FIELD OF INVENTION

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

BACKGROUND OF INVENTION

[002] AIDS is characterized by a progressive decline of CD4 positive immune cells (CD4*) from the host's system. The current antiretroviral drug therapies are effective in containing the viral replication and thus bring up the CD4+ T cell counts but are ineffective in restoring the HIV specific, especially viral envelop gpl20 specific CD4+ Thl responses, that are essential for HIV clearance. The failure of restoration of gpl20 specific CD4+ Thl responses at least in the retroviral drugs treated persons is not entirely due to lack of gpl20 specific CD4+ Th cells. There is extensive published literature supporting a rather unusual negative role for the host produced gpl20 specific antibodies in suppression of gpl20 specific CD4+ Thl responses, despite the presence of good numbers of un¬stimulated gpl20 specific CD4+Thl cells in HIV infected persons.

[003] The neutralizing gpl20 antibodies are well known in containing the HIV replication during early stages of infection. But as the virus mutates, these neutralizing antibodies lose their efficacy and become ineffective overtime. Besides being ineffective, they also suppress gpl20 specific CD4+Thl responses. Gpl20 specific antibodies, especially those that bind to CD4 binding region of gpl20, suppress gpl20 specific CD4+ Thl responses by interfering with antigen presentation by Antigen presenting cells (APC). The gpl20 antibodies specific to CD4 binding region of gpl20, bind to the viral gpl20, and this binding is so strong that the gpl20 antigen processing and presentation to the gpl20 specific CD4+ Thl cells by the antigen presenting cells (APC) is curtailed. Even in the individuals under Retroviral drug treatments, gpl20 antibodies persist as the virus replicates albeit low and these gpl20 antibodies continue to suppress the gpl20 CD4+ Thl responses. This is evident by lack of recovery of gpl20 specific Thl responses while Thl responses against other viral infections could be revoked. These findings suggest that gpl20 antibodies not only fail to contain HIV infection but also seriously curtail cellular immunity against HIV.

[004] Thus, it is imperative to look for strategies that can restore the vital gpl20 specific CD4+ Thl responses, which have the potential to clear the HTV infection and support gpl20 specific CD8+ as well as humoral immune responses against HIV for complete viral control.

[005] As good numbers of gpl20 specific CD4+ T cells are shown to exist in many infected individuals but just are quiescent because of the negative role of gpl20 specific antibodies, elimination of gpl20 antibodies producing B cells and thus gpl20 antibodies can modulate the immune system towards cell mediated immunity in HIV infected persons.

[006] Hence, there is an immediate need for methods for selective targeting and killing of a subset of B cell population to prevent AIDS progression in HTV infected persons.

OBJECT OF INVENTION

[007] The principal object of this invention is to provide prevention and/or treatment of HIV infection by utilizing immunotoxins (or fusion proteins) comprising CD4 binding region of gpl20 fragment and diphtheria toxin.

[008] Another object of this invention is to provide treatment and/or prevention for HIV infection by targeting B cells displaying antibodies specific to gpl20.

BRIEF DESCRIPTION OF FIGURES

[009] 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:

[0010] FIG.l is an illustration of fusion protein TBLIT1, comprising a truncated version of Diptheria toxin AB linked to CD4 binding domain of gpl20 (CD4BD gpl20), according to embodiments as disclosed herein;

[0011]FIG.2 is an illustration of fusion protein TBLIT2, comprising a Diphtheria toxin A chain linked to CD4 binding domain of gpl20, according to embodiments as disclosed herein;

[0012] FIG.3 is an illustration of fusion protein TBLIT3, comprising a Diphtheria toxin B chain linked to CD4 binding domain of gpl20, according to embodiments as disclosed herein;

[0013] FIG.4 depicts the SDS-PAGE gel showing the expression of TBL1T1 fusion protein in E.coli, according to embodiments as disclosed herein;

[0014]FIG.5 depicts the SDS-PAGE gel showing the purified TBL1T1 fusion protein, according to embodiments as disclosed herein;

