WO2024141921A1 - Tumour necrosis factor alpha domain antibody - Google Patents

Abstract

The present disclosure relates to antibodies directed to the tumor necrosis factor alpha (TNF-α) and processes for their production. In one aspect, the present disclosure provides novel tumor necrosis factor alpha domain antibodies (TNF-α DAbs). In another aspect, the present disclosure provides novel polynucleotide sequences encoding polypeptides of TNF-α DAbs. In another aspect, the present disclosure provides compositions or kits comprising the antibodies and their therapeutic uses.

Description

TUMOUR NECROSIS FACTOR ALPHA DOMAIN ANTIBODY

FIELD OF INVENTION:

[0001] The present disclosure is in the broad field of biotechnology and relates to antibody molecules and functional fragments thereof, capable of binding to tumor necrosis factor alpha (TNF-a). In particular, the present disclosure relates to anti-TNF-a domain antibodies, compositions comprising the antibodies, processes for their production, and therapeutic uses of such antibodies.

BACKGROUND OF THE INVENTION:

[0002] Tumor necrosis factor alpha (TNF-a) is a potent pro-inflammatory cytokine which plays a central role in immune responses and inflammatory disorders. It has been described as a key mediator of inflammatory, immunological, and pathophysiological reactions. TNF-a is primarily secreted by monocytes and activated macrophages, but it is also produced by numerous other cell types, including, fibroblasts, neutrophils, eosinophils, and epithelial cells. TNF-a is existed in the two different forms: soluble and transmembrane, and both the forms play role in different inflammatory and immunological disorders. TNF-a exerts its effect by binding with its physiological receptors named TNF receptor-1 (TNF-R1) and TNF receptor-2 (TNF-R2). Binding of TNF-a to its receptors triggers different downstream responses, including the release of different inflammatory mediators and cytokines (like IL-1, IL-6, IL-8); activation of chemokines and neutrophils; expression of adhesion molecules, etc. Increased level of TNF-a plays important roles in initiation and progression of many inflammatory diseases. TNF-a is a master pro-inflammatory cytokine and by blocking the interaction of TNF-a with its receptors, inhibits TNF-a-mediated pro-inflammatory signal activation.

[0003] Many TNF-a-blocking biologies are available for the treatment of a variety of TNF-a- related diseases conditions. These biologies include Adalimumab, Infliximab, Golimumab, Etanercept, and Certolizumab pegol. Adalimumab, Infliximab, and Golimumab are full-length human immunoglobulins, while Etanercept is a TNF-R2 extracellular domain-Fc fusion protein, and Certolizumab pegol is a humanized Fab-PEG conjugate. According to one estimate, Adalimumab is going to be top 7 monoclonal antibody drug in 2024. Different TNF-a-related disease conditions for which these biologies are approved/being developed includes, Ankylosing spondylitis, Crohn disease, Hidradenitis suppurativa, Juvenile idiopathic arthritis, Plaque psoriasis, Polyarticular juvenile idiopathic arthritis, Psoriatic arthritis, Rheumatoid arthritis, Ulcerative colitis and Uveitis, Graft-vs-host disease, Pustular psoriasis, Pyoderma gangrenosum, Sarcoidosis, and Behcet disease, Dupuytren’s disease, Postoperative cognitive dementia (POCD).

[0004] Similarly, TNF-a binding biologies are also used in the development of diagnostic kits.

[0005] Even though there are many TNF-a-blocking biologies available/being developed for clinical and other uses, there is still a need for the development of new TNF-a-blocking biologies. The following art describes the generation and characterization of TNF-a-blocking biologies: US 7,754,853B2; US 2017/0107302A1; US 2005/0042219A1; US 8,703,131B2; US 2006/0024308A1; US 7,560, 108B2; US 7,227,003B2; US 2010/0172894A1; US 9,393,304B2; US 7,744,885B2; US 8,962,807B2; WO 2011/084714A2; US 8,597.648B2; US 2016/0272703 Al; US 2011/0182906A1; US 9,573.996B2; JP2008539772A5; US 2013/0101586A1; US 2017/0137510A1; US 2018/0371074A1; CN103333253B; WO2015173325A2; US 9,321,836B2;, EP 3 191 511B1; US 2017/0107282A1; US 2017 / 0327570A1; CN105753985A; CN106432491A; EP 3 219 726A1; WO 2017/ 102833 Al; CN107365732A; US 2019/0092850A1; US 2019/0345244A1; US 2019/0338022A1; US 2019/0092849; WO2008144753A3; WO 2017/180913 A9; US 10, 233, 238B2; EP 1 534 753B1; AU 2012237287B2; US 2006/0159677A1; US 2017 / 0247444A1; US 2006/0159677A1: US005654407A; US 2001/0027249A1; US 8,652.468B2; W02012009977A1; US 7,612,181B2. Liu et al., Journal of Biotechnology 186 (2014) 1-12; Chiu et al., PLoSONE, 2011, 6:el6373; Morais et al., Nucl Med Biol (2014); Alizadeh et al., Iranian Journal of Pharmaceutical Research (2019), 18 (2): 759-771; Pourtaghi-Anvarian et al., Preparative Biochemistry and Biotechnology, 2019, 38-47, Kim et al., ACS Sens. 2021, 6, 1807-1814; Piguet et al., Immunology 1992 77 510-514: Knight et al., Molecular Immunology, 1443-1453, 1993.

[0006] Conventional antibodies (monoclonal antibodies; mAbs) are made up of 2 heavy (H) and 2 light (L) chains. Camelidae family animals produce antibodies that are made up of only 2H chains and each chain consists of two constant regions, a hinge region and a variable region (i.e., the antigen-binding domain). Such antibodies are called as heavy chain antibodies. Domain antibodies are the fragment of heavy chain antibodies produced by recombinant technology. Because of their smaller size, nano-to-picomolar affinities, excellent chemical and thermal stability and solubility, deep tissue penetration, low immunogenicity, domain antibodies exhibit striking advantages over the conventional antibodies and have emerged as an alternative to conventional antibodies.

