared to when the T cells were activated with the APCs and MOG peptide; Figure 3B).
  • HTLV FP can inhibit the cytosolic activation induced by PMA and IONO, focused the present inventors' attention to the peptide localization site.
  • the present inventors synthesized a peptide with opposite chirality, completely composed of D-amino acids. The results show that both enantiomers inhibited T cell proliferation in a similar manner without a statistical significance between them ( Figure 4E).
  • fluorescence assays were conducted to validate the localization site of the peptide.
  • T-bet and Gata3 expression To determine whether the transition in cytokine pattern driven by the HTLV FP is indicative of a Thl to Th2 shift, the present inventors examined the expression of Thl and Th2 specific transcription factors, T-bet and Gata3 respectively, via FACS analysis. C57BL/6J mMOG(35-55)-specific primary T cells were activated using APC, collected at 0, 24, 48 and 72 hours and stained for T-bet and Gata3. The HIV FP, a known T cell inhibitor [22], was used as a control peptide. Initially, T-bet expressing lymphocytes were gated (Figure 9A).
  • the HIV's FP is known to inhibit T cell proliferation by folding in the membrane into a ⁇ -sheet structure and interact with the a-helical core peptide (CP) of the TCR transmembrane domain. It is a peptide derived from the transmembrane domain of the TCR (CP sequence- GLRILLLLKV, SEQ ID NO: 84) [22, 43, 44].
  • CP a-helical core peptide
  • CP sequence- GLRILLLLKV SEQ ID NO: 84
  • the HIV's FP interacts with the CP, therefore it is only able to inhibit proliferation induced via the TCR.
  • the experiments described herein revealed that unlike HIV's FP, the HTLV's FP inhibits T cell proliferation induced by CD3 and CD28 antibodies and even by PMA and Ionomycin ( Figure 3B). These experiments negate the notion of similar mode of action, as both the ⁇ -sheet structure of the HIV's FP is vital for its function and the different inhibitory abilities imply different counter parts for the HIV and HTLV FP.
  • FPs fusion peptides
  • FPs fusion peptides
  • FPs fusion peptides
  • Table 5 provides a list of fusion peptides (FPs) from various viruses, all of which comprising the FP motif 3 sequence (FP motif 3: xGxxWIPxFGPx; SEQ ID NO: 14).
  • FPs fusion peptides from various viruses, all of which comprising the FP motif 4 sequence (FP motif 4: AVPxAxWLVSALAxGxGxAGx; SEQ ID NO: 15).
  • TMD motif 1 ISxIMGPLxxLLLILLFGPCI; SEQ ID NO: 16
  • TMD motif 2 QTGITxxALxLLxIxxGPC; SEQ ID NO: 17
  • both the HTLV TMD peptide (SEQ ID NO: 22, which includes the consensus sequence set forth by SEQ ID NO: 17) and the MUMPS FP peptide (SEQ ID NO: 18, which includes the consensus sequence set forth by SEQ ID NO: 12) were capable of inhibiting proliferation of T cells.
  • Table 10 shows that both the HTLV TMD peptide (SEQ ID NO: 22, which includes the consensus sequence set forth by SEQ ID NO: 17) and the MUMPS FP peptide (SEQ ID NO: 18, which includes the consensus sequence set forth by SEQ ID NO: 12) were capable of inhibiting proliferation of T cells.
  • the peptides described in Table 10 above were tested for their ability to inhibit T-cell activation.
  • the Mumps FP SEQ ID NO: 18, which includes the consensus sequence set forth by SEQ ID NO: 12
  • Measles FP SEQ ID NO: 86, which includes the consensus sequence set forth by SEQ ID NO: 12
  • Human parainfluenza virus HPIV
  • SEQ ID NO: 87 which includes the consensus sequence set forth by SEQ ID NO: 12
  • the Ebola FP (SEQ ID NO: 60, which includes the consensus sequence set forth by SEQ ID NO: 14) had no inhibitory effect on T-cell proliferation while the HIV FP (SEQ ID NO: 1) which is a known suppressor of T-cell activation inhibited T-cell proliferation (Figure 14A).
  • FP One of the envelope segments utilized by HIV to evade the immune response is its FP [22], thus raising the possibility that FPs from other viruses might posses a similar function.
  • the present inventors aimed to determine whether immune evasion by the envelope FP is a common viral trait.
  • several viral FPs were synthesized and examined for their ability to inhibit T cell activation.
  • the HTLV-1 FP was found to be the most potent suppressor of T cells activation.
  • HTLV-1 FP, HTLV-1 TMD, Mumps FP, Measles FP and hPIV FP were all found to suppressor T cells proliferation without affecting their viability.
  • the active segment of the HTLV's FP was identified at the 5-13 aa region, which similarly to the entire FP possesses mainly a-helical structure. Subsequently, the active segments of HTLV's and HIV's FPs were compared for their ability to inhibit T cell activation in a dose dependent manner and were found to be equally active and that the activity is not due to toxicity. Once the ability of the HTLV FPs_i3 to inhibit T cell proliferation was validated, the present inventors examined its tropism and found that it preferentially binds T cells over B cells.
