FIELD OF INVENTION

[001] The present invention generally relates to the field of immunotherapy, and more particularly to fusion proteins for the treatment of Multiple Sclerosis.

BACKGROUND OF INVENTION

[002] Antibody-secreting B lymphocytes are in the focus in relation to autoimmune diseases, because of their central role in the induction and maintenance of inflammatory reactions. Multiple sclerosis (MS) is an inflammatory demyelinating autoimmune disease of the central nervous system. MS is triggered by the body's own immune system attacking the myelin sheath around the axons. Auto reactive T cells (CD8+ and CD4+ cells) in genetically disposed persons initiate the inflammatory process and through their pro inflammatory cytokines secretion, activate the auto reactive B lymphocytes to secrete myelin specific antibodies.

[003] Auto antibodies against the proteins of the myelin sheath, especially against the Myelin Binding Protein (MBP), Myelin Oligodendrocyte Glycoprotein (MOG) and Proteo-Lipid Protein (PLP) are the biomarkers for clinical prognosis of Multiple Sclerosis. These antibodies (Ab) are involved in complement fixation followed by a subsequent myelin degradation process and the Ab-mediated phagocytosis by activated macrophages. These auto reactive B lymphocytes can further contribute to the pathologic state by presenting the myelin antigens to the activated T lymphocytes. Thus, besides being destructive on their own, the myelin specific auto reactive B lymphocytes help the myelin specific CD4+ T cells to maintain a sustained cell mediated attack on myelin sheath.

[004] Current medical interventions for multiple sclerosis include prevention of infiltration of inflammatory T and B lymphocytes into CNS (Natalizumab) or neutralization of MBP antibodies (Copaxone) or general immune suppression methods (Interferons). Rituximab, a monoclonal antibody that is in use for the treatment of non-Hodgkin's lymphoma is being evaluated for the treatment of Multiple Sclerosis owing to its ability to eliminate mature CD20+ B lymphocytes, which include Myelin antigens specific B lymphocytes. However, this would be a general approach as it targets all mature CD20+ B lymphocytes irrespective of their antigen specificity, and can lead to immunodeficiency, which is also the case with the general immune suppression methods.

[005] Therefore, there is a need for selective removal of myelin specific B lymphocytes while preserving the repertoire of humoral immunity against other antigens.

OBJECT OF INVENTION

[006] The principal object of this invention is to provide a method of treatment for multiple sclerosis using the immunotoxin (or fusion protein) comprising Myelin Binding Protein fragment, Myelin Oligodendrocyte Glycoprotein fragment and a cytotoxic fragment, wherein the cytotoxic fragment may be a diphtheria toxin fragment or an Fc fragment of a IgG.

[007] Another object of the invention is to provide a method of treatment for multiple sclerosis by targeting and killing B cells displaying myelin specific antibodies.

[008] Another object of this invention is to provide a method for killing B cells displaying myelin specific antibodies by administering a fusion protein of SEQ ID NO:2 (TBLMS1) and/or fusion protein of SEQ ID NO:4 (TBLMS2) which comprises of Myelin Binding Protein fragment, Myelin Oligodendrocyte Glycoprotein fragment, and cytotoxic fragment.

STATEMENT OF INVENTION

[009] Accordingly the invention provides a fusion protein comprising Myelin Binding Protein fragment, a Myelin Oligodendrocyte Glycoprotein fragment and a cytotoxic fragment which is characterized in that to target and kill B cells displaying myelin specific antibodies, wherein the cytotoxic fragment may be AB chain of diphtheria toxin or Fc fragment of lgG.

[0010] There is also provided a polypeptide sequence of SEQ ID NO: 2 (TBLMS1) and SEQ ID NO: 4 (TBLMS2) which is encoded by the polynucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 3.

[0011] In another embodiment, the invention provides a vector comprising at least one polynucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 3.

[0012] In another embodiment, the invention provides a pharmaceutical composition comprising at least one polypeptide sequence of SEQ ID NO: 2 and SEQ ID NO: 4, and a pharmaceutical^ acceptable carrier.

[0013] In yet another embodiment, the invention provides a method of killing B cells displaying myelin specific antibodies comprising contacting said B cells with at least one polypeptide sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4.