[0007] Although there are several TNF-blocking biologies that are well-known in the field, it should be understood that each of these biologies is unique. The creation of novel biologies requires a significant amount of human interaction and technical expertise.

[0008] The present disclosure provides novel TNF-a blocking domain antibodies. The TNF-a domain antibodies of the present disclosure can bind the TNF-a antigens with a wide range of affinities. The TNF-a domain antibody of the present disclosure can block or reduce interactions between TNF-a and TNF receptors. These TNF-a antibodies can also neutralize the TNF-a- related activity in a range of in vitro assays, such as cell cytotoxicity, mitogenesis, cytokine induction and induction of adhesion molecules.

SUMMARY OF THE INVENTION:

[0009] This summary is provided to introduce a selection of concepts in a simplified format that are further described in the detailed description of the invention.

[0010] In a first aspect of the present disclosure, there is provided a TNF-a domain antibody (TNF-a DAb) that binds to human TNF-a comprising complementarity determining region 3 (CDR3), wherein the CDR3 comprises an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7.

[0011] In an embodiment of the present disclosure, there is provided a TNF-a domain antibody that binds to human TNF-a, wherein the antibody comprises a variable region of a heavy chain antibody, wherein the variable region comprises: (a) an amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14, or (b) an amino acid sequence with at least 89% identity to the amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14. [0012] In another aspect of the present disclosure, there is provided a TNF-a domain antibody that binds to human TNF-a comprising a polypeptide, wherein the polypeptide comprises: (a) an amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14, or (b) an amino acid sequence with at least 89% identity to the amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, B or 14.

[0013] In an embodiment of the present disclosure, there is provided a polynucleotide encoding the variable region of the heavy chain antibody as described in the present disclosure.

[0014] In an embodiment of the present disclosure, there is provided a polynucleotide encoding the polypeptide as described in the present disclosure.

[0015] In an embodiment of the present disclosure, there is provided a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NO: 15, 16, 17, 18, 19, 20 or 21.

[0016] In an embodiment of the present disclosure, there is provided a vector comprising the polynucleotide as described in the present disclosure.

[0017] In an embodiment of the present disclosure, there is provided a host cell transformed or transduced with the vector as described in the present disclosure.

[0018] In an embodiment of the present disclosure, there is provided a host cell, wherein the host cell is a recombinant host cell.

[0019] In an embodiment of the present disclosure, there is provided a host cell, wherein the host cell is selected from prokaryotic cells, yeast cells, animal cells, plant cells, or insect cells.

[0020] In another aspect of the present disclosure, there is provided a method for the production of the TNF-a domain antibody as described in the present disclosure, wherein the method comprises: a) screening of a TNF-a domain antibody; b) designing of a polynucleotide from the screened TNF-a domain antibody; c) generating a recombinant vector comprising the polynucleotide; d) transforming or transducing a host cell with the recombinant vector; e) culturing the host cell under conditions allowing the expression of the TNF-a domain antibody; and f) recovering the produced TNF-a domain antibody from the culture. [0021] In an embodiment of the present disclosure, there is provided a pharmaceutical composition comprising the TNF-a domain antibody as described in the present disclosure or produced according to the method as described in the present disclosure, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0022] In an embodiment of the present disclosure, there is provided a kit comprising the TNF-a domain antibody as described in the present disclosure or produced according to the method antibody as described in the present disclosure, and reagents.

[0023] In an embodiment of the present disclosure, there is provided a method of treating TNF-a related diseases or conditions comprising administering to a person in need thereof a therapeutically effective amount of the TNF-a domain antibody as described in the present disclosure or produced according to the method as described in the present disclosure.

[0024] In an embodiment of the present disclosure, there is provided a method of treating TNF-a related diseases or conditions, wherein the diseases or conditions is selected from the list comprising of psoriasis, IBD-intestinal bowel disease (ulcerative colitis, Crohn’s disease), multiple sclerosis, viral infections, rheumatoid arthritis, autoimmune diseases, graft-versus-host disease (GVHD), inflammation and related complications, and comorbidities.

[0025] In an embodiment of the present disclosure, there is provided use of the TNF-a domain antibody as described in the present disclosure in the preparation of a medicament for treating TNF-a related diseases or conditions or a diagnostic kit for identifying TNF-a related diseases or conditions.

[0026] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. DESCRIPTION OF THE ACCOMPANYING DRAWINGS OR FIGURES:

[0027] The following drawings or figures form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings or figures in combination with the detailed description of the specific embodiments presented herein.

[0028] Figure 1: Panel A shows a cartoon of designed gene encoding for TNF-a domain antibody (TNF-a DAb) construct of the present disclosure. Panel B depicts the recombinant vector containing the designed gene for expression of TNF-a DAb constructs in E coli. Panel C shows images of western blot analysis of cell lysate of recombinant E. coli expressing TNF-a DAb constructs. The western blots were developed using monoclonal mouse anti-His antibody as a primary antibody and alkaline-phosphatase conjugated anti-mouse antibody as a secondary antibody. Legends: Lane M: Protein molecular weight markers and Lane L: cell lysate of recombinant E. coli expressing TNF-a DAb constructs. Presence of protein bands of expected size indicate production of TNF-a DAb constructs in recombinant E. coli.

[0029] Figure 2: Graphical representation of binding of TNF-a DAb (present in cell lysate of recombinant E. coli) to coated TNF-a as determined by ELISA. Wells of 96-well plates, coated with 20 ng/ml of hTNF-a, were incubated with cell lysate of recombinant E. coli expressing TNF-a DAb constructs and the binding was determined with Immunotag mouse anti- His antibody followed by HRP-tagged anti-mouse secondary antibody. The wells were then developed with substrate, 3.3‘,5,5'-tetramentylbenzidine and the absorbance was measured at 450 nm using microplate reader. A dose-dependent increase signal (of binding) indicates that TNF-a DAb has an antigen (TNF-a) binding activity.

DETAILED DESCRIPTION OF THE INVENTION:

[0030] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps of the process, features of the product, referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features. [0031] Definitions

[0032] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art.

