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  Pharmaceutical Patents  

 

Title:  Method for reducing neuronal degeneration so as to ameliorate the effects of injury or disease
United States Patent: 
7,560,102
Issued: 
July 14, 2009

Inventors:
 Eisenbach-Schwartz; Michal (Rehovot, IL), Hauben; Ehud (Rehovot, IL), Cohen; Irun R. (Rehovot, IL), Beserman; Pierre (Moshav Sitriya, IL), Monsonego; Alon (Rehovot, IL), Moalem; Gila (Petah-Tiqva, IL)
Assignee:
  Yeda Research and Development Co., Ltd (Rehovot, IL)
Appl. No.:
 10/810,653
Filed:
 March 29, 2004


 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

Compositions and methods to promote nerve regeneration or to confer neuroprotection and prevent or inhibit neuronal degeneration within the nervous system, either the central nervous system or the peripheral nervous system, are provided. Treatment involves administering NS-specific activated T cells, or an NS-specific antigen or analog thereof, a peptide derived therefrom or an analog or derivative of said peptide, or a nucleotide sequence encoding such an antigen or peptide, or any combination thereof.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions for promotion of nerve regeneration or for neuroprotection and prevention or inhibition of neuronal degeneration to ameliorate the effects of injury to, or disease of, the nervous system (NS).

The present invention is based in part on the inventors' unexpected discovery that activated T cells that recognize an antigen of the NS of the patient promote nerve regeneration or confer neuroprotection. As used herein, "neuroprotection" refers to the prevention or inhibition of degenerative effects of injury or disease in the NS. Since it was thought until recently that immune cells do not participate in nervous system repair, it was quite surprising to discover that NS-specific activated T cells and also the NS-specific antigens themselves and peptides derived therefrom can be used to promote nerve regeneration or to protect nervous system tissue from secondary degeneration which may follow damage caused by injury or disease of the CNS or PNS.

Thus, in one aspect, the invention relates to a method for promoting nerve regeneration or for conferring neuroprotection and preventing or inhibiting neuronal degeneration in the central nervous system or peripheral nervous system for ameliorating the effects of injury or disease, comprising administering to an individual in need thereof at least one ingredient selected from the group consisting of: (a) NS-specific activated T cells; (b) a NS-specific antigen or an analog thereof; (c) a peptide derived from an NS-specific antigen or from an analog thereof, or an analog or derivative of said peptide; (d) a nucleotide sequence encoding an NS-specific antigen or an analog thereof; (e) a nucleotide sequence encoding a peptide derived from an NS-specific antigen or from an analog thereof, or an analog of said peptide; or (f) any combination of (a)-(e).

In another aspect, the invention relates to a pharmaceutical composition for promoting nerve regeneration or for neuroprotection and prevention or inhibition of neuronal degeneration in the CNS or PNS for ameliorating the effects of injury or disease in the NS, comprising a therapeutically effective amount of at least one ingredient selected from the group consisting of (a) to (e) above or any combination of (a)-(e).

The term "NS-specific antigen" as used herein refers to an antigen of the NS that specifically activates T cells such that following activation the activated T cells accumulate at a site of injury or disease in the NS of the patient. Examples of NS-specific antigens according to the invention include, but are not limited to, myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), S-100, .beta.-amyloid, Thy-1, P0, P2, neurotransmitter receptors, the protein Nogo (Nogo-A, Nogo-B and Nogo-C) and the Nogo receptor (NgR). This definition also includes analogs of said NS-specific antigens as described in the section on NS-specific antigens, analogs thereof, peptides derived therefrom and analogs and derivatives thereof of said peptides hereinafter.

DETAILED DESCRIPTION OF THE INVENTION

As exposed above, the present invention relates to compositions and methods for promoting nerve regeneration or for conferring neuroprotection and preventing or inhibiting neuronal degeneration in the CNS or PNS for ameliorating the effects of injury or disease, comprising administering to an individual in need thereof at least one ingredient selected from the group consisting of: (a) NS-specific activated T cells; (b) a NS-specific antigen or an analog thereof; (c) a peptide derived from an NS-specific antigen or from an analog thereof, or an analog or derivative of said peptide; (d) a nucleotide sequence encoding an NS-specific antigen or an analog thereof; (e) a nucleotide sequence encoding a peptide derived from an NS-specific antigen or from an analog thereof, or an analog of said peptide; or (f) any combination of (a)-(e).

Merely for ease of explanation, the detailed description of the present invention is divided into the following sections: NS-specific activated T cells and T-cell banks; NS-specific antigens, analogs thereof, peptides derived therefrom and analogs and derivatives thereof of said peptides; nucleotide sequences encoding NS-specific antigens, analogs thereof, peptides derived therefrom and analogs thereof; therapeutic uses; and formulations and modes of administration.

NS-Specific Activated T Cells and T-Cell Banks

In one embodiment of the invention, NS-specific activated T cells can be used in an amount which is effective to confer neuroprotection for ameliorating or inhibiting the effects of injury or disease of the CNS or PNS that result in NS degeneration or for promoting regeneration in the NS, in particular the CNS, as described in the section on therapeutic uses hereinafter.