[0014] In another embodiment, the invention provides a method of treating multiple sclerosis comprising administering to a patient therapeutically effective amount of at least one polypeptide sequence of SEQ ID NO: 2 and SEQ ID NO: 4.

[0015] In another embodiment, the invention provides a diagnostic method for diagnosing the presence of myelin specific B cells in a sample, wherein the diagnostic method comprises of contacting the sample with at least one polypeptide sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4 under conditions that allow for formation of a complex between said polypeptide sequence and B cell, and detecting the formation of the complex.

[0016] In yet another embodiment, the invention provides a diagnostic kit for detecting the presence of myelin specific B cells in a sample, wherein said diagnostic kit comprises of at least one polypeptide sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4.

[0017] These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.

BRIEF DESCRIPTION OF FIGURES

[0018] This invention is illustrated in the accompanying drawings, through out which like reference letters indicate corresponding parts in the various figures. The embodiments herein will be better understood from the following description with reference to the drawings, in which:

[0019] Fig. 1A (also referred to as SEQ ID NO:1) is a diagram depicting the nucleotide sequence of the fusion protein, TBLMS1

[0020] Fig. IB (also referred to as SEQ ID NO:2) is a diagram depicting the amino acid sequence of the fusion protein, TBLMS 1

[0021] Fig. 2A (also referred to as SEQ ID NO:3) is a diagram depicting the nucleotide sequence of the fusion protein, TBLMS2

[0022] Fig. 2B (also referred to as SEQ ID NO:4) is a diagram depicting the amino acid sequence of the fusion protein, TBLMS2

DETAILED DESCRIPTION OF INVENTION

[0023] The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.

[0024] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0025] The use of "including", "comprising" or "having" and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms "a" and "an" herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. Further, the use of terms "first", "second", and "third", and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

[0026] Disclosed herein are embodiments that provide a mechanism for targeting and killing of a subset of B cell population with the therapeutic immunotoxin molecules of the described design to treat multiple sclerosis. The focus of the disclosed embodiments is to bring down the myelin antigens specific humoral responses through removal of myelin specific B lymphocytes. Removal of these auto reactive B cells will have a dual impact on disease progression. The immediate effect would be lowering of Myelin specific B lymphocytes and the specific antibodies. As the myelin specific B lymphocytes support and maintain myelin specific CD4+ T lymphocyte functions, the removal of these B lymphocytes will lead to lowering of myelin specific CD4+ T lymphocytes as well. Therefore, eventually, there will be diminished humoral and cell mediated attack on the myelin sheath.

[0027] Currently, MS is being treated with general immune suppression drugs or drugs that prevent infiltration of circulating T and B cells into the CNS. The former therapy can lead to general immune deficiency and the later on compromising of regular T and B cell surveillance of the CNS. The embodiments disclosed herein provide selective removal of the myelin specific B cells without harming other cells of the immune system and therefore circumvent the problems associated with the current medical interventions.

[0028] Clinical evidences show a correlation between the Myelin Oligodendrocyte Glycoprotein (MOG) & Myelin Binding Protein (MBP) auto antibodies and Multiple Sclerosis disease progression. Elimination of either MBP or MOG specific B lymphocytes alone may not be sufficient for complete cure of multiple sclerosis and hence both MOG and MBP specific B lymphocytes are to be removed.

[0029] The embodiments herein disclose designs of therapeutic molecules for the treatment of multiple sclerosis. It aims at removal of myelin antigen specific auto antibodies producing B cells only and thereby effectively brings down the auto reactive B cell and also the auto reactive T lymphocyte populations. The unique design of the therapeutic molecules allow them to target all MBP, MOG reactive B cells without affecting other antibody producing B cells or any other cells of the immune system, thereby being highly target specific. The fusion proteins described herein may also be used to treat autoimmune disease such as Crohn's disease, Rheumatoid arthritis, etc.

Fusion proteins

[0030] In an embodiment the therapeutic molecule is a fusion protein referred to as TBLMS1. TBLMS1 is a bi-specific immunotoxin molecule comprising a targeting domain and a cytotoxic fragment. The targeting domain of TBLMS1 comprises of an MOG fragment and an MBP fragment, and the cytotoxic fragment of TBLMS1 comprises of a truncated diphtheria toxin fragment. The truncated diphtheria toxin may comprise of A chain, B chain or A and B chain of diphtheria toxin. In an embodiment, the diphtheria toxin fragment comprises of the A and B chain of Diphtheria toxin. The therapeutic molecules described herein can be produced and isolated by various methods familiar to those skilled in the art, such as recombinant DNA methods.

