WO2007135684A2 - Method of treatment of anti-cd4 autoimmunity - Google Patents

Abstract

Disclosed by the present application are peptides or polypeptides comprising a stretch of contiguous amino acids consisting of at least 5 amino acids overlapping with a corresponding stretch in CD4 protein, wherein said stretch of contiguous amino acids is characterized by the ability to elicit a T-cell mediated autoimmune response against CD4+ T-cells in a patient with T-cell mediated autoimmunity or to impart on said peptide or polypeptide the ability to elicit a T-cell mediated autoimmune response against CD4+ T-cells in a patient with T-cell mediated autoimmunity. Also disclosed are various applications of these peptides and polypeptides in therapy and diagnostics.

Description

METHOD OF TREATMENT OF ANTI-CD4 AUTOIMMUNITY

FIELD OF THE INVENTION The present invention relates to immunotherapy and in particular to treatment of anti-CD4 autoimmunity, specifically in HIV-infected patients as well as to products for said treatment.

BACKGROUND OF THE INVENTION

The hallmark of HIV-I infection is a continuous attrition of CD4 T-cells, leading progressively to immune deficiency [1, 2]. Infection with HIV leads to direct destruction of T-cells by the virus, however, other indirect effects of HIV on the responding immune system may also participate in the CD4 decline. In several patients, CD4 T-cell leukopenia persists despite a reduction in the viral load achieved by highly active antiretroviral therapy

(HAART) [3]. An additional mechanism of lymphocyte killing by HIV has been attributed to an autoimmune cytotoxic T-cell response against the T-cells' CD4 molecule triggered by HIV infection [4-10].

Salemi et al. [8] suggested that HIVgpl20 activates autoreactive CD4-specific

T-cell responses by unveiling of hidden CD4 peptides during processing. Using overlapping synthetic peptides spanning the entire CD4 molecule they identified two adjacent peptides that were recognized by T-cell clones generated from a CD4 responsive individual (amino acid positions 73-96).

T-cell vaccination has recently evolved as a promising therapeutic modality for the treatment of autoimmune conditions. Immunization with inactivated autoreactive T-cells may induce a reaction to deplete specific subsets of autoreactive T-cells involved in the autoimmune disease. T-cell vaccination has been used clinically to induce down-regulation of the autoimmune activity, e.g. in multiple sclerosis and SLE [11-14].

Atlan et al. [15, 16] have suggested that T-cell vaccination could also have a therapeutic effect in HIV infected patients. Phase I clinical trial of T-cell vaccination (TCV) in HIV-I infected patients manifesting anti-CD4 autoimmunity were performed by Abulafia-Lapid et al [17-18].

Vaccination of these patients with autologous anti-CD4 reactive T-cells resulted in a persistent increase in the patients' blood CD4 T-cell levels and a concomitant decrease in anti-CD4 autoimmunity.

SUMMARY OF THE INVENTION

In accordance with a first aspect, there is disclosed herein a peptide or polypeptide comprising a stretch of contiguous amino acids consisting of at least 5 amino acids overlapping with 5 contiguous amino acids from a CD4 protein, wherein said stretch of contiguous amino acids is characterized by the ability to elicit a T-cell mediated autoimmune response against CD4+ T-cells in a patient with T-cell mediated autoimmunity or to impart on said peptide or polypeptide the ability to elicit a T-cell mediated autoimmune response against CD4+ T-cells in a patient with T-cell mediated autoimmunity. In accordance with a further aspect, there is disclosed herein a method of enriching a T-cell preparation, comprising exposing a population of T-cells from a subject with a T-cell mediated autoimmune response to a peptide or polypeptide disclosed herein and providing conditions for selective cell proliferation of T-cells reactive with said peptide or polypeptide. Further disclosed is an enriched T-cell population obtainable by the method disclosed herein.

Yet further disclosed herein is a T-cell vaccine comprising the enriched T-cell population disclosed herein.

Also disclosed is a method of treating a patient with T-cell mediated autoimmunity, comprising administering to said patient a T-cell vaccine disclosed herein.

Further disclosed is a method for determining whether T-cells from a subject include cells that cause an anti-CD4+ autoimmune response, comprising contacting a sample containing T-cells of said subject with a peptide or polypeptide disclosed herein, and determining either (i) binding of said peptide or polypeptide to T-cells in said sample, or (ij) T-cell proliferation responses.

In addition, disclosed is a conjugate comprising the peptide or polypeptide disclosed herein, conjugated to a cytotoxic moiety. A method of detecting T-cell mediated autoimmunity in a patient is also disclosed, the method comprising: obtaining peripheral mononuclear blood cells (PMBC) from said patient; culturing said PMBC in the presence of a peptide or polypeptide disclosed herein. providing said cells with conditions for selective cell proliferation of T-cells reactive with said peptide or polypeptide; wherein a proliferation response indicates a T-cell specific autoimmune response in said patient.

Yet further, disclosed is a kit for detecting T-cell mediated autoimmune response in a subject comprising at least one peptide or polypeptide as disclosed herein.

Finally, there is disclosed a T-cell receptor (TCR) peptide complementary with said peptide or polypeptide as well as a vaccine comprising the TCR peptide for use in therapeutic vaccination of immune deficient patients, in particular, HIV 1 infected patients.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1A-1B are graphs showing proliferation of T-cells obtained from HIV-I patients detailed in Table 3 (Fig. IA) and healthy subjects (Fig. IB) in response to different CD4 peptides. S.I. represents Stimulation Index.

DETAILED DESCRIPTION The present invention is based on the finding that peptides derived from the CD4 protein evoke an autoimmune response in HIV-I infected patients. The immunogenic peptides were identified by analyzing T-cell proliferation responses towards a set of synthetic CD4-derived peptides. T-cells were obtained from several HIV infected patients and healthy subjects and the proliferation responses were compared. Specifically, a set of autoimmune peptides, specific to HIV-I infected patients were identified.

Thus, according to a first of its aspects there are disclosed isolated peptides or polypeptides comprising a stretch of contiguous amino acids, the stretch consisting of at least 5 amino acids overlapping with a stretch of contiguous amino acids from the CD4 protein (preferably the human CD4 protein), the peptide or polypeptide capable of eliciting a T-cell mediated autoimmune response against CD4⁺ T-cells in patients with anti CD4 autoimmunity. In accordance with yet another embodiment of the invention, there are disclosed isolated peptides or polypeptides comprising a stretch of contiguous amino acids consisting of at least 5 overlapping amino acids from the CD4 protein, said stretch imparting on said peptide or polypeptide the ability to elicit a T-cell mediated autoimmune response against CD4+ T-cells in a patient with T-cell mediated autoimmunity.