In the practice of the invention, administration of NS-specific activated T cells may optionally be in combination with an NS-specific antigen or an analog thereof or a peptide derived therefrom or an analog or derivative of said peptide. Additionally, oral administration of NS-specific antigen or an analog thereof or a peptide derived therefrom or an analog or derivative thereof, can be combined with active immunization to build up a critical T-cell response immediately after injury.

Activation of T cells is initiated by interaction of a TCR complex with a processed antigenic peptide bound to a MHC molecule on the surface of an antigen-presenting cell (APC). As used herein, the term "activated T cells" includes both (i) T cells that have been activated by exposure to a cognate antigen or peptide derived therefrom or derivative thereof; and (ii) progeny of such activated T cells. As used herein, a "cognate antigen" is an antigen that is specifically recognized by the TCR of a T cell that has been previously exposed to the antigen. Alternatively, the T cell which has been previously exposed to the antigen may be activated by a mitogen, such as phytohemagglutinin (PHA) or concanavalin A (Con A).

The term "NS-specific activated T cell" as used herein refers to an activated T cell having specificity for an antigen of the NS, said NS-specific antigen being an antigen of the NS that specifically activates T cells such that these activated T cells will accumulate at a site of injury or disease in the NS of the patient. The NS-specific antigen used to confer the specificity to the T cells may be a self NS-antigen of the patient or a non-self NS-antigen of another individual or even of another species, or an analog of said NS-antigen, or a peptide derived from said NS-antigen or from said analog thereof, or an analog or derivative of said peptide, all as described in the section on NS-specific antigens, analogs thereof, peptides derived therefrom and analogs and derivatives thereof of said peptides hereinafter, as long as the activated T cell recognizes an antigen in the NS of the patient.

If the disease being treated by the NS-specific activated T cells of the invention is an autoimmune disease, in which the autoimmune antigen is an NS antigen, the T cells which are used in accordance with the present invention for the treatment of neural damage or degeneration caused by such disease are preferably not activated against the same autoimmune antigen involved in the disease. While the prior art has described methods of treating autoimmune diseases by administering activated T cells to create a tolerance to the autoimmune antigen, the T cells of the present invention are not administered in such a way as to create tolerance, but are administered in such a way as to create accumulation of the T cells at the site of injury or disease so as to facilitate neural regeneration or to inhibit neural degeneration.

The prior art also discloses uses of immunotherapy against tumors, including brain tumors, by administering T cells specific to an NS antigen in the tumor so that such T cells may induce an immune system attack against the tumors. The present invention is not intended to comprehend such prior art techniques. However, the present invention is intended to comprehend the inhibition of neural degeneration or the enhancement of neural regeneration in patients with brain tumors by means other than the prior art immunotherapy of brain tumors. Thus, for example, NS-specific activated T cells, which are activated to an NS-antigen of the patient other than an antigen which is involved in the tumor, would be expected to be useful for the purpose of the present invention and would not have been suggested by known immunotherapy techniques.

The NS-specific activated T cells are preferably autologous, most preferably of the CD4 and/or CD8 phenotypes, but they may also be semi-allogeneic T cells or allogeneic T cells from related donors, e.g., siblings, parents, children, or from donors with the same HLA type (HLA-matched) or a very similar HLA type (HLA-partially matched), or even from unrelated donors.

Thus, in addition to the use of autologous T cells isolated from the subject, the present invention also comprehends the use of semi-allogeneic T cells for neuroprotection. The T cells may be prepared as short- or long-term lines and stored by conventional cryopreservation methods for thawing and administration, either immediately or after culturing for 1-3 days, to a subject suffering from injury to the CNS and in need of T-cell neuroprotection.

The use of semi-allogeneic T cells is based on the fact that T cells can recognize a specific antigen epitope presented by foreign APC, provided that the APC expresses the MHC molecule, class I or class II, to which the specific responding T-cell population is restricted, along with the antigen epitope recognized by the T cells. Thus, a semi-allogeneic population of T cells that can recognize at least one allelic product of the subject's MHC molecules, preferably a class II HLA-DR or HLA-DQ or other HLA molecule, and that is specific for a NS-associated antigen epitope, will be able to recognize the NS antigen in the subject's area of NS damage and produce the needed neuroprotective effect. There is little or no polymorphism in the adhesion molecules, leukocyte migration molecules, and accessory molecules needed for the T cells to migrate to the area of damage, accumulate there, and undergo activation. Thus, the semi-allogeneic T cells will be able to migrate and accumulate at the CNS site in need of neuroprotection and will be activated to produce the desired effect.

It is known that semi-allogeneic T cells will be rejected by the subject's immune system, but that rejection requires about two weeks to develop. Hence, the semi-allogeneic T cells will have the two-week window of opportunity needed to exert neuroprotection. After two weeks, the semi-allogeneic T cells will be rejected from the body of the subject, but that rejection is advantageous to the subject because it will rid the subject of the foreign T cells and prevent any untoward consequences of the activated T cells. The semi-allogeneic T cells thus provide an important safety factor and are a preferred embodiment.