[0031] In another embodiment the therapeutic molecule is a fusion protein referred to as TBLMS2. TBLMS2 is a bi-specific immunotoxin molecule comprising a targeting domain and a cytotoxic fragment. The targeting domain of TBLMS2 comprises of an MOG fragment and an MBP fragment, and the cytotoxic fragment of TBLMS2 comprises of the Fc fragment of an immunoglobulin. In an embodiment, the cytotoxic fragment is Fc fragment of IgG. The therapeutic molecules described herein can be produced and isolated by various methods familiar to those skilled in the art, such as recombinant DNA methods.

[0032] Referring now to the drawings, and more particularly to FIGS. 1 A, IB, 2A and 2B, there are shown preferred embodiments.

[0033] Fig. 1A (also referred to as SEQ ID NO:1) is the nucleotide sequence of TBLMS1 which is a design of the bi-specific immunotoxin molecule that can specifically target MBP as well as MOG antibody producing B cells. The targeting domain is a fusion of extracellular domains of MOG and MBP that is linked to the diphtheria toxin by a linker sequence. This design is well suited for expression in E.coli, mammalian cells or yeast as a monomer. The targeting domain (MOG+MBP) targets all MBP and MOG specific B lymphocytes, which are killed by the Diphtheria toxin after the target cells internalize the whole immunotoxin molecule.

[0034] Fig. IB (also referred to as SEQ ID NO:2) is the amino acid sequence of TBLMS1. SEQ ID NO:2 is encoded by the nucleotide sequence of SEQ ID NO: 1.

[0035] Fig. 2A (also referred to as SEQ ID NO:3) is the nucleotide sequence of TBLMS2 which is the design of the bi-specific immunotoxin molecule that can specifically target MBP as well as MOG antibody producing B cells. In this design, the targeting domain (MOG+MBP) is linked to the Fc fragment of human IgG by a linker sequence. This molecule can be expressed in mammalian system like CHO as a dimer with an IgG like structure. The targeting domain of the immunotoxin molecule binds to the MOG and MBP specific B lymphocytes and via Fc portion of the immunotoxin, these B lymphocyte complexes are recognized and internalized by the macrophages which subsequently dispose them effectively.

[0036] Fig. 2B (also referred to as SEQ ID NO:4) is the amino acid sequence of TBLMS2. SEQ ID NO:4 is encoded by the nucleotide sequence of SEQ ID NO:3.

[0037] In an embodiment, a vector comprising the polynucleotide sequence of SEQ ID NO:1 and/or SEQ ID NO:3 is provided. In another embodiment, a cell line comprising the vector, wherein the vector further comprises of polynucleotide sequence of SEQ ID NO:1 and/or SEQ ID NO:3, is provided . The cell line may be used to produce the fusion proteins TBLMSI and/or TBLMS2.

Pharmaceutical compositions

[001] In an embodiment, the fusion protein(s) TBLMSI and/or
TBLMS2 may be administered to a subject with multiple sclerosis by way of a pharmaceutical composition. The pharmaceutical composition may comprise of TBLMSI and/or TBLMS2, and a pharmaceutically acceptable carrier.

[002] The pharmaceutically acceptable carriers in the pharmaceutical composition include generally used carriers well known in the art including water, salt solutions, gelatins, oils, alcohols, and other excipients and auxiliaries that facilitate processing of the active compounds into preparations that may be used pharmaceutically.

[003] In addition to the fusion protein(s) disclosed herein, the pharmaceutical composition may also comprise of pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or other carriers well known in the art.

Treatment

[004] The disclosed embodiments of the fusion protein(s) may be used for the treatment of autoimmune disease including Multiple sclerosis, Crohn's disease and Rheumatoid arthritis.

[005] In an embodiment the fusion protein(s) of the present invention may be used for the treatment of multiple sclerosis. The method for treating multiple sclerosis comprises of administering a therapeutically effective amount of TBLMS1 and/or TBLMS2 to the subject.

[006] The term "therapeutically effective amount" includes an amount of the composition having therapeutic effect on a subject upon administration of the composition to the subject.