  • the present inventors conducted T cell proliferation assays while activating the T cells in different steps of their signaling cascade.
  • the data demonstrates that the HTLV FP inhibits T cell proliferation induced by antigen presentation, CD3 and CD28 antibodies and even the cytosolic activation induced by PMA and IONO.
  • the present inventors assessed whether it acts in the membrane. It has been suggested in several studies that interactions within the membrane can be chiraly independent [36, 39, 40], therefore, the present inventors synthesized an HTLV FPs_i3 peptide with opposing chirality.
  • Both HTLV FPs_i3 enantiomers inhibited T cell proliferation in a similar manner without a statistical significance between them, suggesting that the HTLV FPs_i3 is active in the membrane.
  • the present inventors thereafter preformed image stream analysis to visualize the site of the peptide interaction with the cells.
  • Both HTLV FP and the control LL37 peptide were found to have similar distribution compared to either a membrane dye (DID) or CD3 receptor. As LL37 is known to associate with the membrane, these results further support the notion that the HTLV FP site of action is in the membrane.
  • the present inventors have shown that not only HIV, but HTLV as well is able to utilize its FP in order to modulate the immune response. Nonetheless, the FP is not the only region in the envelope with immunosuppressive properties. Without being bound by any theory, other elements in the envelope may also contribute to the virus' ability to modulate the immune response. Additionally, without being bound by any theory the efficacy of the HTLV FPs_i 3 D-enantiomer may have clinical importance in treating T cell mediated autoimmune disease, since the D-enantiomer is not cleaved by endogenous proteases and might have a prolonged half-life to exert its therapeutic effect.
  • a peptide comprising residues 35-55 of mouse myelin oligodendrocyte glycoprotein was injected to mice subcutaneously above the lumbar spinal cord with 100 ⁇ of emulsion containing 200 ⁇ g/mouse of the encephalitogenic peptide in complete Freund's adjuvant enriched with 250 ⁇ g/mouse of heat-inactivated Mycobacterium tuberculosis at 0 days post-induction (DPI).
  • Pertussis toxin at a dose of 300 ng per mouse was injected intraperitoneally immediately after the encephalitogenic injection, as well as at 0 DPI. It should be noted that symptoms of multiple sclerosis often appear about 5-10 days after administration of the MOG 3 5_55 peptide.
  • mice Two groups of 10 mice each were examined where one group received the HTLV FPi_ 33 fusion peptide (FP) together with EAE induction (i.e. MOG 3 5_5s) and the second group received PBS (phosphate buffered saline) with MOG 3 5_55.
  • FP HTLV FPi_ 33 fusion peptide
  • PBS phosphate buffered saline
  • mice treated with HTLV FPi_ 33 peptide (as set forth in SEQ ID NO: 2) along with EAE induction showed a marked reduction in disease progression and in disease severity as compared to mice not treated with the HTLV FPi_ 33 peptide (i.e. saline group). This is evident by both low clinical score and very low weight loss ( Figures 13A-B, respectively).
  • a comparison of the cumulative EAE score, maximal EAE score and cumulative initial weight further revealed the HTLV FP's clinical efficacy in treating EAE in mice ( Figure 13C-E).
  • the adjuvant arthritis animal model (AA, a model of rheumatoid arthritis) includes the rat heat-killed Mt strain H37Ra-induced AA [previously described in e.g. Kannan, Theor Biol Med Model. (2005) 2: 17].
  • mice and rat models of Type-l-Diabetes-Melitus are used, such as by administration of toxins, e.g. streptozotocin and alloxan, which induce hyperglycaemia [previously described in Rees et al. Diabet Med. 2005 Apr;22(4):359-70].
  • toxins e.g. streptozotocin and alloxan
  • a psoriasis mouse model(s) is used such as described in Boehncke and Schon, Clin Dermatol. 2007 Nov-Dec;25(6):596-605.

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Abstract

L'invention concerne des procédés d'inhibition de la prolifération d'une cellule T (ou lymphocytes T) par mise en contact de la cellule T avec un peptide comprenant une séquence d'acides aminés qui comprend un motif de séquence consensus choisi dans le groupe constitué de SEQ ID NO: 12-17 et 85, ladite séquence d'acides aminés étant capable d'inhiber la prolifération des lymphocytes T. L'invention concerne également des procédés et des compositions pharmaceutiques pour le traitement d'une maladie auto-immune à médiation par les lymphocytes T. L'invention concerne également un peptide comprenant une séquence d'acides aminés qui comprend un motif de séquence consensus choisi dans le groupe constitué par les SEQ ID NO: 12-17 et 85, le peptide étant capable d'inhiber la prolifération des lymphocytes T.
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