[0015] FIG.6 depicts the cytotoxic effects of the fusion protein TBL1T1 on B cells bearing gpl20 antibody by viable cell count method, according to embodiments as disclosed herein;

[0016] FIG.7 is a graph depicting the cytotoxic effects of the fusion protein TBL1T1 on bearing B cells bearing gpl20 antibody as assayed by XTT method, according to embodiments as disclosed herein;

[0017] FIG.8 (referred to as SEQ ID NO: 1) depicts a polynucleotide sequence of fusion protein TBLIT1, according to embodiments as disclosed herein;

[0018] FIG.9 (referred to as SEQ ID NO: 2) depicts a polypeptide sequence of fusion protein TBLIT1, according to embodiments as disclosed herein;

[0019] FIG. 10 (referred to as SEQ ID NO: 3) depicts a polynucleotide sequence of fusion protein TBLIT2, according to embodiments as disclosed herein;
[0020] FIG.l 1 (referred to as SEQ ID NO: 4) depicts a polypeptide sequence of fusion protein TBLIT2, according to embodiments as disclosed herein;

[0021] HG.l2 (referred to as SEQ ID NO: 5) depicts a polynucleotide sequence of fusion protein TBLIT3, according to embodiments as disclosed herein; and

[0022] HG.l3 (referred to as SEQ ID NO: 6) depicts a polypeptide sequence of fusion protein TBLIT3, according to embodiments as disclosed herein.

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] [0054] 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] The embodiments herein disclose fusion proteins of the described design that are directed towards targeting and/or killing of a subset of B cell population to prevent HIV infection or AIDS progression in HIV infected persons, and/or to treat HIV infection. The disclosed fusion proteins are instrumental in restoring gpl20 specific CD4+ helper cell responses through selective elimination of gpl20 antibody producing B cells and stimulation of CD4+ T cell responses in the HIV infected individuals.

Fusion proteins

[0027] The disclosed embodiments herein provide three designs of fusion protein which can target and kill gpl20 antibody producing B cells. The fusion protein comprises of a gpl20 fragment and a toxin fragment, wherein the toxin fragment may be at least one of chain A of diphtheria toxin, chain B of diphtheria toxin, chain A and B (chain AB) of diphtheria toxin, anthrax toxin and pseudomonas toxin.

[0028] The designs designated TBLIT1, TBLIT2 and TBLIT3 comprises of chain AB of diphtheria toxin, chain A of diphtheria toxin and chain B of diphtheria toxin, respectively, linked to gpl20 fragments. The chain AB of diphtheria toxin, chain A of diphtheria toxin and chain B of diphtheria toxin may be truncated such that the fusion molecules are able to kill the gpl20 specific B cells. In an embodiment, the fusion protein (TBLIT1) comprises of chain AB of diphtheria toxin linked to gpl20 fragment which may be administered to a subject such that it targets B cells producing gpl20 specific antibodies. TBLIT1 may further bring about killing of the gpl20 producing B cells.

[0029] In an embodiment, die fusion protein (TBLIT2) comprises of: A chain of diphtheria toxin linked to gpl20 fragment. In another embodiment, the fusion protein (TBLIT3) comprises of: chain B of diphtheria toxin linked to gpl20 fragment. The fusion proteins TBLIT2 and TBLIT3 may be administered together to a subject, in an embodiment, in order to bring about targeting and/or killing of gpl20 antibody producing B cells.

[0030] Referring now to the drawings FIGS. 1 through 7, there are shown preferred embodiments.

[0031] FIG.1 illustrates the fusion protein TBLIT1 which comprises of a truncated version of AB chain of Diptheria toxin linked to CD4 binding domain of gpl20 (CD4BD gpl20). In an embodiment, the CD4BD gpl20 of the fusion protein molecule binds to the specific B cells bearing antibodies to CD4BD gpl20 on their surface. In another embodiment, the fusion protein upon binding to the specific B cell is internalized via endosomal pathway. The diphtheria toxin chain A is then released from the rest of the fusion protein molecule in the endosome due to cleavage actions of endosomal enzymes. The Diphtheria toxin chain B forms a channel in the endosomal membrane and translocates the diphtheria chain A into cytosol where it induces cell death. CD4BD gpl20 antibody producing B cells are capable of binding and internalizing this therapeutic fusion protein. The other cells of the immune system, more importantly CD4+ T cells are incapable of internalizing this fusion protein and hence are not affected.