It is known that a relatively small number of HLA class II molecules are shared by most individuals in a population. For example, about 50% of the Jewish population express the HLA-DR5 gene. Thus, a bank of specific T cells reactive to NS-antigen epitopes that are restricted to HLA-DR5 would be useful in 50% of that population. The entire population can be covered essentially by a small number of additional T cell lines restricted to a few other prevalent HLA molecules, such as DR1, DR4, DR2, etc. Thus, a functional bank of uniform T cell lines can be prepared and stored for immediate use in almost any individual in a given population. Such a bank of T cells would overcome any technical problems in obtaining a sufficient number of specific T cells from the subject in need of neuroprotection during the open window of treatment opportunity. The semi-allogeneic T cells will be safely rejected after accomplishing their role of neuroprotection. This aspect of the invention does not contradict, and is in addition to the use of autologous T cells as described herein.

The NS-specific activated T cells are preferably non-attenuated, although attenuated NS-specific activated T cells may be used. T cells may be attenuated using methods well-known in the art including, but not limited to, by gamma-irradiation, e.g., 1.5-10.0 Rads (Ben-Nun et al, 1981; Ben-Nun and Cohen, 1982); and/or by pressure treatment, for example as described in U.S. Pat. No. 4,996,194 (Cohen et al); and/or by chemical cross-linking with an agent such as formaldehyde, glutaraldehyde and the like, for example as described in U.S. Pat. No. 4,996,194 (Cohen et al); and/or by cross-linking and photoactivation with light with a photoactivatable psoralen compound, for example as described in U.S. Pat. No. 5,114,721 (Cohen et al); and/or by a cytoskeletal disrupting agent such as cytochalsin and colchicine, for example as described in U.S. Pat. No. 4,996,194 (Cohen et al). In a preferred embodiment the NS-specific activated T cells are isolated as described below. T cells can be isolated and purified according to methods known in the art (Mor and Cohen, 1995). For an illustrative example, see Example 1, Materials and Methods.

Circulating T cells of a subject which recognize an NS-antigen are isolated and expanded using known procedures (Burns et al, 1983; Pette et al, 1990; Martin et al, 1990; Schluesener et al, 1985; Suruhan-Dires Keneli et al, 1993, which are incorporated herein by reference in their entirety). In order to obtain NS-specific activated T cells, T cells are isolated and the NS-specific activated T cells are then expanded.

The isolated T cells may be activated by exposure of the cells to one or more of a variety of natural or synthetic NS-specific antigens or epitopes as described in section on NS-specific antigens, analogs thereof, peptides derived therefrom and analogs and derivatives thereof of said peptides hereinafter. During ex vivo activation of the T cells, the T cells may be activated by culturing in medium to which at least one suitable growth promoting factor has been added, such as cytokines, e.g., TNF-.alpha., IL-2 and/or IL-4.

In one embodiment, the NS-specific activated T cells endogenously produce a substance that ameliorates the effects of injury or disease in the NS.

In another embodiment, the NS-specific activated T cells endogenously produce a substance that stimulates other cells, including, but not limited to, transforming growth factor-.beta. (TGF-.beta.), nerve growth factor (NGF), neurotrophic factor 3(NT-3), neurotrophic factor 4/5 (NT-4/5), brain derived neurotrophic factor (BDNF); IFN-.gamma. and IL-6, wherein the other cells, directly or indirectly, ameliorate the effects of injury or disease.

Following their proliferation in vitro, the T cells are administered to a mammalian, preferably a human, subject. T cell expansion is preferably performed using peptides corresponding to sequences in a non-pathogenic, NS-specific, self-protein.

A subject can initially be immunized with an NS-specific antigen using a non-pathogenic peptide of the self-protein. A T-cell preparation can be prepared from the blood of such immunized subjects, preferably from T cells selected for their specificity towards the NS-specific antigen. The selected T cells can then be stimulated to produce a T cell line specific to the self-antigen (Ben-Nun and Cohen, 1982).

NS-specific antigen activated T cells, obtained as described above, can be used immediately or may be preserved for later use, e.g., by cryopreservation as described below. NS-specific activated T cells may also be obtained using previously cryopreserved T cells, i.e., after thawing the cells, the T cells may be incubated with NS-specific antigen, optimally together with thymocytes, to obtain a preparation of NS-specific activated T cells.

As will be evident to those skilled in the art, the T cells can be preserved, e.g., by cryopreservation, either before or after culture.

Cryopreservation agents which can be used include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959; Ashwood-Smith, 1961), polyvinylpyrrolidone (Rinfret, 1960), glycerol, polyethylene glycol (Sloviter and Ravdin, 1962), albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol (Rowe et al, 1962), D-sorbitol, i-inositol, D-lactose, choline chloride (Bender et al, 1960), amino acids (Phan The Tran and Bender, 1960), methanol, acetamide, glycerol monoacetate (Lovelock, 1954), inorganic salts (Phan The Tran and Bender, 1960 and 1961) and DMSO combined with hydroxyethyl starch and human serum albumin (Zaroulis and Leiderman, 1980).

A controlled cooling rate is critical. Different cryoprotective agents (Rapatz et al, 1968) and different cell types have different optimal cooling rates. See, e.g., Rowe and Rinfret, 1962; Rowe, 1966; Lewis et al, 1967; Mazur, 1970) for effects of cooling velocity on survival of cells and on their transplantation potential. The heat of fusion phase where water turns to ice should be minimal. The cooling procedure can be carried out by use of, e.g., a programmable freezing device or a methanol bath procedure.