[0033] The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0034] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0035] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”. Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps. The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[0036] The term “polypeptide” as used herein refers to two or more polymers of natural or unnatural amino acids.

[0037] The term “heterologous proteins” refers to those proteins that are foreign to the host cells used, such as human proteins produced in E. coli.

[0038] The terms “polynucleotide sequence”, “nucleic acid” and “gene” mean a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid).

[0039] The term “optimized polynucleotide sequence” refers to a synthetically synthesized nucleic acid (gene) optimized for high level expression of recombinant protein in host cells. [0040] The term “recombinant expression plasmid” or “recombinant expression vector” refers to plasmid in which polynucleotide sequence encoding for target heterologous proteins can be placed and ferried into the suitable host cell where it can be copied or expressed.

[0041] The term “recombinant host cell” refers to host cells that contain recombinant expression plasmid or vector and that can express the target proteins into them. The host cell is preferably selected from the group consisting of prokaryotic cells, bacterial cells such as, for example, E. coli, yeast cells such as, for example, S. cerevisiae, P. pastoris, animal cells, plant cells, mammal cells, or insect cells.

[0042] The term “functionally active” refers to the ability of the polypeptide to exert one or more activities known to be associated with antibody, such as the ability to bind to human TNF-a and inhibit their binding to their respective receptor.

[0043] The term “immunizes”, “immunization”, or “immunizing” refers to exposing the immune system of an animal to an antigen or to an epitope thereof as illustrated in more detail below. The antigen may be introduced into the animal using a desired route of administration, such as injection, inhalation or ingestion. Upon a second exposure to the same antigen, the adaptive immune response, in particular T cell and B cell responses, is enhanced.

[0044] The term “antigen” refers to a substance that is capable of interacting with an antibody and in the context of the present disclosure is tumor necrosis factor alpha (TNF-a), preferably human TNF-a or any TNF-a. The TNF-a may be soluble TNF-a or membrane associated TNF- a.

[0045] The term “immunoglobulin” is used herein to refer to a protein comprising of one or more polypeptides encoded by immunoglobulin genes. The recognized immunoglobulin heavy chain constant region gene isotypes include IgG, (subtypes IgGl, IgG2, IgG3, and IgG4), IgM, IgA (sub types IgAl and IgA2), IgD, and IgE. Multiple immunoglobulin variable region genes are utilized in the production of natural antibodies. One natural form of immunoglobulin is a tetramer comprising two identical pairs in which each pair has one light (L) chain and one heavy (H) chain. In each pair the heavy and light chain variable regions together provide the binding surface capable of interacting with the antigen. [0046] The term “conventional antibody” or “monoclonal antibody” is used herein to refer one form of immunoglobulin which is tetrameric in nature and comprises of two identical pairs in which each pair has one light (L) chain and one heavy (H) chain. In each pair the heavy and light chain variable regions together provide the binding surface capable of interacting with the antigen.

[0047] The term “antibody fragment” refers to a portion of a full-length antibody, generally the target binding or variable region. Examples of antibody fragments include Fab, Fab’, F(ab’)2 and Fv fragments. An “Fv” fragment is the minimum antibody fragment which contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, noncovalent association (VH-VE dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VE dimer. Often, the six CDRs confer target binding specificity to the antibody. However, in some instances even a single variable domain (or half of an FV comprising only three CDRs specific for a target) can have the ability to recognize and bind a target. “Single chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of an antibody in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for target binding.

[0048] The term “scFv” refers to a single chain fragment variable antibody in which the variable domains of the heavy chain and the light chain from a conventional antibody have been joined to form one chain.

[0049] ‘ ‘VH” refer to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain variable fragment, scFv, or Fab. References to “VL” refer to the variable region of an immunoglobulin light chain, including the light chain variable fragment, scFv, dsFv or Fab. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at the amino terminus a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at the amino terminus (VL) and a constant domain at the carboxy terminus.

[0050] The term “heavy chain antibody” refers to antibody produced by Camelidae family animals in which the antibody is made up of only 2H chains and each chain consists of two constant regions, a hinge region and a variable region (i.e., the antigen-binding domain).

[0051] The term “domain antibody (DAb)” or “single domain antibody” is used herein to refer fragment or portion of heavy chain antibodies produced by recombinant technology. Because of their smaller size, nano-to-picomolar affinities, excellent chemical and thermal stability and solubility, deep tissue penetration, low immunogenicity, domain antibodies exhibit striking advantages over the conventional antibodies and have emerged as an alternative to conventional antibodies. In a specific embodiment, the antibody is a TNF-a domain antibody. In another embodiment, the antibody is a TNF-a neutralizing domain antibody.

[0052] The term “TNF-a” is tumor necrosis factor alpha and is a cytokine involved in systematic inflammation. TNF-a is known in the art to be associated with inflammatory disorders such as rheumatoid arthritis, Crohn's disease, ulcerative colitis, and multiple sclerosis. Both TNF-a and its receptors (CD 120a andCD120b) have been studied in detail. TNF-a in its bioactive form is a trimer. Several strategies to antagonize the action of TNF-a using anti-TNF-a antibodies or TNF- a blocking biologies have been developed and are currently commercially available, such as Remicade* and Humira*. Numerous examples of TNF-a binding molecules are disclosed in WO 04/041862, WO 04/041865, and W006/122786, the contents of all of which are incorporated by reference herein in their entirety.

[0053] The term “transformation” as used herein refers to a change in a cell’s genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA. For example, a cell is transformed where it is genetically modified from its native state. Following transformation, the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, or may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. A cell is considered to have been stably transformed when the DNA is replicated with the division of the cell. The term “transfection” as used herein refers to the uptake of foreign or exogenous DNA by a cell, and a cell has been “transfected” when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art. Such techniques can be used to introduce one or more exogenous DNA molecules into suitable host cells. The term “transduction” as used herein refers to the process of inserting a foreign nucleotide sequence into a cell using a viral vector.

[0054] ‘ ‘Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. It means a sequence which presents a high sequence identity (more than 80%, 85%, 80%, 90%, 95% or 98% sequence identity) with the parent sequence and is preferably characterized by similar properties of the parent sequence, namely affinity, said identity calculated using known methods.