[0032] FIG. 2 illustrates the fusion protein TBLIT2 which comprises of CD4 binding domain of gpl20 linked to Diphtheria toxin chain A.

[0033] FIG. 3 illustrates the fusion protein TBLIT3 which comprises of CD4 binding domain of gpl20 linked to Diphtheria toxin chain B.

[0034] In another embodiment, the two fusion proteins depicted in FIG. 2 & FIG. 3 are used in conjunction. The fusion proteins TBLIT2 and TBLIT3 are capable of targeting the CD4BD gpl20 antibody producing B cells. Both of these fusion protein molecules bind to the B cells bearing gpl20 CD4 binding domain antibodies on their surface and are internalized independently. Killing of the target B cells, i.e. B cells bearing gpl20 CD4 binding domain antibodies, occurs only when the B cells co-ingests both fusion protein molecules. Both chains of diphtheria toxin (A&B) are required for killing, and the target B cells alone are capable of internalizing both fusion proteins. This added safety design of the fusion protein molecules makes them harmless on off target cells such as macrophages, which may accidentally ingest one of these molecules. These features of the therapeutic fusion protein molecules make them safe on the immune system.

[0035] Furthermore, the receptor less, truncated Diphtheria toxin A and B chains can associate inside the targeted cells (whether toxin chains are provided in cis or trans) and kill them effectively even at low doses as one molecule of truncated Diphtheria toxin B chain is sufficient to translocate many truncated Diphtheria toxin A chain. Even low doses of fusion proteins are sufficient to target CD4BD gpl20 antibody producing B cells. So the present therapeutic tool results in a cost effective treatment of AIDS with a fewer side effects.

[0036] FIG. 4 is a diagram depicting a SDS-PAGE gel showing expression of TBL1T1 protein in E.coli. According to an embodiment, the vector construct containing TBLIT1 gene is introduced into E.coli strain BL21-DE3 by transformation and a colony producing high amounts of selective protein is selected through screening. Protein production in the selected strain is induced by IPTG and as depicted in FIG.4, there is a high amount of protein expression and all of the proteins are in insoluble form as Inclusion bodies. The band marked with a molecular weight 80 KD indicates the protein expressed by the TBL1T1 gene.

[0037] FTG.5 is a diagram depicting a SDS-PAGE gel showing the purified TBL1T1 protein. In an embodiment, TBL1T1 protein in the insoluble fraction is refolded in an optimized buffer containing arginine and then purified by Anion exchange and size exclusion chromatographies. The fusion protein is obtained in its purest form in the band marked with 80 KD.

[0038] FIG.6 is a graph depicting cytotoxic effects of TBL1T1 protein on gpl20 antibody bearing B cells by a viable cell count method. The B lymphocytes producing gpl20 antibodies are incubated with different concentrations (1 µg/ml, 2 µg/ml, 4 µg/ml and the like) of the fusion protein, TBLIT1, up to 72 hrs. The viable cells are counted at 0, 24, 48 and 72 hours by trypan blue method. It is observed that there is a significant decrease in the viable cell count of B lymphocytes in millions per ml treated with the toxin protein, TBLIT1, at 48 hours and beyond. This further indicates that the protein is cytotoxic even at a concentration of 1 µg/ml to gpl20 antibody bearing B cells.

[0039] FIG.7 is a graph depicting cytotoxic effects of TBL1T1 protein on gpl20 antibody bearing B cells by XTT assay method. In an embodiment, B cells producing gpl20 antibodies are chosen as target cells and incubated with different concentrations (1 µg/ml, 2 µg/ml and 4 µg/ml)
of the fusion protein, TBLIT1, up to 72 hours. Cell proliferation assay is done using XTT assay Kit (Biological Industries, Israel) at 0, 24,48 and 72 hours. The toxin treated samples show a significant decrease from the controls at 48 hours and beyond, similar to the results of the viable count method, confirming the cytotoxicity of the tested protein, TBLIT1 on gpl20 antibody specific B cells.