Programmable freezing apparatuses allow determination of optimal cooling rates and facilitate standard reproducible cooling. Programmable controlled-rate freezers such as Cryomed or Planar permit tuning of the freezing regimen to the desired cooling rate curve.

After thorough freezing, cells can be rapidly transferred to a long-term cryogenic storage vessel. In one embodiment, samples can be cryogenically stored in mechanical freezers, such as freezers that maintain a temperature of about -80.degree. C. or about -20.degree. C. In a preferred embodiment, samples can be cryogenically stored in liquid nitrogen (-196.degree. C.) or its vapor. Such storage is greatly facilitated by the availability of highly efficient liquid nitrogen refrigerators, which resemble large Thermos containers with an extremely low vacuum and internal super insulation, such that heat leakage and nitrogen losses are kept to an absolute minimum.

Considerations and procedures for the manipulation, cryopreservation, and long term storage of T cells can be found, for example, in the following references, incorporated by reference herein: Gorin, 1986; Bone-Marrow Conservation, Culture and Transplantation, 1968.

Other methods of cryopreservation of viable cells, or modifications thereof, are available and envisioned for use, e.g., cold metal-mirror techniques. See Livesey and Linner, 1987; Linner et al, 1986; see also U.S. Pat. No. 4,199,022 by Senken et al, U.S. Pat. No. 3,753,357 by Schwartz, U.S. Pat. No. 4,559,298 by Fahy.

Frozen cells are preferably thawed quickly (e.g., in a water bath maintained at 37-47.degree. C.) and chilled immediately upon thawing. It may be desirable to treat the cells in order to prevent cellular clumping upon thawing. To prevent clumping, various procedures can be used, including but not limited to the addition before or after freezing of DNAse (Spitzer et al, 1980), low molecular weight dextran and citrate, citrate, hydroxyethyl starch (Stiff et al, 1983), or acid citrate dextrose (Zaroulis and Leiderman, 1980), etc.

The cryoprotective agent, if toxic in humans, should be removed prior to therapeutic use of the thawed T cells. One way in which to remove the cryoprotective agent is by dilution to an insignificant concentration.

Once frozen T cells have been thawed and recovered, they are used to promote neuroprotection as described herein with respect to non-frozen T cells. Once thawed, the T cells may be used immediately, assuming that they were activated prior to freezing. Preferably, however, the thawed cells are cultured before injection to the patient in order to eliminate non-viable cells. Furthermore, in the course of this culturing over a period of about one to three days, an appropriate activating agent can be added so as to activate the cells, if the frozen cells were resting T cells, or to help the cells achieve a higher rate of activation if they were activated prior to freezing. Usually, time is available to allow such a culturing step prior to administration as the T cells may be administered as long as a week after injury, and possibly longer, and still maintain their neuro-regenerative and neuroprotective effect.

To minimize secondary damage after nerve injury, patients can be treated by administering autologous or semi-allogeneic T lymphocytes sensitized to at least one appropriate NS-antigen. As the window of opportunity has not yet been precisely defined, therapy should be administered as soon as possible after the primary injury to maximize the chances of success, preferably within about one week.

To bridge the gap between the time required for activation and the time needed for treatment, a bank with autologous, semi-allogeneic or allogeneic T cells can be established for future use.

Thus, in another embodiment, the invention provides cell banks that can be established to store NS-sensitized T cells for neuroprotective treatment of individuals at a later time, as needed.

In one embodiment, autologous T cells may be obtained from an individual and the cell bank will contain personal vaults of autologous T lymphocytes prepared for future use for neuroprotective therapy against secondary degeneration in case of NS injury. T lymphocytes are isolated from the blood, sensitized to a NS-antigen, and the cells are then frozen and suitably stored under the person's name, identity number, and blood group, in a cell bank until needed.

Additionally, autologous stem cells of the CNS can be processed and stored for potential use by an individual patient in the event of traumatic disorders of the NS such as ischemia or mechanical injury, as well as for treating neurodegenerative conditions such as Alzheimer's disease or Parkinson's disease.

Alternatively, allogeneic or semi-allogeneic T cells may be stored such that a bank of T cells of each of the most common MHC-class II types are present. The semi-allogeneic or allogeneic T cells are stored frozen for use by any individual who shares one MHC type II molecule with the source of the T cells.

In case an individual is to be treated for an injury, preferably autologous stored T cells are used, but, if autologous T cells are not available, then cells should be used which share an MHC type II molecule with the patient, and these would be expected to be operable in that individual.

The cells are preferably stored in an activated state after exposure to an NS-antigen or peptide derived therefrom. However, the cells may also be stored in a resting state and activated once they are thawed and prepared for use. The cell lines of the bank are preferably cryopreserved. The cell lines are prepared in any way which is well known in the art. Once the cells are thawed, they are preferably cultured prior to injection in order to eliminate non-viable cells. During this culturing, the cells can be activated or reactivated using the same NS-antigen or peptide as used in the original activation. Alternatively, activation may be achieved by culturing in the presence of a mitogen, such as phytohemagglutinin (PHA) or concanavalin A (preferably the former). This will place the cells into an even higher state of activation. The few days that it takes to culture the cells should not be detrimental to the patient as the treatment in accordance with the present invention may occuo still be effective. Alternatively, if time is of the essencer any time up to a week or more after the injury in order t, the stored cells may be administered immediately after thawing.