According to a preferred embodiment, said CD4 protein is human CD4 protein.

The term "T-cell mediated autoimmunity" denotes a condition where a subject exhibits a T-cell mediated immune response against self antigens. A non-limiting list of conditions manifesting a T-cell mediated autoimmune response include, without being limited thereto, human immunodeficiency virus (HIV) infection, multiple sclerosis (MS), diabetes (type 1 diabetes), systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA). According to one particular embodiment, T-cell mediated autoimmunity comprises an immune response against a subjects own CD4⁺ T-cells.

According to one embodiment, said patient is an HIV-I subtype B or C infected patient, although other subtypes are also included within the scope of the present invention.

The term "peptide" or "polypeptide" as used herein denote any amino acid sequence comprising a stretch of contiguous amino acids consisting of at least 5 overlapping amino acids from CD4 protein (the "original" native sequence). The stretch of amino acids may also include modifications of one or more amino acids from the CD4 protein sequence as long as the resulting stretch, and consequently, the resulting peptide or polypeptide exhibits a similar immunogenic character (the immunogenic character may be stronger or lower in the resulting peptide or polypeptide). According to one embodiment, the CD4 protein is a human CD4 protein.

The term "at least S overlapping amino acids " denotes a sequence of at least 5 contiguous amino acids within said peptide or polypeptide which overlap (have per residue sequence identity) with at least 5 contiguous amino acids from the CD4 protein, preferably front the human CD4 protein. The term "immunogenic character" concerns at least a capacity of the peptide or polypeptide to elicit, under suitable conditions, a T-cell proliferation response towards the peptide or polypeptide. The conditions may be in vitro conditions as well as within a mammal's (e.g. human) body.

The term "substitution" includes generally the replacement of one or more amino acid residues either by other naturally occurring amino acids, (conservative and non- conservative substitutions), by non-naturally occurring amino acids (conservative and non-conservative substitutions), or with organic moieties which serve either as true peptidomimetics (i.e., having the same steric and electrochemical properties as the replaced amino acid), or merely serve as spacers in lieu of an amino acid, so as to keep the spatial relations between the amino acid spanning this replaced amino acid.

The term "conservative substitution" in the context of the present invention refers to the replacement of an original amino acid present in the identified peptide with a naturally or non-naturally occurring amino or a peptidomimetic residue having similar steric properties. Where the side-chain of the original amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side- chain of the replaced amino acid). However where the original amino acid to be replaced is charged, the conservative substitution according to the definition of the invention may be with a naturally occurring amino acid, a non-naturally occurring amino acid or a peptidomimetic moiety which are charged, or with non-charged (polar, hydrophobic) amino acids that have the same steric properties as the side-chains of the replaced amino acids. The purpose of such a procedure of maintaining the steric properties but decreasing the charge is to decrease the total charge of the compound, for example for improving its membrane penetrating properties.

For example in accordance with the invention the following substitutions are considered as conservative: replacement of arginine by cytroline; arginine by glutamine; aspartate by asparagine; glutamate by glutamine.

As the naturally occurring amino acids are grouped according to their properties, conservative substitutions by naturally occurring amino acids can be easily determined bearing in mind the fact that in accordance with the invention replacement of charged amino acids by sterically similar non-charged amino acids are considered as conservative substitutions.

The following are some non-limiting examples of groups of naturally occurring amino acids or of amino acid analogs. Replacement of one member in the group by another member of the group will be considered herein as conservative substitutions:

Group I includes leucine, isoleucine, valine, methionine, phenylalanine, serine, cysteine, threonine and modified amino acids having the following side chains: ethyl, n- butyl, -CH₂CH₂OH, -CH₂CH₂CH₂OH, -CH₂CHOHCH₃ and-CH₂SCH₃. Preferably Group I includes leucine, isoleucine, valine and methionine.

Group II includes glycine, alanine, valine, serine, cysteine, threonine and a modified amino acid having an ethyl side chain. Preferably Group II includes glycine and alanine.

Group III includes phenylalanine, phenylglycine, tyrosine, tryptophan, cyclohexylmethyl, and modified amino residues having substituted benzyl or phenyl side chains. Preferred substituents include one or more of the following: halogen, methyl, ethyl, nitro, methoxy, ethoxy and -CN. Preferably, Group III includes phenylalanine, tyrosine and tryptophan.

Group IV includes glutamic acid, aspartic acid, a substituted or unsubstituted aliphatic, aromatic or benzylic ester of glutamic or aspartic acid (e.g., methyl, ethyl, n- propyl iso-propyl, cyclohexyl, benzyl or substituted benzyl), glutamine, asparagine, CO- NH-alkylated glutamine or asparagine (e.g., methyl, ethyl, n-propyl and iso-propyl) and modified amino acids having the side chain -(CH₂)₃COOH, an ester thereof (substituted or unsubstituted aliphatic, aromatic or benzylic ester), an amide thereof and a substituted or unsubstituted N-alkylated amide thereof. Preferably, Group IV includes glutamic acid, aspartic acid, glutamine, asparagine, methyl aspartate, ethyl aspartate, benzyl aspartate and methyl glutamate, ethyl glutamate and benzyl glutamate.

Group V includes histidine, lysine, arginine, N-nitroarginine, β-cycloarginine, μ- hydroxyarginine, N-amidinocitruline and 2-amino-4-guanidinobutanoic acid, homologs of lysine, homologs of arginine and ornithine. Preferably, Group V includes histidine, lysine, arginine, and ornithine. A homolog of an amino acid includes from 1 to about 3 additional methylene units in the side chain.

Group VI includes serine, threonine, cysteine and modified amino acids having C1-C5 straight or branched alkyl side chains substituted with -OH or -SH. Preferably, Group VI includes serine, cysteine or threonine.

The term "non-conservative substitutions" concerns replacement of one or more amino acid residues present in the original molecule by another naturally or non-naturally occurring amino acid, having a different size, configuration and/or electronic properties compared with the amino acid being substituted. Thus, the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the original amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted.

According to the invention modification may also involve non-conservative substitutions, as long as the immunogenic character of the peptide is retained. Conservative substitutions are nevertheless preferable.