[0040]FIG.8 (referred to as SEQ ID NO: 1) depicts the polynucleotide sequence of the fusion protein TBLIT1.

[0041 ] FIG.9 (referred to as SEQ ID NO: 2) depicts the polypeptide sequence of the fusion protein TBLIT1.

[0042] FIG. 10 (referred to as SEQ ID NO: 3) depicts the polynucleotide sequence of the fusion protein TBLIT2.

[0043] FIG. 11 (referred to as SEQ ID NO: 4) depicts the polypeptide sequence of the fusion protein TBLIT2.

[0044] FIG. 12 (referred to as SEQ ID NO: 5) depicts the polynucleotide sequence of the fusion protein TBLIT3.

[0045] FIG. 13 (referred to as SEQ ID NO: 6) depicts the polypeptide sequence of the fusion protein TBLIT3.

[0046] In an embodiment, a vector comprising the polynucleotide sequence of SEQ ID NO:l, SEQ ID NO:3 and/or SEQ ID NO:5 is provided. In another embodiment, a cell line comprising the vector, wherein the vector comprises of polynucleotide sequence of SEQ ID

NO:1, SEQ ID N0:3 and/or SEQ ID NO:5, is provided. The cell line may be used to produce the fusion protein(s) TBL1T1, TBL1T2 and/or TBL1T3. In an embodiment, the cell line is a bacterial cell line. In a preferred embodiment, the cell line is a E.coli strain BL21-DE3.

[0047] According to another embodiment, the present method for eliminating B cell sub populations producing CD4BD gpl20 antibodies can be adopted with suitable changes in the targeting domain for selective elimination of any sub population of B cells with minimal or no killing of other cell types. The disclosed fusion proteins are best suited for treatment of diseases such as autoimmune diseases, pathogen-mediated autoimmune conditions and the like.
Pharmaceutical compositions

[0048] In an embodiment, the fusion protein(s) TBL1T1, TBL1T2 and/or TBL1T3 may be administered to a subject by way of a pharmaceutical composition. The pharmaceutical composition may comprise of at least one of the fusion proteins TBL1T1, TBL1T2 and TBL1T3, and a pharmaceutically acceptable carrier.

[0049] 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.

[0050] In addition to the fusion proteins 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

[0051] The disclosed embodiments of the fusion proteins may be used for the treatment of autoimmune disease; pathogen-mediated autoimmune conditions including HIV mediated AIDS progression.

[0052] In an embodiment, the disclosed fusion proteins may be used for the treatment of HIV infection. In another embodiment the disclosed fusion proteins may be used for the prevention of HIV infection. The method for treatment or prevention of HIV infection comprises of administering a therapeutically effective amount of at least one of the fusion proteins TBL1T1, TBL1T2 and TBL1T3 to the subject.

[0053] In an embodiment, TBL1T1 is administered into the subject for treatment or prevention of HIV infection. In another embodiment, TBL1T2 and TBL1T3 is administered into the subject for treatment or prevention of HIV infection.

[0054] 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.

[0055] In an embodiment, the fusion protein(s) TBL1T1, TBL1T2 and/or TBL1T3 are instrumental in targeting gpl20 specific B cell by contacting at least one of TBL1T1, TBL1T2 and TBL1T3 with gpl20 specific B cell. In another embodiment, the targeted B cells, i.e. gpl20 specific B cell, are inactivated or killed by fusion protein(s) TBL1T1, TBL1T2 and/or TBL1T3.

[0056] In another embodiment, the fusion protein(s) TBL1T1, TBL1T2 and/or TBL1T3 may be used to detect the presence of B cells displaying gpl20 specific antibodies in a sample. The diagnostic method for detecting the presence of B cells displaying gpl20 specific antibodies in a sample comprises of: contacting the sample with the fusion protein(s) TBL1T1, TBL1T2 and/or TBL1T3, under conditions that allow for formation of a complex between the polypeptide sequence(s) of fusion protein(s) TBL1T1, TBL1T2 and/or TBL1T3 and B cell; and detecting the formation of the complex.