[007] In an embodiment the fusion protein(s) TBLMS1 and/or TBLMS2 are used to target B cells displaying myelin specific antibodies by contacting TBLMS1 and/or TBLMS2 with myelin specific B cells. In another embodiment, the targeted B cells are inactivated or killed by the cytotoxic fragments of fusion protein(s) TBLMS1 and/or TBLMS2

[008] In another embodiment, the fusion protein(s) TBLMS1 and/or TBLMS2 may be used to detect the presence of B cells displaying myelin specific antibodies in a sample. The diagnostic method for detecting the presence of B cells displaying myelin specific antibodies in a sample comprises of: contacting the sample with the fusion protein(s) TBLMS1 and/or TBLMS2, under conditions that allow for formation of a complex between the polypeptide sequence(s) of fusion protein(s) TBLMS1 and /or TBLMS2 and B cell; and detecting the formation of the complex.

[009] In an embodiment, the complex formed between the fusion protein(s) TBLMS1 and/or TBLMS2, and the B cells displaying myelin specific antibodies may further be detected by methods known in the art.

[0010] The "sample" may originate from a mammal, and includes tissue or body fluids (such as bone marrow tissue, colon tissue, blood sample etc.) or an extract of any tissue suspected of having B cells displaying myelin specific antibodies.

[0011] The detection of the complex as described herein can be performed by apparatus capable of detecting specific signals emitted by detectable labels generally known in the art such as radiation emission, color change, fluorescence, etc.

[0012] In another embodiment, the fusion protein(s) TBLMS1 and/or TBLMS2 may be provided in a diagnostic kit for detecting the presence of B cells displaying myelin specific antibodies.

[0013] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.

WE CLAIM:

1. A fusion protein for targeting B cells displaying myelin specific antibodies, wherein said fusion protein comprises of a Myelin Binding Protein fragment, a Myelin Oligodendrocyte Glycoprotein fragment and a cytotoxic fragment.

2. The fusion protein for targeting B cells displaying myelin specific antibodies as claimed in claim 1, wherein said cytotoxic fragment comprises of a toxin selected from the group consisting of A chain of diphtheria toxin, B chain of diphtheria toxin, A and B chain of diphtheria toxin, anthrax toxin and pseudomonas toxin.

3. The fusion protein for targeting B cells displaying myelin specific antibodies as claimed in claim 1, comprising a polypeptide sequence of SEQ ID NO: 2.

4. The fusion protein for targeting B cells displaying myelin specific antibodies as claimed in claim 1, wherein said cytotoxic fragment comprises of an Fc fragment.

5. The fusion protein for targeting B cells displaying myelin specific antibodies as claimed in claim 1, wherein said cytotoxic fragment comprises of an Fc fragment of IgG.

6. The fusion protein for targeting B cells displaying myelin specific antibodies as claimed in claim 1, comprising a polypeptide sequence of SEQ ID NO: 4.

7. A polynucleotide sequence of SEQ ID NO: 1 that encodes the polypeptide sequence of SEQ ID NO: 2.

8. A polynucleotide sequence of SEQ ID NO: 3 that encodes the polypeptide sequence of SEQ ID NO: 4.

9. A vector for expressing a fusion protein for targeting B cells displaying myelin specific antibodies, wherein said vector comprises of at least one polynucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3.

10. A cell line comprising a vector, for expressing a fusion protein for targeting B cells displaying myelin specific antibodies, wherein said vector comprises of at least one polynucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3.

11. A pharmaceutical composition for targeting B cells displaying myelin specific antibodies, wherein said composition comprises of: at least one polypeptide sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4; and a pharmaceutically acceptable carrier.

12. A method of targeting B cells displaying myelin specific antibodies, wherein said method comprises of contacting said B cells with at least one polypeptide sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4.

13. A method of treating multiple sclerosis comprising administering a therapeutically effective amount of at least one polypeptide sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ED NO: 4.

14. A diagnostic method for diagnosing the presence of B cells displaying myelin specific antibodies in a sample, wherein said diagnostic method comprises of:

contacting said sample with at least one polypeptide sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4 under conditions that allow for formation of a complex between said polypeptide sequence and B cell; and detecting the formation of said complex.

15. A diagnostic kit for detecting the presence of B cells displaying myelin specific antibodies in a sample, wherein said diagnostic kit comprises of at least one polypeptide sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4.