NS-Specific Antigens, Analogs therof, Peptides Derived Therefrom, and Analogs and Derivatives Thereof

The term "NS-specific antigen" as used herein refers to an antigen of the NS that specifically activates T cells such that following activation the activated T cells accumulate at a site of injury or disease in the NS of the patient.

The NS-specific antigen used according to the present invention may be an antigen obtained from NS tissue, preferably from tissue at a site of CNS injury or disease. It may be a crude NS-tissue preparation, e.g., derived from NS tissue obtained from mammalian NS that may include cells, both living or dead cells, membrane fractions of such cells or tissue, etc., and may be obtained by an NS biopsy or necropsy from a mammal, preferably human, tissue including, but not limited to, from a site of CNS injury; from cadavers; and from cell lines grown in culture.

In one embodiment, the NS-specific antigen is an isolated or purified antigen. The NS-specific antigen may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of antigens. The functional properties may be evaluated using any suitable assay. Additionally, an NS-specific antigen may be a protein obtained by genetic engineering, chemically synthesized, etc.

In the practice of the invention, natural or synthetic NS-specific antigens are preferred and include, without being limited to, myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), myelin-associated glycoprotein (MAG), S-100, .beta.-amyloid, Thy-1, P0, P2, neurotransmitter receptors, Nogo and Nogo receptor (NgR).

Specific illustrative examples of useful NS-specific antigens include but are not limited to, human MBP, depicted in FIG. 21 (SEQ ID NO:12, see Original Patent); human PLP, depicted in FIG. 22 (SEQ ID NO:13, see Original Patent); human MOG, depicted in FIG. 23 (SEQ ID NO:14, see Original Patent), rat Nogo A, B and C (Chen et al, 2000; WO 00/31235) (SEQ ID NOs:18, 20 and 21), peptide p472 (SEQ ID NO:19), human Nogo A, B and C (Prinjha et al, 2000) (SEQ ID NOs:23-25), and human or mouse Nogo receptor (NgR) (Fournier et al, 2001) (SEQ ID NOs:26 and 27, respectively).

Also encompassed by the present invention are analogs of NS-specific antigens including, but not being limited to, those molecules comprising regions that are substantially homologous to the full-length NS-specific antigen, or fragments thereof. In various embodiments, these analogs will have at least 60% or 70% or 80% or 90% or 95% identity over an amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art or whose encoding nucleic acid is capable of hybridizing to a coding nucleotide sequence of the full-length NS-specific antigen, under high stringency, moderate stringency, or low stringency conditions. Computer programs for determining homology may include, but are not limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988; Altschul et al, 1990; Thompson, et al, 1994; Higgins, et al, 1996).

The NS-specific antigen analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, a cloned gene sequence can be modified by any of numerous strategies known in the art (Maniatis, 1990). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.

Additionally, the coding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson, et al, 1978), etc.

Manipulations may also be made at the protein level. Included within the scope of the invention are NS-specific antigen derivatives which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH.sub.4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.

In a preferred embodiment, the invention relates to peptides derived from NS-specific antigens or from analogs thereof and to analogs or derivatives of said peptides, which are functionally active, i.e., they are capable of displaying one or more known functional activities associated with a full-length NS-specific antigen. Such functional activities include, but are not limited to, antigenicity (ability to bind, or compete with an NS-antigen for binding, to an anti-NS-specific antibody), immunogenicity (ability to generate antibody which binds to an NS-specific protein), and ability to interact with T cells, resulting in activation comparable to that obtained using the corresponding full-length NS-specific antigen. The crucial test is that the antigen which is used for activating the T cells causes the T cells to be capable of recognizing an antigen in the NS of the mammal (patient) being treated.

The NS-antigen derived peptide may be either: (1) an immunogenic peptide, i.e., a peptide that can elicit a human T-cell response detected by a T-cell proliferation assay or by cytokine, e.g., IFN-.gamma., IL-2, IL-4 or IL-10, production, or (2) a "cryptic epitope" (also designated herein as "immunosilent" or "non-immunodominant" epitope), i.e., a peptide that by itself can induce a T-cell immune response that is not induced by the whole antigen protein (see Moalem et al, 1999).

A peptide derived from a NS-specific antigen preferably has a sequence comprised within the NS-specific antigen sequence and has at least 10, 13, 15, 18, 20 or 50 contiguous amino acids of the NS-specific antigen sequence. In one embodiment, the peptide derived from an NS-specific antigen is a "cryptic epitope" of the antigen. A cryptic epitope activates specific T cells after an animal is immunized with the particular peptide, but not with the whole antigen. Cryptic epitopes for use in the present invention include, but are not limited to, peptides of the MBP sequence: peptides p11-30, p51-70, p87-99, p91-110, p131-150, and p151-170. Such cryptic epitopes are particularly preferred as T cells activated thereby will accumulate at the injury site, but are particularly weak in autoimmunity. Thus, they would be expected to have fewer side effects.