[0057] In an embodiment, the complex formed between the fusion protein(s) TBL1T1, TBL1T2 and/or TBL1T3, and the B cells displaying gpl20 specific antibodies may further be detected by methods known in the art.

[0058] 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 gpl20 specific antibodies.

[0059] 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.

[0060] In another embodiment, the fusion protein(s) TBL1T1, TBL1T2 and/or TBL1T3 may be provided in a diagnostic kit for detecting the presence of B cells displaying gpl20 specific antibodies.

[0061] As will be appreciated by a person skilled in the art the present invention provides a variety of advantages. The method of restoring gpl20 specific CD4+ T cell responses by selective annihilation of CD4BD gpl20 antibody producing B cells is a novel immunomodulation approach which has a potential to replace the current antiretroviral drug /Interleukin therapies. Majority of the conventional therapeutics are aimed at strengthening either CTLs or Humoral Immunity, where the conventional therapeutics neglected the fact that antigen specific CD4+ T cell supportive functions are the key factors behind both forms of immune responses. Stimulation of CD4+ T cell responses is a must to bring about the two different forms of immune responses for an effective control of HIV. As discussed, the gpl20 specific CD4+ T responses that are essential for comprehensive immune responses are severely affected by antibodies specific to CD4 binding domain of gpl20. The various embodiments of the disclosed fusion proteins on elimination of CD4BD gpl20 antibodies through selective destruction of CD4BD gpl20 antibody producing B cells will modulate the immune system towards cell mediated immunity. As anti-retroviral drug treatments alone are not so effective in restoring the vital gpl20 specific CD4+ T cell responses against HIV, the Immune-modulation therapy utilizing the disclosed fusion proteins may be used in conjunction with anti-retroviral drug treatment to initiate the restoration of antigen specific T cell responses. Subsequently, the disclosed fusion proteins can be used, without the need for anti-retroviral drug therapy, to continue the restoration of healthy T cell responses.

[0062] While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.

[0063] 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 antibodies specific to gpl20, wherein said fusion protein comprises of a gpl20 fragment and a toxin fragment.

2. The fusion protein for targeting B cells displaying antibodies specific to gpl20 as claimed in claim 1, wherein the toxin 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 antibodies specific to gpl20 as claimed in claim 1, wherein said gpl20 fragment comprises of CD4 binding region.

4. The fusion protein for targeting B cells displaying antibodies specific to gp 120 as claimed in claim 1, wherein said fusion protein comprises of a polypeptide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.

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

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

7. A polynucleotide sequence of SEQ ID NO: 5 that encodes the polypeptide sequence of SEQ ID NO: 6.

8. A vector for expressing a fusion protein for targeting B cells displaying antibodies specific to gpl20, wherein said vector comprises at least one polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5.

9. A cell line comprising a vector for expressing a fusion protein for targeting B cells displaying antibodies specific to gpl20, wherein said vector comprises at least one polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5.

10. The cell line as claimed in claim 9, wherein the cell line is a bacterial cell line.

11. A pharmaceutical composition for targeting B cells displaying antibodies specific to gpl20 comprising:

at least one fusion protein of polypeptide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6; and a pharmaceutically acceptable carrier.

12. A method of targeting B cells displaying antibodies specific to gpl20, wherein said method comprises of: contacting said B cells with at least one fusion protein of polypeptide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.

13. A method of treating HIV infection comprising:

administering a therapeutically effective amount of at least one fusion protein of polypeptide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.

14. A diagnostic method for diagnosing the presence of gpl20 specific B cell in a sample, wherein said diagnostic method comprises of:

contacting said sample with at least one fusion protein of polypeptide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 under conditions that
allow for formation of a complex between said polypeptidesequence and B cell; and

detecting the formation of said complex.

15. A diagnostic kit for detecting the presence of gpl20 specific B cell in a sample, wherein said diagnostic kit comprises of at least one fusion protein of polypeptide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.