[0055] The term “analogs” or “fragments” or “portion” used herein refers to an amino acid sequence comprising at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 89% 90%, 95%, 96%, 97%, 98%, 99% or 100% of a naturally occurring said protein or mutant thereof.

[0056] The term “polypeptide or protein” used in this invention collectively refers to the domain antibody of TNF-a polypeptide of the present disclosure. Accordingly, the term “protein or peptide” encompasses amino acid sequences comprising at least one of the 20 common amino acids found in naturally occurring proteins (Table 1).

Table 1: Naturally occurring amino acids

[0057] The term “antibody” is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments, regions or derivatives thereof, provided by any known technique, such as, but not limited to enzymatic cleavage, peptide synthesis or recombinant techniques. Such anti-TNF-a antibodies of the present disclosure are capable of binding portions of TNF-a that inhibit the binding of TNF-a to TNF -receptors.

[0058] The term “neutralizing antibody” as used herein refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen. It refers to an antibody that blocks or reduces at least one activity of a polypeptide comprising an epitope to which the antibody specifically binds. The neutralizing antibody decreases activity in vitro and / or in vivo. In a specific embodiment, the antibody is a TNF-a neutralizing antibody.

[0059] ‘ Inflammation” is a biological response of the immune system that can be triggered by a variety of factors, including pathogens, damaged cells and toxic compounds. Inflammation is the immune system’s response to harmful stimuli, such as pathogens, damaged cells, toxic compounds, or irradiation, and acts by removing injurious stimuli and initiating the healing process. Inflammation is therefore a defense mechanism that is vital to health. Usually, during acute inflammatory responses, cellular and molecular events and interactions efficiently minimize impending injury or infection.

[0060] “Inflammatory disorder” means a disorder or pathological condition where the pathology results, in whole or in part, from, e.g., a change in number, change in rate of migration, or change in activation, of cells of the immune system. Cells of the immune system include, e.g., T cells, B cells, monocytes or macrophages, antigen presenting cells (APCs), dendritic cells, microglia, NK cells, NKT cells, neutrophils, eosinophils, mast cells, or any other cell specifically associated with the immunology, for example, cytokine -producing endothelial or epithelial cells.

[0061] The term “TNF-a related diseases or conditions” refers to the diseases or conditions in which TNF-a inhibition has therapeutic effects. This can be achieved by administering certain drugs and anti-TNF-a antibodies. Examples of such diseases or conditions include, but are not limited to:

(A) acute and chronic immune and autoimmune pathologies, Such as systemic lupus erythematosus, rheumatoid arthritis, thyroiditis, graft versus host disease, scleroderma, diabetes mellitus, Grave’s disease, and the like;

(B) infections, including, but not limited to, sepsis syndrome, cachexia, circulatory collapse and shock resulting from acute or chronic bacterial infection, acute and chronic parasitic and/or bacterial, viral or fungal infectious diseases, such as AIDS (including sequelae such as cachexia, autoimmune disorders, AIDS, dementia complex and infections); (C) inflammatory diseases, such as chronic inflammatory pathologies and vascular inflammatory pathologies, including chronic inflammatory pathologies such as sarcoidosis, chronic inflammatory bowel disease, ulcerative colitis, and Crohn’s pathology and vascular inflammatory pathologies, such as, but not limited to, disseminated intravascular coagulation, atherosclerosis, and Kawasaki’s pathology.

(D) neurodegenerative diseases, including, but are not limited to, demyelinating diseases, such as multiple sclerosis and acute transverse myelitis; extrapyramidal and cerebellar disorders such as lesions of the corticospinal system; disorders of the basal ganglia or cerebellar disorders; hyperkinetic movement disorders such as Huntington’s Chorea and senile chorea; drug-induced movement disorders, such as those induced by drugs which block CNS dopamine receptors; hypokinetic movement disorders, such as Parkinson's disease; Progressive Supranucleopalsy, Cerebellar and Spinocerebellar Disorders, such as astructurallesions of the cerebellum; spinocerebellar degenerations (spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations, multiple systems degenerations (Mencel, Dejerine-Thomas, Shi-Drager, and Machado-Joseph); and systemic disorders (Refsum’s disease, abetalipoprotemia, ataxia, telangiectasia, and mitochondrial multi. System disorder); demyelinating core disorders, such as multiple sclerosis, acute transverse myelitis; disorders of the motor unit, such as neurogenic muscular atrophies (anterior horn cell degeneration, Such as amyotrophic lateral Sclerosis, infantile spinal muscular atrophy and juvenile spinal muscular atrophy); Alzheimer’s disease, Down’s Syndrome in middle age, Diffuse Lewy body disease, Senile Dementia of Lewy body type, Wernicke-Korsakoff syndrome, chronic alcoholism, Creutzfeldt- Jakob disease, subacute sclerosing panencephalitis, Hallerrorden-Spatz disease, and dementia pugilistica, or any subset thereof;

(E) malignant pathologies involving TNF-a-secreting tumors or other malignancies involving TNF-a, such as, but not limited to leukemias (acute, chronic myelocytic, chronic lymphocytic and/or myelodyspastic syndrome); lymphomas (Hodgkin’s and nonHodgkin’s lymphomas, such as malignant lymphomas (Burkitt’s lymphoma or Mycosis fungoides); and

(F) alcohol-induced hepatitis. [0062] In a specific embodiment, TNF-a related diseases or conditions refer to psoriasis, IBD- intestinal bowel disease (Ulcer colitis, Crohn’s disease), multiple sclerosis, viral infections, rheumatoid arthritis, autoimmune diseases, graft-versus-host disease (GVHD), inflammation and related complications and comorbidities.

[0063] The term “diagnosis” used herein refers to a process of identifying a disease condition by detecting the serum level of antigen TNF-a with the help of the TNF-a domain antibody of the present disclosure. The term “detecting” used herein encompasses qualitative and / or quantitative detection (level measurement) with or without reference to a control. In one aspect, the present disclosure provides a method for detecting antigen TNF-a in a biological sample using the antibody or fragment thereof of the present disclosure.