In another embodiment, the peptide derived from an NS-specific antigen is an immunogenic epitope of the antigen.

Examples of further peptides according to the invention are immunogenic peptides derived from the Nogo protein sequence such as, but not being limited to, the 18-mer p472 Nogo peptide (SEQ ID NO:19) and peptides derived from the Nogo receptor (Fournier et al, 2001) such as the 15-mer peptides of the sequences -- see Original Patent.

In still another embodiment of the invention, the peptide is an analog of a peptide derived from an NS-specific antigen that is immunogenic but not encephalitogenic. The most suitable peptides for this purpose are those in which an encephalitogenic self-peptide is modified at the T-cell receptor (TCR) binding site and not at the MHC binding site(s), so that the immune response is activated but not anergized (Karin et al, 1998; Vergelli et al, 1996).

These analogs, also referred herein as modified peptides or altered peptides, may be produced by replacement of one or more amino acid residues of the peptide by other amino acid residues, preferably in their TCR binding site. Suitable replacements are those in which charged amino residues like lysine, proline or arginine are replaced by glycine or alanine residues. For example, altered peptides can be produced from peptides p11-30, p51-70, p87-99, p91-110, p131-150, and p151-170 of human MBP, for example from the p87-99 peptide in which the lysine 91 is replaced by glycine and/or the proline 96 is replaced by an alanine residue, thus converting an encephalitogenic peptide in immunogenic but non-encephalitogenic peptide that still recognizes the TCR. In the same way, altered peptides can be produced from the encephalitogenic p472 Nogo peptide (Nogo p623-640) by replacement of the lys 628 residue and from the Nogo receptor peptides above by replacement of the arg (R) residue by Val or Ala or another similar residue.

In addition, the analogs also comprise replacement of one or more amino acid residues of the peptide or addition to the peptide of non-natural amino acids including, but not limited to, the D-isomers of the common amino acids, .alpha.-aminoisobutyric acid; 4-aminobutyric acid (Abu); 2-Abu (.gamma.-Abu); 6-amino hexanoic acid (.epsilon.-Ahx); 2-aminoisobutyric acid (Aib); 3-aminopropionic acid; ornithine; norleucine (Nle); norvaline (Nva); hydroxyproline; sarcosine; citrulline; cysteic acid; t-butylglycine; t-butylalanine; phenylgylcine; cyclohexylalanine; .beta.-alanine; fluoro-amino acids; designer amino acids such as .beta.-methyl amino acids, C.alpha.-methyl amino acids, N.alpha.-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

Furthermore, the invention also comprises chemical derivatives of the peptides of the invention including, but not being limited to, esters of both carboxylic and hydroxy groups, amides, and the like.

The NS-specific antigen peptides of the invention can be chemically synthesized. For example, a peptide corresponding to a portion of an antigen which comprises the desired domain or which mediates the desired activity can be synthesized by use of a peptide synthesizer.

The functional activity of NS-specific antigens and peptides derived therefrom and analogs and derivatives thereof can be assayed by various methods known in the art, including, but not limited to, T-cell proliferation assays (Mor and Cohen, 1995) and cytokine production assays.

An NS-specific antigen or peptide derived therefrom or derivative thereof may be kept in solution or may be provided in a dry form, e.g., as a powder or lyophilizate, to be mixed with appropriate solution prior to use. They may be used both as ingredients of pharmaceutical compositions for neuroprotection and preventing or inhibiting the effects of injury or disease that result in NS degeneration or for promoting nerve regeneration in the NS, particularly in the CNS as well as for in vivo or in vitro activation of T cells.

Nucleotide Sequences Encoding NS-Antigens and Peptides Derived Therefrom

The present invention further provides pharmaceutical compositions comprising a therapeutically effective amount of a nucleotide sequence encoding an NS-specific antigen or a peptide derived therefrom or an analog thereof and methods of use of such compositions to promote nerve regeneration or for neuroprotection and prevention or inhibition of neuronal degeneration in the CNS or PNS in which the amount is effective to ameliorate the effects of an injury or disease of the NS.

Specific illustrative examples of useful nucleotide sequences encoding NS-specific antigens or peptides derived from an NS-specific antigen include, but are not limited to, nucleotide sequences encoding rat MBP, depicted in FIG. 15 (SEQ ID NO:1, see Original Patent); human MBP, depicted in FIG. 16 (SEQ ID NO:2, see Original Patent); human PLP, depicted in FIG. 17(A-F) (SEQ ID NOs:3-8, see Original Patent); human MOG, depicted in FIG. 18 (SEQ ID NO:9); rat PLP and variant, depicted in FIG. 19 (SEQ ID NO:10); rat MAG, depicted in FIG. 20 (SEQ ID NO:11); rat Nogo (SEQ ID NO:17); and human Nogo (SEQ ID NO:22). Other illustrative examples are the nucleotide sequences disclosed in Chen et al (2000), Prinjha et al (2000) and Fournier et al (2001) (the contents of each of which being hereby incorporated herein by reference) encoding rat and human Noga A, B and C and mouse and human NgR.