"Peptidomimetic organic moiety" can be substituted for amino acid residues in the peptides of the invention both as conservative and as non-conservative substitutions. The peptidomimetic organic moieties often have steric, electronic or configurational properties similar to the replaced amino acid. The peptidomimetics can be produced by organic synthetic techniques. Examples of suitable peptidomimetics include D amino acids of the corresponding L amino acids, tetrazol [Zabrocki et al, J. Am. Chem. Soc. 110:5875-5880 (1988)]; isosteres of amide bonds [Jones et al, Tetrahedron Lett. 29: 3853-3856 (1988)]; LL-3-amino-2- propenidone-6-carboxylic acid (LL-Acp) [Kemp et al, J. Org. Chem. 50:5834-5838 (1985)]. Similar analogs are shown in Kemp et al, Tetrahedron Lett. 29:5081-5082 (1988) as well as Kemp et al, Tetrahedron Lett. 29:5057-5060 (1988), Kemp et al, Tetrahedron Lett. 29:4935-4938 (1988) and Kemp et al, J. Org. Chem. 54:109-115 (1987). Other suitable peptidomimetics are shown in Nagai and Sato, Tetrahedron Lett. 26:647-650 (1985); Di Maio et al, J. Chem. Soc. Perkin Trans., 1687 (1985); Kahn et al, Tetrahedron Lett. 30:2317 (1989); Olson et al, J. Am. Chem. Soc. 112:323-333 (1990); Garvey et al, J. Org. Chem. 56:436 (1990). Further suitable peptidomimetics include hydroxy- 1,2,3,4-tetrahydroisoquinoline- 3-carboxylate (Miyake et al, J. Takeda Res. Labs 43:53-76 (1989)); 1,2,3,4-tetrahydro- isoquinoline-3-carboxylate [Kazmierski et al, J. Am. Chem. Soc. 133:2275-2283 (1991)]; histidine isoquinolone carboxylic acid (HIC) [Zechel et al, Int. J. Pep. Protein Res. 43 (1991)]; (2S, 3S)-methyl-phenylalanine, (2S, 3R)-methyl-phenylalanine, (2R, 3S)-methyl- phenylalanine and (2R, 3R)-methyl- phenylalanine [Kazmierski and Hruby, Tetrahedron Lett. (1991)].

The stretch may also include chemically modified amino acids. The term "chemically modified" includes modification at the side chain of the amino acid residue, as well as modification of the peptidic bond. Accordingly, a functional group may be added to the side chain, deleted from the side chain or exchanged with another functional group. Typically, the modifications are conservative modifications resulting in conservative substitution. Examples of conservative modifications of this type include adding an amine or hydroxyl, carboxylic acid to the aliphatic side chain of valine, leucine or isoleucine, exchanging the carboxylic acid in the side chain of aspartic acid or glutamic acid with an amine or deleting the amine group in the side chain of lysine or ornithine.

Other chemical modifications known in the art include arboxymethylation, ^' acylation, phosphorylation, glycosylation or fatty acylation, and others. The amino acid stretch may also include modifications with respect to the original sequence at the peptide backbone, i.e. that the bond between the N- of one amino acid residue to the C- of the next has been altered to non-naturally occurring bonds. Examples of such modifications include, without being limited thereto, reduction (to -CH₂-NH-), alkylation (methylation) on the nitrogen atom, or the bonds have been replaced by amidic bond, urea bonds, or sulfonamide bond, etheric bond (-CH₂-O-), thioetheric bond (-CH₂-S-), or to -CS-NH-; The side chain of the residue may be shifted to the backbone nitrogen to obtain N-alkylated-Gly (a peptidoid).

The peptide or polypeptide disclosed herein may be a linear sequence as well as a cyclic peptide. Cyclization of the amino acid molecule may be performed by S-S bonds. S-S bonds may be formed via the inclusion of sulphor-containing amino acid residues, such as cysteine at each terminus of the amino acid molecule. Cyclic peptides have been shown to be more stable and with higher biological activity than the corresponding linear molecule [Tibbetts S. et al. Peptides. 21(8)1161-7 (2000)]. According to a preferred embodiment, the peptide or polypeptide comprises one or more amino acid sequence comprising a stretch of at least five contiguous amino acids derived or comprise a peptide marked in the following Table 1 as Pl, P4, P21, P28 and P29 and which comprise the following respective sequences:

Pl: MNRGVPFRHLLLVLQLALLP (SEQ ID NO:1) P4: KKSIQFHWKNSNQIKILGNQ (SEQ ID NO :4)

P14: KIDIVVLAFQKASSIVYKKE (SEQ IDNO:14)

P21: LEAKTGKLHQEVNLVVMRAT (SEQ ID NO:21)

P28: AGLLLFIGLGIFFCVRCRHR (SEQ ID NO:28)

P29: RCRHRRRQAERMSQIKRLLS (SEQ ID NO:29) More preferably, the peptide or polypeptide disclosed herein is that comprising a stretch of at least 5 contiguous amino acids comprises one or more of a SEQ ID NO: 1, 4, 14, 21, 28 or 29. AU these sequences were shown to elicit a proliferation response in T- cells obtained from HIV-infected patients and not in T-cells obtained from healthy (namely, HIV-negative) subjects. Also disclosed herein is a method of enriching a T-cell preparation, comprising expqsing a population of peripheral mononuclear blood cells (PMBC) from a patient T- cell mediated autoimmunity to a peptide or polypeptide as disclosed herein and providing conditions allowing for selective cell proliferation of T-cells reactive with said peptide or polypeptide.

In the following description T-cells reactive with said peptide or polypeptide may, at times, be generally referred to as CD4 peptide-recognizing T-cells or in short, CD4 recognizing T-cells.

The enrichment method in accordance with one embodiment disclosed herein comprises the following steps: providing peripheral mononuclear blood cells (PBMC) from a subject; culturing said PBMC with one or more peptides or polypeptides as disclosed herein under conditions allowing selective cell proliferation of T-cells reactive with said peptide or polypeptide. The term " conditions for selective cell proliferation" includes the addition to the

T-cell culture a media including substances required for the growth of said cells. The media should preferably include at least one growth stimulating factor for stimulating the growth of said cells. Growth factors for stimulating cell proliferation and cell expansion are known to those versed in the art . Such growth factors will be selected according to their ability to facilitate cell expansion essentially without inducing cell differentiation.