[0064] A “biological sample” encompasses a variety of sample types obtained from a subject or person and can be used in a diagnostic or monitoring assay. Biological samples include, but are not limited to, blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom, and their progeny. Thus, biological samples include clinical samples, cells in culture, cell supernatants, cell lysates, serum, plasma, urine, cerebrospinal fluid, body fluids, and tissue samples.

[0065] The term “pharmaceutical composition” describes that the antibodies of the present disclosure can be combined with a pharmaceutically acceptable carrier, excipient, or diluent to form therapeutic compositions or a medicament. The pharmaceutical composition of the present disclosure can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration.

[0066] The term “pharmaceutically acceptable” used herein is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the antibody without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. A pharmaceutically acceptable carrier or excipient or diluent refers to a non-toxic solid, semi-solid or liquid bulking agent, encapsulating material or any type of formulation aid. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers, such as sterile phosphate buffered saline solutions, pneumatic fungi, and the like (refer to Remington’s Pharmaceutical Sciences 16^th Edition, A. Osal., Ed. 1980). Acceptable carriers, excipients, or stabilizers or diluents are non-toxic to the recipient at the dosages and concentrations employed and include buffers such as phosphate, citrate, acetate and other organic acids; Antioxidants including ascorbic acid and methionine; Preservatives such as octadecyl dimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butylbenzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclo Hexanol, 3-pentanol, and m-cresol); Low molecular weight (less than about 10 residues) polypeptides; Proteins, such as serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; Monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; Chelating agents such as EDTA; Sugars such as sucrose, mannitol, trehalose or sorbitol; Sweeteners and other flavorings; Fillers such as microcrystalline cellulose, lactose, corn and other starches; Binder; additive; coloring agent; Salt-forming counterions such as sodium; Metal complexes (e. G., Zn-protein complexes); And / or non-ionic surfactants such as TWEEN TM, PLURONICS TM or polyethylene glycol (PEG). The pharmaceutical composition comprising an antibody of the present disclosure may be present in a water-soluble form, e.g., as a pharmaceutically acceptable salt, which means that it includes both acid addition salts and base addition salts.

[0067] “Therapeutically effective amount”, “therapeutically effective dose” and “effective amount” used herein mean the amount needed to achieve the desired result or results (modulating TNF-alpha binding; treating or preventing inflammation). One of ordinary skill in the art will recognize that the potency and, therefore, an “effective amount” can vary for the various compounds that modulate TNF-alpha binding used in the invention. One skilled in the art can readily assess the potency of the compound.

[0068] The term “kit” or “diagnostic kit” used herein refers to a product containing detecting reagents like TNF-a domain antibody and other items necessary to conduct a test to detect the presence of or to measure an antigen TNF-a in a given patient sample. As with the article of manufacture, the kit comprises a container and a label or package insert on or associated with the container. The container holds a composition comprising at least one protein (e.g., TNF-a domain antibody) of the present disclosure. Additional containers may be included that contain reagents, e.g., diluents and buffers or control antibodies. The label or package insert may provide a description of the composition as well as instructions for the intended in vitro or diagnostic use.

[0069] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0070] The present disclosure relates to polypeptides of novel TNF-a domain antibodies and polynucleotides encoding the polypeptides of the TNF-a domain antibodies. The TNF-a domain antibodies of the present disclosure comprise a VHH domain of the TNF-a polypeptide identified de novo and a (His)e-tag. In addition, the TNF-a domain antibody of the present disclosure can neutralize the TNF-a antigen and be used in TNF-a-related inflammatory disorders or conditions.

[0071] The TNF-a domain antibody of the present disclosure can bind the TNF-a antigens with a wide range of affinities. Exemplary TNF-a antibodies or domain antibodies bind with the TNF-a antigen. The affinity of TNF-a antibodies for TNF-a can be determined experimentally using any suitable methods known in the art, e.g., Biacore or KinEXA instrumentation, ELISA and competitive binding assays.

[0072] The TNF-a domain antibody of the present disclosure can block or reduce interactions between TNF-a and TNF receptors. These TNF-a antibodies neutralize the TNF-a-related activity in a range of in vitro assays, such as cell cytotoxicity, mitogenesis, cytokine induction and induction of adhesion molecules.

[0073] There is provided a TNF-a domain antibody comprising an VHH domain of TNF-a polypeptide and (His)g-tag, wherein the said domain antibody is suitable for end application without being processed for removal of (His)e-tag.

[0074] There is provided a method for expressing at least one TNF-a domain antibody comprising culturing the cell lines under conditions, wherein the TNF-a domain antibody is expressed in detectable or recoverable amounts. The present disclosure also provides methods for generating at least one TNF-a domain antibody comprising translating the TNF-a domain antibody encoding nucleic acids under conditions in vitro or in situ, such that the TNF-a domain antibody is expressed in detectable or recoverable amounts.

[0075] The TNF-a domain antibody of the present disclosure is useful diagnostic purposes (example: in preparation of ELISA kit).

[0076] The present disclosure also provides polynucleotide sequences encoding the TNF-a antibodies, vectors containing these polynucleotide sequences, host cells, compositions and methods of making and using the TNF-a antibodies.

[0077] In the detailed description of the present invention, numerous specific details are described to provide a thorough understanding of the various embodiments of the present invention. However, a person skilled in the relevant art will recognize that an embodiment of the present invention can be practiced without one or more of the specific details, or with other apparatus, systems, assemblies, methods, components, materials, parts, and/or the like. In other instances, well-known structures, materials, or operations are not specifically shown or described in detail to avoid obscuring aspects of embodiments of the present invention.

[0078] In one aspect of the present disclosure, there is provided a TNF-a domain antibody (TNF- a DAb) that binds to human TNF-a and comprising complementarity determining region 3 (CDR3), wherein the CDR3 comprises an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7.

[0079] In an embodiment of the present disclosure, there is provided a TNF-a domain antibody that binds to human TNF-a, wherein the antibody comprises a variable region of a heavy chain antibody, wherein the variable region comprises: (a) an amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14, or (b) an amino acid sequence with at least 89% identity to the amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14.