Therapeutic Uses

The T cells, NS-specific antigens, analogs thereof, peptides derived therefrom and analogs and derivatives thereof, and nucleotide sequences described in the previous sections and compositions comprising them may be used to promote nerve regeneration or to confer neuroprotection and prevent or inhibit secondary degeneration which may otherwise follow primary NS injury, e.g., spinal cord injury, blunt trauma, penetrating trauma, hemorrhagic stroke, ischemic stroke or damages caused by surgery such as tumor excision.

In addition, such compositions may be used to ameliorate the effects of disease that result in a degenerative process, e.g., degeneration occurring in either gray or white matter (or both) as a result of various diseases or disorders, including, without limitation: diabetic neuropathy, senile dementias, Alzheimer's disease, Parkinson's disease, facial nerve (Bell's) palsy, glaucoma, Huntington's chorea, amyotrophic lateral sclerosis (ALS), non-arteritic optic neuropathy, intervertebral disc herniation, vitamin deficiency, prion diseases such as Creutzfeldt-Jakob disease, carpal tunnel syndrome, peripheral neuropathies associated with various diseases, including but not limited to, uremia, porphyria, hypoglycemia, Sjorgren Larsson syndrome, acute sensory neuropathy, chronic ataxic neuropathy, biliary cirrhosis, primary amyloidosis, obstructive lung diseases, acromegaly, malabsorption syndromes, polycythemia vera, IgA- and IgG gamma-pathies, complications of various drugs (e.g., metronidazole) and toxins (e.g., alcohol or organophosphates), Charcot-Marie-Tooth disease, ataxia telangectasia, Friedreich's ataxia, amyloid polyneuropathies, adrenomyeloneuropathy, Giant axonal neuropathy, Refsum's disease, Fabry's disease, lipoproteinemia, etc.

In a preferred embodiment, the NS-specific activated T cells, the NS-specific antigens, peptides derived therefrom, analogs and derivatives thereof or the nucleotides encoding said antigens, or peptides or any combination thereof of the present invention are used to treat diseases or disorders where promotion of nerve regeneration or prevention or inhibition of secondary neural degeneration is indicated, which are not autoimmune diseases or neoplasias. In a preferred embodiment, the compositions of the present invention are administered to a human subject.

While activated NS-specific T cells may have been used in the prior art in the course of treatment to develop tolerance to autoimmune antigens in the treatment of autoimmune diseases, or in the course of immunotherapy in the treatment of NS neoplasms, the present invention can also be used to ameliorate the degenerative process caused by autoimmune diseases or neoplasms as long as it is used in a manner not suggested by such prior art methods. Thus, for example, T cells activated by an autoimmune antigen have been suggested for use to create tolerance to the autoimmune antigen and, thus, ameliorate the autoimmune disease. Such treatment, however, would not have suggested the use of T cells directed to other NS antigens or NS antigens which will not induce tolerance to the autoimmune antigen or T cells which are administered in such a way as to avoid creation of tolerance. Similarly, for neoplasms, the effects of the present invention can be obtained without using immunotherapy processes suggested in the prior art by, for example, using an NS antigen which does not appear in the neoplasm. T cells activated with such an antigen will still accumulate at the site of neural degeneration and facilitate inhibition of this degeneration, even though it will not serve as immunotherapy for the tumor per se.

Nogo protein or a fragment thereof which are active in inhibiting cell proliferation have been disclosed as useful for treatment of a neoplastic disease of the CNS such as glioma, glioblastoma, medulloblastoma, craniopharyngioma, ependyoma, neuroblastoma and retinoblastoma. The present invention does not encompass the use of Nogo or a peptide derived therefrom for treatment of neoplasias in general, and for treatment of a neoplastic disease of the CNS, in particular.

Formulations and Administration

The present invention also provides pharmaceutical compositions useful in methods to promote nerve regeneration or to confer neuroprotection and prevent or inhibit neuronal degeneration in the CNS or PNS, comprising a therapeutically effective amount of at least one ingredient selected from the group consisting of: (a) NS-specific activated T cells; (b) a NS-specific antigen or an analog thereof; (c) a peptide derived from an NS-specific antigen or from an analog thereof, or an analog or derivative of said peptide; (d) a nucleotide sequence encoding an NS-specific antigen or an analog thereof; (e) a nucleotide sequence encoding a peptide derived from an NS-specific antigen or from an analog thereof, or an analog of said peptide; or (f) any combination of (a)-(e).

The compositions comprising ingredients (b) and/or (c) above are also effective to activate T cells in vitro, wherein the activated T cells inhibit or ameliorate the effects of an injury or disease of the NS.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. The carriers in the pharmaceutical composition may comprise a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monochydrate; a disintegrating agent, such as alginic acid, maize starch and the like; a lubricant or surfactant, such as magnesium stearate or sodium lauryl sulphate; a glidant, such as colloidal silicon dioxide; a sweetening agent, such as sucrose or saccharin; and/or a flavoring agent, such as peppermint, methyl salicylate, or orange flavoring.

Methods of administration include, but are not limited to, parenteral, e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, mucosal (e.g., oral, intranasal, buccal, vaginal, rectal,, intraocular), intrathecal, topical and intradermal routes. Administration can be systemic or local.

For oral administration, the pharmaceutical preparation may be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The pharmaceutical compositions may take the form of, for example, tablets, lozenges or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well-known in the art.