A non-limiting list of growth factors suitable for stimulating T-cell production includes phytohemmaglutinin (PHA, a mitogen known to induce non-specific T-cell proliferation), anti-CD3, and anti-CD28 (antibodies known to induce non-specific T-cell proliferation, [see, 25, incorporated herein in its entirety by reference]) IL-2 (specific for T-cell expansion) and any combinations of same as well as combinations of same with other growth factors.

According to one preferred embodiment, the stimulating factor is phytohemmaglutinin (PHA). The addition of PHA to the T-cell culture is preferably followed by the addition of other factors which facilitate expansion of the cells. For example, IL-2 may be added to the T-cell culture after initial cell growth stimulation with PHA or the like, so as to facilitate expansion of the PHA-activated T-cells.

According to a one embodiment, the conditions include the addition of irradiated CD4-pulsed antigen presenting cells (APCs) so as to "boost" the proliferation of the T- cells in the culture.

In addition or alternatively, the conditions may include the addition of beads to which the peptide or polypeptide of the invention are bound, optionally in the presence of MHC molecules [23, incorporated herein by reference, in its entirety]. Such beads may replace the need to use irradiated APCs. Also disclosed herein is an enriched CD4-recognizing T-cell preparation obtainable by the method described above. It has already been shown that during HIV infection, CD4+ T-cells become the target of CD8+ T-cells [4,5,17,18, each incorporated herein by reference, in its entirety]. Thus, according to one embodiment, the enriched CD4 recognizing T-cell preparation is enriched in CD8+ T-cells and manifest high INFγ and low IL-IO expression.

The enriched CD4-recognizing T-cell preparation may be inactivated to be used as part of a vaccine to trigger an immune response in the patient against the autoimmune T-cells Thus, also disclosed is a method of producing an enriched, inactivated CD4- recognizing T-cell preparation comprising: - providing an enriched CD4 recognizing T-cell preparation obtainable by the method as disclosed above, and inactivating said T-cells with an inactivating agent.

According to one embodiment, inactivation is performed by the use of a fixation agent. Non-limiting examples of fixation agents include glutaraldehyde, formaldehyde or paraformaldehyde. The fixating agent is then neutralized. According to one embodiment neutralization is performed in the presence of glycine. Preferably, the inactivated T-cell preparation is then washed at least once with a suitable washing solution, e.g. saline.

Alternatively, inactivation may be performed by irradiating the T-cells population (6,000-7,000 rads) prior to injection. The thus formed enriched, inactivated, T-cell preparation may then be further treated by washing and re-suspending the cells with a washing medium, such as injection- grade saline solution. The preparation may also be divided into aliquots for storage. For example, the preparation may be divided into aliquots of between 10⁶-10⁸ cells for freezing in liquid nitrogen.

Also disclosed herein is a T-cell vaccine comprising an enriched, inactivated CD4-recognizing T-cell preparation obtainable by the method disclosed above, together with a pharmaceutically acceptable carrier.

The vaccine may be combined with other active agents, such as drugs used to treat an autoimmune disorder, anti-HIV drugs, antibiotics, vitamins etc. The preparation of vaccines is well known in the art and has been described in many articles and text books see e.g. Remington's pharmaceutical sciences, Gennaro A.R. ed. Mack Publishing Co.

Easton PA, 1995.

Also disclosed is a method of treating a patient with CD4 mediated autoimmunity, comprising administering to said patient a vaccine comprising an enriched preparation of autologous inactivated CD4-recognizing T-cells obtainable by the method of the invention. As a result of treatment, an immune response is generated against the T-cell recognizing CD4 or the peptide or polypeptide derived therefrom.

Also disclosed is a method of treating an HIV-I infected patient, comprising administering to said patient a vaccine comprising an enriched preparation of autologous inactivated CD4-recognizing T-cells obtainable by the method of the invention. As a result of treatment, an immune response is generated against the T-cell recognizing CD4 or the peptide derived therefrom.

According to one embodiment, the vaccine is administered to the HIV infected patient by injection, preferably subcutaneous injection. Each injection may comprise from about 10⁶ to about 10⁸ cells/ml in saline. According to one embodiment, the vaccine comprises 10⁷ T-cells/ml saline.

Following injection the immune response is triggered as immune cells recognize the injected (autologous) autoimmune cells as "foreign". As a consequence the patient's immune system eliminates these pathologic cells which are involved in eliciting the auto mmunity from the patients blood.

The HIV patient may be provided with a single dose vaccination which may be followed by one or more doses so as to increase the response to the CD4-recognizing T- cells (boosters).

The effectiveness of treatment may be evaluated and monitored. Accordingly, HIV-infected patients exhibiting an auto-reactive response and treated in accordance with the invention are clinically followed, the follow-up including, for example, analysis of viral load, plasma CD4 counts, immunological follow-up including, for example, anti- clonotypic assay [20, 21, each incorporated herein by reference, in their entirety], anti- CD4 autoimmune assay, cytotoxicity assay and anti-recall antigens assay. All assays are known to those versed in the art and are inter alia described by Abulafia-Lapid R et al. [17-18, each incorporated herein by reference, in their entirety].

It is noted that pre-clinical and clinical studies for determining therapeutic protocols of human subjects are well known to those versed in the art. In the context of the present disclosure, pre-clinical studies may involve assaying one or more (e.g. combinations) peptides as disclosed herein in an animal model.

In accordance with one embodiment, developing an animal model may comprise the use of transgenic mice carrying the human CD4 coding sequences and expressing the same, such as those described by Blum MD et al. [Reconstitution of the subclass-speciβc expression ofCD4 in thymocytes and peripheral T cells of transgenic mice: identification of a human CD4 enhancer J Exp Med. 1993 ; 177(5): 1343-58]. The mice may be induced to develop auto-immunity to the human CD4 by injecting thereto one or more of the peptides disclosed herein. Selected peptides, such as pi, p4, pl4, p28 and/or p29 may be used as a first tier of peptides in the pre-clinical evaluation. The peptide or a combination of peptides, in a suitable carrier and in combination with a suitable adjuvant, may be injected intravenously (or by other conventional means), with optionally a subsequent booster injection (after several days or weeks, e.g. two weeks). An adjuvant may include, without being limited thereto heat killed M. tuberculosis strain H37RA (Difco) emulsified in Incomplete Freund's adjuvant (Difco). In accordance with another embodiment, anti-CD4 autoimmunity may be induced by injecting mice with one or more of the disclosed peptide, a mixture of peptides, or with rCD4 protein with an appropriate adjuvant.