[0080] In an embodiment of the present disclosure, there is provided a TNF-a domain antibody that binds to human TNF-a, wherein the antibody comprises a variable region of a heavy chain antibody, wherein the variable region comprises an amino acid sequence with at least 89%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14.

[0081] In an embodiment of the present disclosure, there is provided a TNF-a domain antibody that binds to human TNF-a, wherein the antibody comprises a variable region of a heavy chain antibody, wherein the variable region comprises an amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14.

[0082] In another aspect of the present disclosure, there is provided a TNF-a domain antibody that binds to human TNF-a comprising a polypeptide, wherein the polypeptide comprises: (a) an amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14, or (b) an amino acid sequence with at least 89% identity to the amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, B or 14.

[0083] In another aspect of the present disclosure, there is provided a TNF-a domain antibody that binds to human TNF-a comprising a polypeptide, wherein the polypeptide comprises an amino acid sequence with at least 89%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14.

[0084] In another aspect of the present disclosure, there is provided a TNF-a domain antibody that binds to human TNF-a comprising a polypeptide, wherein the polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14.

[0085] The TNF-a domain antibody polypeptides according to the present disclosure are proteins created which originally encoded the TNF-a domain antibody polypeptide. Translation of TNF-a domain antibody polynucleotide sequence will result in a single polypeptide sequence which may have functional ability to bind with TNF-a protein. Polynucleotide sequences encoding TNF-a domain antibody polypeptide may be created artificially by standard molecular biology methods. The resulting polynucleotide sequence may be inserted into an appropriate expression vector that supports the heterologous TNF-a domain antibody polypeptide construct expression in a standard host organism.

[0086] In an embodiment of the present disclosure, there is provided a polynucleotide encoding the variable region of the heavy chain antibody as described in the present disclosure. [0087] In an embodiment of the present disclosure, there is provided a polynucleotide encoding the polypeptide as described in the present disclosure.

[0088] In an embodiment of the present disclosure, there is provided a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NO: 15, 16, 17, 18, 19, 20 or 21.

[0089] The present disclosure provides methods of designing the vector of all above mentioned TNF-a domain antibody polypeptide comprising of different genetic information required to develop TNF-a domain antibody polypeptide under suitable conditions for expression of recombinant polypeptides. In certain embodiments, the method further comprises the step of obtaining the recombinant polypeptides from the cell culture. The recombinant polypeptides can be obtained from the cell culture by any methods of protein isolation or purification known in the art, including without limitation, collecting recombinant cells, freezing/thawing, centrifugation, cell lysis.

[0090] In an embodiment of the present disclosure, there is provided a vector comprising the polynucleotide as described in the present disclosure.

[0091] In an embodiment of the present disclosure, there is provided a vector, wherein the vectors can be selected among conventional vector types including insects, e.g., baculovirus expression, or yeast, fungal, bacterial or viral expression systems. In one embodiment, the expression vector is pET23a(+). Numerous types of appropriate expression vectors are known in the art for protein expression by standard molecular biology techniques.

[0092] In an embodiment of the present disclosure, there is provided a host cell transformed or transduced with the vector as described in the present disclosure.

[0093] In an embodiment of the present disclosure, there is provided a host cell, wherein the host cell is a recombinant host cell.

[0094] In an embodiment of the present disclosure, there is provided a host cell, wherein the host cell is selected from prokaryotic cells, yeast cells, animal cells, plant cells, or insect cells. [0095] In an embodiment of the present disclosure, there is provided a host cell, wherein the host cell is a prokaryotic cell, bacterium such as, for example, E. coli (e.g., HB101, MC1061, BL21(DE3) etc.) B. subtilis, Pseudomonas ssp., Streptomyces ssp. In one embodiment, the host cell is a mammalian cell, such as, for example, human 293 cells, Chinese hamster ovary cells (CHO), monkey COS-1 cells, murine 3T3 cells, and other cell lines.

[0096] In another aspect of the present disclosure, there is provided a method for the production of the TNF-a domain antibody as described in the present disclosure, wherein the method comprises: a) screening of a TNF-a domain antibody; b) designing of a polynucleotide from the screened TNF-a domain antibody; c) generating a recombinant vector comprising the polynucleotide; d) transforming or transducing a host cell with the recombinant vector; e) culturing the host cell under conditions allowing the expression of the TNF-a domain antibody; and f) recovering the produced TNF-a domain antibody from the culture.

[0097] In an embodiment of the present disclosure, the recombinant host cells are grown under the conditions favorable to express the heterologous TNF-a domain antibody polypeptide. One or more favorable experiment conditions may include effective media, temperature, pH, oxygen condition, shaking time and speed, and like. In another embodiment, various chemical or physical agents, such as IPTG, lactose, low or high temperature change, and like, are used to express the heterologous recombinant polypeptides in recombinant host cells. The recombinant host cells were lysed by using either physical or chemical methods such as sonication, French press, lysozyme- and detergent-treatment, and like, to release the recombinant polypeptides.

[0098] In an embodiment of the present disclosure, there is provided a pharmaceutical composition comprising the TNF-a domain antibody as described in the present disclosure or produced according to the method as described in the present disclosure, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0099] In an embodiment of the present disclosure, there is provided a kit comprising the TNF- a domain antibody as described in the present disclosure or produced according to the method antibody as described in the present disclosure, and reagents. [0100] In an embodiment of the present disclosure, there is provided a method of treating TNF-a related diseases or conditions comprising administering to a person in need thereof a therapeutically effective amount of the TNF-a domain antibody as described in the present disclosure or produced according to the method as described in the present disclosure.

[0101] In an embodiment of the present disclosure, there is provided a method of treating TNF-a related diseases or conditions, wherein the diseases or conditions is selected from the list comprising of psoriasis, IBD-intestinal bowel disease (ulcerative colitis, Crohn’s disease), multiple sclerosis, viral infections, rheumatoid arthritis, autoimmune diseases, graft-versus-host disease (GVHD), inflammation and related complications, and comorbidities.