Preparations for oral administration may be also suitably formulated to give controlled release of the active compound.

The compositions may be formulated for parenteral administration by injection, 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 an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compositions may also be formulated as rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

For administration by inhalation, the compositions for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol 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 an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

In one embodiment, compositions comprising NS-specific activated T cells, an NS-specific antigen or peptide derived therefrom, or derivative thereof, or a nucleotide sequence encoding such antigen or peptide, are formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous or intraperitoneal administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water or saline for injection can be provided so that the ingredients may be mixed prior to administration.

Pharmaceutical compositions comprising NS-specific antigen or peptide derived therefrom or derivative thereof may optionally be administered with an adjuvant.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.

In a preferred embodiment, the pharmaceutical compositions of the invention are administered to a mammal, preferably a human, shortly after injury or detection of a degenerative lesion in the NS. The therapeutic methods of the invention may comprise administration of an NS-specific activated T cell or an NS-specific antigen or peptide derived therefrom or derivative thereof, or a nucleotide sequence encoding such antigen or peptide, or any combination thereof. When using combination therapy, the NS-specific antigen may be administered before, concurrently or after administration of NS-specific activated T cells, a peptide derived from an NS-specific antigen or derivative thereof or a nucleotide sequence encoding such antigen or peptide.

In one embodiment, the compositions of the invention are administered in combination with one or more of the following: (a) mononuclear phagocytes, preferably cultured monocytes (as described in PCT publication No. WO 97/09985, which is incorporated herein by reference in its entirety), that have been stimulated to enhance their capacity to promote neuronal regeneration; (b) a neurotrophic factor such as acidic fibroblast growth factor; and (c) an anti-inflammatory therapeutic substance, e.g., an anti-inflammatory steroid, such as dexamethasone or methyl-prednisolone, or a non-steroidal anti-inflammatory peptide, such as Thr-Lys-Pro (TKP)).

In another embodiment, mononuclear phagocyte cells according to PCT Publication No. WO 97/09985 and U.S. Pat. No. 6,267,955, are injected into the site of injury or lesion within the CNS, either concurrently, prior to, or following parenteral administration of NS-specific activated T cells, an NS-specific antigen or peptide derived therefrom or derivative thereof, or a nucleotide sequence encoding such antigen or peptide

In another embodiment, administration of NS-specific activated T cells, NS-specific antigen or peptide sequence encoding such antigen or peptide, may be administered as a single dose or may be repeated, preferably at 2-week intervals and then at successively longer intervals once a month, once a quarter, once every six months, etc. The course of treatment may last several months, several years or occasionally also through the lifetime of the individual, depending on the condition or disease which is being treated. In the case of a CNS injury, the treatment may range between several days to months or even years, until the condition has stabilized and there is no or only a limited risk of development of secondary degeneration. In chronic human disease or Parkinson's disease, the therapeutic treatment in accordance with the invention may be for life.

As will be evident to those skilled in the art, the therapeutic effect depends at times on the condition or disease to be treated, on the individual's age and health condition, on other physical parameters (e.g., gender, weight, etc.) of the individual, as well as on various other factors, e.g., whether the individual is taking other drugs, etc.

The optimal dose of the therapeutic compositions comprising NS-specific activated T cells of the invention is proportional to the number of nerve fibers affected by NS injury or disease at the site being treated. In a preferred embodiment, the dose ranges from about 5.times.10.sup.6 to about 10.sup.7 for treating a lesion affecting about 10.sup.5 nerve fibers, such as a complete transection of a rat optic nerve, and ranges from about 10.sup.7 to about 10.sup.8 for treating a lesion affecting about 10.sup.6 -10.sup.7 nerve fibers, such as a complete transection of a human optic nerve. As will be evident to those skilled in the art, the dose of T cells can be scaled up or down in proportion to the number of nerve fibers thought to be affected at the lesion or site of injury being treated.
 

Claim 1 of 27 Claims

1. A method for reducing secondary neuronal degeneration that follows neuronal damage caused by an injury, disease, disorder or condition in the central or peripheral nervous system of an individual in need thereof, comprising: causing T cells activated against a nervous system (NS)-specific antigen which, in its native state, is present at the site of secondary neuronal degeneration, to accumulate at the site of secondary neuronal degeneration in the individual in need, thereby reducing secondary neuronal degeneration at that site, wherein, when the individual in need has an autoimmune disease, the NS-specific antigen is not the autoimmune antigen of that disease, and when the individual in need has a neoplasm, the NS-specific antigen is one that does not appear in the neoplasm, wherein said causing step is accomplished by-- administering an effective amount of said NS-specific antigen, or an immunogenic epitope thereof, in such a manner as to cause a T cell response thereto, such that T cells become activated against the NS-specific antigen which is present at the site of secondary neuronal degeneration, wherein said NS-specific antigen or immunogenic epitope thereof that is administered is myelin basic protein (MBP), the peptide p51-70 of MBP, or Nogo-A p472 peptide (SEQ ID NO:19); or administering an effective amount of T cells that are activated against said NS-specific antigen or said immunogenic epitope thereof, wherein said activated T-cells are T-cells activated against MBP or the peptide p51-70 of MBP.

 

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