Once an anti-CD4 autoimmunity model is established, the therapeutic effect of the T-cell vaccine disclosed herein may be evaluated.

The vaccine is administered in an effective amount, being an amount that is effective in achieving the desired therapeutic effect, which may be manifested by a decrease in autoimmune response, as determined by, e.g. increase CD4 cell counts in the peripheral blood. The effective amount may be determined in dose-finding clinical studies in patients with T-cell mediated autoimmunity, such as HIV patients which developed the said autoimmune response, or through extrapolation from animals (such as the mice model discussed above) using one of many such extrapolation methods readily known to those of skill in the art of clinical studies. The effective amount may depend on factor known per se such as weight, body surface, gender, disease history and status, concomitant medicines taken by the patient, severity of disease, frequency of administration, drug residence time in the plasma or blood, extent of binding of the drug by blood proteins and others.

Further disclosed is a diagnostic method, namely a method of determining whether an HIV-infected patient has an auto-reactive CD4-specific T-cell response, the method comprises contacting a sample of peripheral mononuclear blood cells (PBMC) containing T-cells of said patient with a peptide or polypeptide as disclosed herein and determining either (i) binding of said peptide or polypeptide to T-cells in said sample, or (ii) a T-cell proliferation response in the presence of said peptide or polypeptide.

In addition, disclosed herein is a conjugate comprising a peptide or polypeptide according to the invention conjugated to a cytotoxic moiety. The peptide or polypeptide will thus function as a carrier delivering the cytotoxic moiety to autoreactive T-cells. The cytotoxic moiety may be a low molecular weight compound such as a radioactive label, or a high molecular weight substance such as a toxin [24, 25, 26, each incorporated herein by reference, in their entirety]. Cytotoxic compounds which may be employed in accordance with the present disclosure will be readily appreciated by those versed in the art. .

Alternatively, the peptide or polypeptide may be associated with a liposome, e.g. by conjugating (chemically or physically) to a component at the outer surface of a liposome, or by having the liposome carrying the peptide or polypeptide its intra- liposomal compartment, or by incorporating of same in the liposomal bilayer, etc. as appreciated by those versed in biochemistry, wherein the liposome also carries said cytotoxic compound.

In one embodiment, the peptides and polypeptides disclosed herein may also be utilized to diagnose a T-cell mediated autoimmune response in a subject. Specifically, the peptides and polypeptides can be used to profile the patients' autoimmune CD4 peptide recognition pattern/profile and allow the development of a "tailor made" vaccine. There is thus also provided herein a method for detecting T-cell autoimmunity in a subject, the method comprising: (a) obtaining peripheral mononuclear blood cells (PMBC) from said subject;

(b) exposing said PMBC to a peptide or polypeptide according to the invention;

(c) providing said cells with conditions for selective cell proliferation of T- cells reactive with said peptide or polypeptide; whereby a proliferation response indicates a T-cell specific autoimmune response in said subject.

According to one embodiment, the subject is an HIV infected patient. According to another embodiment, the T-cell mediated autoimmunity is anti-CD4 mediated autoimmunity. Within this diagnostic aspect of the invention, there is also provided a kit for profiling anti CD4 autoimmunity in a subject, the kit comprising a CD4 peptide or polypeptide as disclosed herein.

In another aspect, there is provided a T-cell receptor (TCR) peptide complementary with the immunogenic CD4 peptides or polypeptides as disclosed herein. Such complementary TCR peptides can be easily identified using bioinformatics tools well, known in the art and further used for therapeutic vaccination. Thus, also disclosed herein is a composition comprising the complementary TCR peptides for use in therapeutic vaccination of immune deficient HIV 1 infected patients. Further, disclosed is thus a method of vaccination of such patients using the vaccine composition, optionally in combination with anti-retroviral treatment or the T-cell vaccine of the invention.

DESCRIPTION OF SOME EXEMPLARY EMBODIMENTS EXAMPLE 1 - In vitro study Materials And Methods Study participants

Twenty five HIV-I subtype B infected patients (Pl to P25) with age range of 24 to 63 (median age 40 years) were recruited from the Kaplan Hospital AIDS Center (Rehovot, Israel) and the Hadassah University Hospital in Jerusalem, Israel. The duration of their infection from time of diagnosis ranged from 0.3 months to 16 years (median, 7.0 years). All of the patients (but one) were treated according to the (HAART regimen, receiving a highly active anti-retroviral therapy), and the duration of treatment varied (median, 2.3 years). Their CD4 T- cell numbers ranged from 93 to 679 cells/ml blood (mean 387, medial 376) and their plasma viral loads (Amplicor, Hoffman-LaRoche) from <400 to 59,300 copies/ml (mean 16,307, median 14,450). Blood was also obtained from 24 HIV-sero-negative (Cl to C24) healthy donors, through the Tel Hashomer Central Blood Bank, to serve as controls.

Epitope mapping

For epitope mapping, 31 CD4 overlapping peptides spanning the entire human CD4 molecule and overlapping in 5 amino acids at each side were used The 31 CD4-derived sequences are provided in the following Table 1 : TABLE 1: CD4 peptides used for screening