[0102] In an embodiment of the present disclosure, there is provided use of the TNF-a domain antibody as described in the present disclosure in the preparation of a medicament for treating TNF-a related diseases or conditions or a diagnostic kit for identifying TNF-a related diseases or conditions.

EXAMPLES:

[0103] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. Person skilled in the art will be aware of the fact that the present examples will further subject to variations and modifications specifically described herein based on the technical requirement of the experiment and shall not be limiting what specifically mentioned.

Example 1: Identification of TNF-a domain antibody (TNF-a DAb) candidates.

[0104] The service of Diaclone SAS, Besancon Cedex, France, which utilizes Diaclone Phage Display Platform, was used to identify TNF-a DAb constructs. Briefly, the RNA was isolated from the two Lamas immunized with human TNF-a and its quality was confirmed on the Experion platform. The RNA was then cloned and the size of final VHH phagemid library was found to be a size of >10⁶ cfu. The colony PCR was performed to evaluate the percentage insertion of PCR products in the phagemid and the percentage of insertion for the final library was found to be >95%. In order to confirm the good insertion and a good reading frame in the phagemid, a final sequencing control was made on 48 randomly chosen clones coming from the final library and all analyzed clones showed a good reading frame. The final library (size >10⁶ cfu and >80% of insertion) was then cultured and infected by phage and the bank of phage obtained was stored at -80°C with glycerol until the panning steps.

[0105] Phages expressing at their surface anti-TNF-a VHHs were selected and amplified by biopanning. Two successive rounds of biopanning were performed by ELISA using hTNF-a recombinant protein (from Diaclone). Briefly, plates were coated with 1 pg, 0.1 pg or 0 pg of TNF-a protein per well. One pl / well of phage expressing VHH were then incubated for 30 min in wells containing PBS-BSA 1%. The remaining phages in the supernatants were incubated for Ih with the TNF-a protein coated. After elution, infection and overnight incubation, a more than 100-fold amplification was observed between the conditions at Ipg and the conditions without proteins. Two rounds of panning allowed the amplification of TNF-a-specific phages, and the libraries were subjected to screening steps.

[0106] The screen was performed by ELISA on periplasmic extract of VHH. Briefly, ELISA 96- well plates were coated with anti-TNF-a antibody (1 pg/well) for 2h at RT. Plates were washed twice and saturated for 2h at RT with PBS containing 1% BSA and 10% sucrose. Then, 100 pL of TNF-a in PBS containing 1% BSA (10 ng/well) were added to two ELISA plates (conditions with protein). In parallel, 100 pL of PBS containing 1% BSA were added to the other two ELISA plates (conditions without protein). Plates were then incubated Ih at RT and washed four times. 20 pL of periplasmic supernatant containing VHHs and 80 pL of PBS containing 1% BSA were then added to each well. Plates were incubated Ih at RT and washed 4 times. Then, lOOpL of anti-Myc-HRP antibody was added to each well and incubated Ih at RT. Plates were washed 4 times, then revealed by adding lOOpL of TMB per well. After 10 min the reaction was stopped by adding lOOpL of H2SO4 and absorbance was read at 450nm with an ELISA plate reader BioTek ELx 808. After the screening, clones identified as positives on the recombinant (hTNF- a) protein from Peprotech (Catalog # 300-01 A) were subjected to sequencing and final 7 candidates were identified and their amino acid sequences were determined.

Example 2: Cloning and expression of TNF-q DAb:

[0107] Amino acid sequences of 07 candidates were used to design TNF-a DAb constructs. The amino acid sequence TNF-a DAb constructs (SEQ ID No. 8 to 14) were further used to design respective gene (polynucleotides constructs of SEQ ID No. 15 to 21) encoding for respective TNF-a DAb polypeptides. The designed genes were codon-optimized for high level expression in E. coli and custom synthesized from Gene Script, NJ. In the designed gene, the 5'end of the open reading frame (ORF) is flanked by a Nde \ restriction site. The 3'end of the ORF is flanked by nucleotides encoding for a (His)e-tag, a stop codon and a EcoRl restriction site (Figure 1A). The designed genes were cloned in pET23a(+) expression plasmid (vector), between Nde \ and EcoRl restriction sites, to generate recombinant expression plasmids encoding for particular TNF-a DAb construct of this invention (Figure IB).

[0108] The recombinant expression plasmid was transformed into E. coli BL21 (DE3), by following standard molecular biology technique known in the art, to generate recombinant E. coli cells for the production of TNF-a DAb polypeptides. The recombinant E. coli cells were then stored as a glycerol stock at -80°C for further use. For expression of TNF-a DAb polypeptides, glycerol stock of recombinant E. coli BL21 (DE3) (Cat # EC0114, THERMO SCIENTIFIC, INVITROGEN) cells was streaked on Luria Bertani (LB)-agar plate containing 50 pg/ml carbenicillin and the plates were incubated overnight at 37 °C. A single colony from the plate was used to initiate a seed culture in LB-broth supplemented with 50 pg/ml carbenicillin and the seed culture was grown at 37°C overnight at 200 rpm. One percent of this seed culture was then inoculated into fresh LB-broth supplemented with 50 pg/ml carbenicillin and the main culture was grown at 30°C till ODgoo reached 0.4-0.6. The culture was then induced with 0.5 mM isopropyl P-D-l -thiogalactopyranoside (IPTG) and was allowed to grow further at 20°C for 32 h at 200 rpm. The wet cell mass (WCM) of E. coli was then harvested from the culture by centrifugation (6500xg, 10 min, 4°C) and re-suspended in ice-cold lysis buffer (50 mM Tris- HC1, pH 8.0 containing 150 mM NaCl, 1 mM P-ME and 1 mM PMSF). The cell suspensions were gently stirred at RT for 1 h and the cells were subjected to sonication (25% amplitude, 10 pulses of 1 min each with 1 min break after each pulse on ice) using Ultrasonics, Inc. model W830 ultrasonic processor with a macro-probe tip. To the sample was then added DNase (1 pg/ml) and 300 mM MgC^ and the sample was further incubated on ice for 2 h. The samples were then centrifuged (10,000xg, 20 min, 4°C) to separate supernatant cell lysate fractions from insoluble cell debris fractions. These fractions were then analyzed for the presence of TNF-a DAb polypeptides by western blot analysis.