Peptide Number Position in CD4 Sequence pl(SEQIDNo. 1) 1-20 MNRGVPFRHLLLVLQLALLP p2 (SEQ ID No. 2) 16-35 LALLPAATQGKKVVLGKKGD p3 (SEQ ID No. 3) 31-50 GKKGDTVELTCTASQKKSIQ p4 (SEQ ID No. 4) 46-65 KKSIQFHWKNSNQIKILGNQ p5 (SEQIDNo. 5) 61-80 ILGNQGSFLTKGPSKLNDRA p6 (SEQ ID No. 6) 76-95 LNDRADSRRSLWDQGNFPLI p7 (SEQ ID No. 7) 91-110 NFPLIIKNLKIEDSDTYICE p8 (SEQ ID No. 8) 106-125 TYICEVEDQKEEVQLLVFGL p9 (SEQ ID No. 9) 121-140 LVFGLTANSDTHLLQGQSLT plO(SEQ ID No. 10) 136-155 GQSLTLTLESPPGSSPSVQC pll (SEQIDNo. 11) 151-170 PSVQCRSPRGKNIQGGKTLS pl2(SEQIDNo. 12) 166-185 GKTLSVSQLELQDSGTWTCT p 13 (SEQ IDNo. 13) 181-199 TWTCTVLQNQKKVEFKIDIV pl4(SEQIDNo. 14) 195-214 KIDIVVLAFQKASSIVYKKE pi 5 (SEQ IDKo. 15) 210-229 VYKKEGEQVEFSFPLAFTVE pl6(SEQIDNo. 16) 225-244 AFTVEKLTGSGELWWQAERA pl7(SEQIDNo. 17) 240-259 QAERASSSKSWITFDLKNKE pi 8 (SEQ IDNo. 18) 255-274 LKNKEVSVKRVTQDPKLQMG pl9(SEQIDNo. 19) 271-290 KLQMGKKLPLHLTLPQALPQ p20(SEQIDNo. 20) 286-305 QALPQYAGSGNLTLALEAKT p21 (SEQIDNo. 21) 301-320 LEAKTGKLHQEVNLVVMRAT p22(SEQIDNo. 22) 316-335 VMRATQLQKNLTCEVWGPTS p23(SEQIDNo. 23) 331-350 WGPTSPKLMLSLKLENKEAK p24(SEQIDNo. 24) 346-365 NKEAKVSKREKAVWVLNPEA p25(SEQIDNo. 25) 361-380 LNPEAGMWQCLLSDSGQVLL p26(SEQIDNo. 26) 376-395 GQVLLESNIKVLPTWSTPVQ p27(SEQIDNo. 27) 391-410 STPVQPMALIVLGGVAGLLL Peptide Number Position in CD4 Sequence

> p28 (SEQ ID No. 28) 406-425 AGLLLFIGLGIFFCVRCRHR p29 (SEQ ID No. 29) 421 -440 RCRHRRRQAERMSQIKRLLS p30 (SEQ ID No. 30) 436-455 KRLLSEKKTCQCPHRFQKTC

P31 (SEQ ID No. 31) 451-458 FQKTCSPI

Peptides were synthesized at the Biological Services Laboratory of the Weizmann Institute of Science, Rehovot, Israel and were used at a concentration of 20μg/ml. Culturing conditions were as described previously [17, 18, each being incorporated herein by reference, in its entirety].

T-cell proliferation

Peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation on Ficoll Paque (Pharmacia Biotech, Uppsala, Sweden) from 30-40ml of heparinized venous blood, obtained from patients (see above). Cells were cultured in RPMI 1640 medium (Biological Industries Ltd., Beit Haemek, Israel), supplemented with 10% fetal calf serum (FCS) (Gibco BRL, Buffalo, NY, USA), 1% sodium-pyruvate, 1% L-glutamine, 1% Penicillin/Streptomycin (10,000 U/ml/10,000 mg/ml) (Seromed, Berlin, Germany) and 2% Hepes (IM, pH7.3) (Biological Industries, Beit Haemek, Israel). Specific cell proliferation was assayed by ³H-thymidine incorporation, as described previously [19].

In a first assay, the antigens used were soluble recombinant human CD4 and HIV- 1, gpl20 proteins (2.5μg/ml each), expressed in baculovirus (Intracell Corp., Cambridge, MA, USA, or other source), tetanus toxoid (5μg/ml, Connaught Lab. Inc., PA, USA, or other source), Candida (lOμg/ml, Hollister-Stier, Toronto, Canada, or other source) (data not shown, but discussed), and PHA (0.3 μg/ml, Murex Diagnostics Ltd., Dartford, UK, or other source).

In a second assay, each of the selected CD4 derived peptides were used at a concentration of 20μg/ml. In any case, the cells were plated in 96-well round bottom microplates (Falcon, Linooln Park, NJ, USA), at a cell concentration of 2xlO⁵ cells/well in medium, with or without the test antigens or mitogen. The plates were incubated at 37°C in a 5% CO₂ humidified incubator for 7 days. On day 6, the cells were labeled overnight with 1 μCi/well of 3H-thymidine and counted in a Matrix 96 -counter (Packard, Meriden, CT, USA). The results of the proliferation assays are presented as stimulation indexes (S. L). A stimulation index of above 2 was considered as a positive response. This value was chosen for two reasons: (1) the minimum concentration of CD4 was used (2μg), and (2) the low frequency of these cells as a result of immunosuppression. Preparation of a T-cell vaccine

For T-cell vaccine preparation, CD4 peptide-reactive T-cells are generated from a patient's blood sample as generally described in Materials and Methods. Briefly, PBMC are isolated from the patient's blood sample (about 30-40 ml blood is obtained from each patient), cultured with 3 μg/ml phytohemaglutinin (PHA) for 3 days, and expanded with 5-20 U/ml recombinant interleukin-2 (rIL-2) for 11 days. The cultures are re-stimulated on day 14, with irradiated CD4 peptide-pulsed antigen presenting cells (APC) [24, incorporated herein by reference, in its entirety], and are further grown for another 7 days.

On day 21, the cells are inactivated for 5 minutes, at room temperature with 1.0% glutaraldehyde and prepared for injection. Aliquots of 10x10⁶ CD4 peptide-reactive T- cells are frozen in liquid nitrogen, to be used later for second and third booster vaccinations, at two to six months intervals. For pure lines of anti CD4 peptide cells, several rounds of CD4 peptide re-stimulation are necessary, preferably between 4 to 8 rounds. All cultures are performed according to the procedures required for the preparation of biological products to be used in humans.

Statistical analysis

Statistical analysis was performed using the InStat 2.01 computer program, and P values were calculated using the Mann- Whitney non-parametric test. RESULTS

T-cell reactivity to recombinant CD4 (rCD4), recombinant gpl20 (rgpl20), and to the recall antigens Tetanus toxin and Candida was studied in HIV infected patients and in healthy donors. The experiments measuring the response towards the recall antigens were conducted in order to examine parameters of the immune response of the study subjects .

The results of the T-cell proliferation responses are summarized in Table 2A for the HIV-I infected patients (HIV subtype B) and in Table 2B, for the healthy subjects.

Table 2A: T-cell proliferative responses in HIV infected patients

Table

^: ot statstca y sgn icant; = . n : not etermne ; As evidenced from Tables 2A and 2B, T-cell proliferation responses to rCD4 were significantly higher in HIV patients in comparison to the healthy controls. In 20 of the 25 HIV patients (80%), the Stimulation Index (S.I.) in response to rCD4 had a value above 2 (mean S.I. 3.6+ 2.1), whereas in the controls, only 4 out of 24 (25%) had an S.I. above 2 (mean S.I. 1.6+0.9).

The major immunogenic epitopes of the CD4 molecule were then mapped by testing T-cell proliferation responses to 31 synthetic overlapping peptides obtained from the human CD4 molecule.

Eight HIV-infected patients were further tested for their immunogenic responses to the 31 overlapping peptides. All the tested patients showed responses to certain peptides, sometimes at a higher S.I. than that observed against the whole molecule. The reactivity of each patient to the different peptides is summarized in Table 3 and

Figures IA- IB. Positive responses were marked with grey background.

TABLE 3: Mosaic display of peptide responses in HlV infected patients (PT) or in healthy subjects

Peptides PT15 PT16 PT17 PT18 PT19 PT20 PT24 PT25 Pep. (C21) (C22) (C23)

_P1 3.8 2.9 1 1 2.7 2.3 1 3.4 1 2.3 1

P2 1 1 2.1 1 1 1 1 1 1 1 2.8

^ , p3 1 2.3 1 1 1 1 1 1 p3 1 1 2.7 p4 2.4 3.5 1 1 6.3 2.4 1 1 p4 1 1 2.3 p5 1 2.4 1 1 4.7 1 4.2 1 p5 1 1 1 p6 1 3 1 1 1 1 1 1 p6 1 2.7 1

P7 1 6.1 1 1 1 1 2.2 1 P7 1 1 3.2 p8 1 1 1 1 1 1 1 1 p8 1 1 1 p9 1 1 1 1 1 1 1 1 p9 1 1 2.4

IO p10 1 1 1 1 3.7 1 1 1 p10 1 1 3.3 p11 1 1 1 1 1 1 1 1 p11 1 2.3 2.3 p12 1 1 6.6 1 1 1 1 1 p12 1 1 1 p13 1 1 1 1 1 1 1 1 p13 1 1 1 p14 7.1 1 1 1 2.4 4.9 5.4 1 p14 2 6.3 2.1 p15 1 4 1 1 1 1 1 1 p15 1 1 1 p16 1 1 1 1 1 1 3.3 2.1 p16 1 1 2.2 p17 1 4.2 1 1 1 1 1 1 p17 1 1 1 p18 1 3.5 1 1 1 1 1 1 p18 1 1 1 p19 1 1 1 1 1 1 1 1 p19 1 1 1 p20 1 5.7 1 2.2 4.3 1 1 1 p20 1 1 1

Peptides PT15 PT16 PT17 PT18 PT19 PT20 PT24 PT25 Pep. (C21) (C22) (C23) p21 1 2.7 2.6 1 3_;4 1 2.1 1 p21 1 1 1 p22 1 1 1 1 2.2 1 1 1 p22 1 1 1 p23 1 1 1 3.4 1 1 1 2 p23 1 1 1 p24 1 1 1 1 1 1 1 1 p24 1 1 1 p25 1 1 1 1 1 1 1 1 p25 1 1 1 p26 1 4.4 1 1 5.4 1 1 1 p26 1 1 4.4 p27 1 1 5.1 1 1 1 1 1 p27 4 3.3 3.15 p28 2.7 4.8 1 2.4 1 2.7 2.4 2.2 p28 1 1 1 p29 3.4 2.7 1 2.1 3 1 1 2.2 p29 1 1 1 p30 1 1 1 1 1 1 1 1 p30 1 1 1 to p31 1 1 1 1 1 1 1 1 p31 1 1 1 rCD4* 6 5.1 1 2.1 5.2 2 2 2.7 rCD4* 2.6 3.4 5.2

* Response to the rCD4 molecule

A peptide was defined as immunogenic only when at least 50% of the tested HIV- infected patients showed positive proliferation responses to this peptide (in terms of Simulation index, S.I.)- Tables 4A-4B show the mean responses of HIV patients (Table 4A) and HIV negative subjects (Table 4B) and the percentage responding to the six major immunogenic CD4 peptides.

Table 4A- responsiveness to immunogenic CD4 peptides in HIV patients (S.I.)

Table 4B- responsiveness to immunogenic CD4 peptides in HIV-negative subjects

As can be understood from the above, six peptides met this criterion: pi, p4, pl4, p21, p28 and p29. Peptides pi, p28 and p29 were thus determined as major epitopes as they elicited a response in more than 50% of the patients, in the specific example, in 62.5% (Pl and P29) to 75% (P28) of the patients. Proliferation responses were observed in 4 distinct clusters (1-7, 11-15, 16-22, 25-30) with the most significant being p28 and p29.

In contrast, the three HIV-negative controls responded mainly to peptides p27, pl4 and pl l.

It is noted that no significant response was obtained in the present studies towards the p6 peptide that largely corresponds with the peptides identified by Salemi et al. [8, incorporated herein by reference, in its entirety]

The p4 and p5 peptides are located in the amino acid sequence found in CD4 domain 1, which is the HIV gpl20-binding site. Epitopes p28 and p29 are distant from the binding site. Each patient responded to at least 2 of the immunogenic peptides (except for patient P 17, see below).

Patient Pl 7 responded to p2, pi 2, p21 and p27. Of these 4 peptides, only p21 is considered autoimmune. Interestingly, the other 3 peptides were also responded to by the healthy subjects. This unusual response of P 17, whose disease duration is 2.6 years, may be an indication of a transition from healthy to autoimmune response. The proliferation response to a mixture of 5 peptides was also tested. The mixture contained the immunogenic peptides pi, p4, pl4, p28 and p29 (p21 was excluded since its mean SI was below 2 (namely, 1.8)) at a concentration of 20 μg/ml each. The responses towards the mixture were compared to responses towards the full CD4 molecule.

Table 5A and 5B show responses to the peptide mixture in HIV positive (Table 5A) and HIV negative (Table 5B) donors. As a comparison, T cells were cultured with recombinant CD4 at a concentration of 2 μg/ml. Among the 12 patients tested, 6 patients responded to CD4 and the peptide mixture. 4 out of the 6 responders responded at a higher stimulation index (SI) to the mixture than to the CD4. Patients P5, P6 and P8 showed low blood CD4 counts and did not manifested a response to either the CD4 molecule or to the peptide. These patients also failed to respond to tetanus toxoid.