[0109] Protein bands of expected molecular weights were observed in the western blot analysis of cell lysate of recombinant E. coli (Figure 1C) confirming the production of TNF-a DAb in recombinant E. coli.

Example 3: Binding of TNF-a DAb to hTNF-a as determined by a solid-phase ELISA.

[0110] E. coli cell lysate containing particular TNF-a DAb were used in the solid-phase ELISA. Wells of 96-well plates were coated overnight at 25°C with 20 ng/ml of hTNF-a (Elabsciences Biotechnology Inc, Cat # PKSH033490) in phosphate buffer, pH 7.4. After washing the wells with PBST (phosphate buffered saline containing 0.05% (w/v) Tween-20), the wells were blocked with PBS containing 3% (w/v) bovine serum albumin (BSA) for 2 h at 25°C. After washing, wells were incubated with different dilutions (in PBS buffer) of E. coli cell lysates for 2h at 25°C. Appropriate negative controls were also used in the experiment. Subsequently, wells were then washed with PBST (thrice) and further incubated with Immunotag mouse anti-His antibody (Cat. No. M 1001020, G-Bioscineces, US A; 1:5000 diluted in PBST) for 2h. Following another washing step (with PBST), the wells were probed for 1.5 h with 100 pl/well of HRP- tagged anti-mouse secondary antibody (Cat. No. 1089153, Roche Applied Science, Indianapolis, Ind.; diluted 1 :40000 in the PBST buffer). Wells were further washed with PBST and then developed by incubating with substrate, 3,3',5,5'-tetramentylbenzidine (Cat. No. Abl71523, Abeam, UK) for 30 min. Color development was stopped by adding 100 pl/well of stop solution (0.2 M H2SO4) and the absorbance was measured at 450 nm using a microplate reader (Infinite 200 PRO).

[0111] Histogram showing binding of representative TNF-a DAb polypeptides to soluble hTNF- a coated in 96-well plates are shown in Figure 2. A dose-dependent increase in signal (of binding) indicates that TNF-a DAb has an antigen (TNF-a) binding activity. [0112] It will thus be seen that the objects set forth above, among those made apparent from the preceding description, are efficiently attained, and since certain changes may be made in the constructions set forth without departing from the spirit and scope of the invention, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense. The invention has been described with reference to preferred and alternate embodiments. Modifications and alterations will become apparent to those skilled in the art upon reading and understanding the detailed discussion of the invention provided herein. This invention is intended to include all such modifications and alterations insofar as they come within the scope of the present invention. These and other modifications of the preferred embodiments as well as other embodiments of the invention will be obvious from the disclosure herein, whereby the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.

[0113] Finally, to the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated.

[0114] Acknowledgement: The inventors thank the CRG, SERB, Department of Science and Technology (New Delhi, Government of India; grant ## No. CRG/2020/000865) and NIPER SAS Nagar for providing support through research grants.

Claims

We Claim:

1. A TNF-a domain antibody (TNF-a DAb) that binds to human TNF-a and comprising complementarity determining region 3 (CDR3), wherein the CDR3 comprises an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7.

2. The TNF-a domain antibody as claimed in claim 1, wherein the antibody comprises a variable region of a heavy chain antibody, wherein the variable region comprises:

(a) an amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14, or

(b) an amino acid sequence with at least 89% identity to the amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14.

3. A TNF-a domain antibody that binds to human TNF-a comprising a polypeptide, wherein the polypeptide comprises:

(a) an amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14, or

(b) an amino acid sequence with at least 89% identity to the amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14.

4. A polynucleotide encoding the variable region of the heavy chain antibody as claimed in claim 2.

5. A polynucleotide encoding the polypeptide as claimed in claim 3.

6. The polynucleotide as claimed in any of claims 4-5, wherein the polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NO: 15, 16, 17, 18, 19, 20 or 21.

7. A vector comprising the polynucleotide as claimed in any of claims 4-6.

8. A host cell transformed or transduced with the vector as claimed in claim 7.

9. The host cell as claimed in claim 8, wherein the host cell is a recombinant host cell.

10. The host cell as claimed in any of claims 8-9, wherein the host cell is selected from prokaryotic cells, yeast cells, animal cells, plant cells, or insect cells.

11. A method for the production of the TNF-a domain antibody as claimed in any of claims 1-3, wherein the method comprises: a) screening of a TNF-a domain antibody; b) designing of a polynucleotide from the screened TNF-a domain antibody; c) generating a recombinant vector comprising the polynucleotide; d) transforming or transducing a host cell with the recombinant vector; e) culturing the host cell under conditions allowing the expression of the TNF-a domain antibody; and f) recovering the produced TNF-a domain antibody from the culture.

12. A pharmaceutical composition comprising the TNF-a domain antibody as claimed in any of claims 1 -3 or produced according to the method as claimed in claim 11 , and a pharmaceutically acceptable carrier, excipient, or diluent.

13. A kit comprising the TNF-a domain antibody as claimed in any of claims 1-3 or produced according to the method as claimed in claim 11, and reagents.

14. A method of treating TNF-a related diseases or conditions comprising administering to a person in need thereof a therapeutically effective amount of the TNF-a domain antibody as claimed in any of claims 1-3 or produced according to the method as claimed in claim 11.

15. The method as claimed in claim 14, wherein the diseases or conditions is selected from the list comprising of psoriasis, IBD-intestinal bowel disease (ulcerative colitis, Crohn’s disease), multiple sclerosis, viral infections, rheumatoid arthritis, autoimmune diseases, graft-versus-host disease (GVHD), inflammation and related complications, and comorbidities.

16. Use of the TNF-a domain antibody as claimed in any of claims 1-3 in the preparation of a medicament for treating TNF-a related diseases or conditions or a diagnostic kit for identifying TNF-a related diseases or conditions.