This may be an indication of immuno-suppression in these patients and that immuno-suppression may have influenced the results in these patients, as these patients are not responding to any antigen. With these 3 patients removed, the overall response rate rises to 67%, providing a closer match to the earlier results shown in Table 2A, in which an 80% response rate was found.

TABLE 5A: Proliferation responses to a peptide mixture in HIV patients

TABLE 5A: Proliferation responses to a peptide mixture in HIV patients

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Claims

1. A peptide or polypeptide comprising a stretch of contiguous amino acids consisting of at least 5 overlapping amino acids from a CD4 protein, wherein said stretch of amino acids is characterized by the ability to elicit a T-cell mediated autoimmune response against CD4+ T-cells in a patient with T-cell mediated autoimmunity or to impart on said peptide or polypeptide the ability to elicit a T-cell mediated autoimmune response against CD4+ T-cells in a patient with T-cell mediated autoimmunity.

2. A peptide or polypeptide according to Claim 1 , wherein said stretch comprises a sequence selected from SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 14, SEQ ID No. 28 and SEQ ID No. 29.

3. A polypeptide comprising a conjugate of two or more of the peptides or polypeptides of Claim 1.

4. A peptide or polypeptide according to Claim 1, wherein said stretch of amino acids impart on said peptide or polypeptide the ability to elicit a T-cell mediated autoimmune response against CD4+ T-cells in an HIV infected patient.

5. A method of enriching a T-cell preparation, comprising exposing a population of T-cells from a subject with a T-cell mediated autoimmune response to a peptide or polypeptide of Claim 1, and providing conditions for selective cell proliferation of T-cells reactive with said peptide or polypeptide.

6. An enriched T-cell population obtainable by the method of Claim 6.

7. A T-cell vaccine comprising the enriched T-cell population of Claim 7.

8. A method of treating a patient with T-cell mediated autoimmunity, comprising administering to said patient a T-cell vaccine according to Claim 7.

9. A method for determining whether T-cells from a subject include cells that cause an anti-CD4+ autoimmune response, comprising contacting a sample containing T-cells of said subject with a peptide or polypeptide according to Claim 1, and determining either (i) binding of said peptide or polypeptide to T-cells in said sample, or (ii) T-cell proliferation responses.

10. A conjugate comprising a peptide or polypeptide according to Claim 1, conjugated to a cytotoxic moiety.

11. The conjugate of Claim 11, wherein the cytotoxic moiety is selected from a radioactive label, or a toxin.

12. The conjugate of embodiment 11, wherein said cytotoxic moiety comprises a liposome conjugated to said peptide or polypeptide and a cytotoxic drug associated with said liposome.

13. A method of detecting T-cell mediated autoimmunity in a patient comprising:

(i) obtaining peripheral mononuclear blood cells (PMBC) from said patient;

(ii) culturing said PMBC in the presence of a peptide or polypeptide ^N according to Claim 1 ;

(iii) providing said cells with conditions for selective cell proliferation of T- cells reactive with said peptide or polypeptide; wherein a proliferation response indicates a T-cell specific autoimmune response in said patient.

14. A kit for detecting T-cell mediated autoimmune response in a subject comprising at least one peptide or polypeptide according to Claim 1.

15. A kit for detecting T-cell mediated autoimmune response in a subject having an anti-CD4 autoimmunity comprising at least one peptide or polypeptide according to Claim 1.

16. A T-cell receptor (TCR) peptide complementary with a peptide or polypeptide according to Claim 1.

17. A vaccine comprising the TCR peptide of Claim 16 for use in therapeutic vaccination of immune deficient HIV 1 infected patients.

18. A peptide or polypeptide comprising a stretch of contiguous amino acids comprising a sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 14, SEQ ID No. 28 and SEQ ID No. 29, wherein said stretch of contiguous amino acids is characterized by the ability to elicit a T-cell mediated autoimmune response against CD4+ T-cells in a patient with T-cell mediated autoimmunity or to impart on said peptide or polypeptide the ability to elicit a T-cell mediated autoimmune response against CD4+ T-cells in a patient with T-cell mediated autoimmunity.

19. A polypeptide comprising a conjugate of two or more of the peptides or polypeptides of Claim 18.

20. A peptide or polypeptide according to Claim 18, wherein said stretch of contiguous amino acids impart on said peptide or polypeptide the ability to elicit a T-cell mediated autoimmune response against CD4+ T-cells in an HIV infected patient.

21. A method of enriching a T-cell preparation, comprising exposing a population of T-cells from a subject with a T-cell mediated autoimmune response to a peptide or polypeptide of Claim 18, and providing conditions for selective cell proliferation of T- cells reactive with said peptide or polypeptide.

22. An enriched T-cell population obtainable by the method of Claim 21.

23. A T-cell vaccine comprising the enriched T-cell population of Claim 22.

24. A method of treating a patient with T-cell mediated autoimmunity, comprising administering to said patient a T-cell vaccine according to Claim 23.

25. A method for determining whether T-cells from a subject include cells that cause an anti-CD4+ autoimmune response, comprising contacting a sample containing T-cells of said subject with a peptide or polypeptide according to Claim 18, and determining either (i) binding of said peptide or polypeptide to T-cells in said sample, or (ii) T-cell proliferation responses.

26. A conjugate comprising a peptide or polypeptide according to Claim 18, conjugated to a cytotoxic moiety.

27. The conjugate of Claim 26, wherein the cytotoxic moiety is selected from a radioactive label, or a toxin.

28. The conjugate of embodiment 26, wherein said cytotoxic moiety comprises a liposome conjugated to said peptide or polypeptide and a cytotoxic drug associated with said liposome.

29. A method of detecting T-cell mediated autoimmunity in a patient comprising:

(i) obtaining peripheral mononuclear blood cells (PMBC) from said patient;

(ii) culturing said PMBC in the presence of a peptide or polypeptide according to Claim 18; (iii) providing said cells with conditions for selective cell proliferation of T- cells reactive with said peptide or polypeptide; wherein a proliferation response indicates a T-cell specific autoimmune response in said patient.

30. A kit for detecting T-cell mediated autoimmune response in a subject comprising at least one peptide or polypeptide according to Claim 18.

31. A kit for detecting T-cell mediated autoimmune response in a subject having an anti-CD4 autoimmunity comprising at least one peptide or polypeptide according to Claim 18.