A bispecific domain antibody and method for the preparation thereof

Abstract

The present invention relates to the field of biotechnology, specifically recombinant antibodies. The present invention provides an engineered bispecific domain antibody "BiSpekDAb" construct comprising anti-IL-23 domain antibody and anti-TNF-α domain antibody linked to a non-antibody polypeptide via linker peptides. Further, the present invention provides novel polynucleotide sequence encoding BiSpekDAb. Furthermore, the present invention provides a method to produce BiSpekDAb construct. Also, the BiSpekDAb polypeptide construct disclosed in the present invention can block both TNF-α and IL-23 simultaneously, and can be used in various inflammatory diseases or conditions.

Description

A BISPECIFIC DOMAIN ANTIBODY AND METHOD FOR THE PREPARATION THEREOFFIELD OF THE INVENTION

[0001] The present invention relates to the field of biotechnology. Particularly, the present invention relates to a bispecific domain antibody, which is a bispecific anti-IL-23 / TNF-a domain antibody capable of inhibiting interleukin 23 (IL-23) and tumor necrosis factor-alpha (TNF-a). More particularly, the present invention relates to a method for the preparation of bispecific domain antibody. Further, the antibody of the present invention is useful in the treatment of various inflammatory and autoimmune diseases like Inflammatory Bowel Disease (IBD) including both Ulcer Colitis (UC) and Crohn’s Disease (CD), Lung inflammation (Asthma, SARS-Covid), Psoriasis, Psoriatic Arthritis (PA), Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE) and Ankylosing Spondylitis (AS).BACKGROUND OF THE INVENTION

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Inflammation is the body's immunological self-defense response against various external and internal harmful stimuli, but if it remains uncontrolled, it turns chronic which results in various inflammatory and autoimmune disorders such as Ulcer Colitis (UC), Crohn's Disease (CD), Psoriasis, Psoriatic Arthritis (PA), Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), Ankylosing Spondylitis (AS), Pulmonary inflammation (Asthma), etc. These immunological responses, being characterized by the activation and differentiation of various innate and adaptive immune cells, lead to the release of pro-inflammatory and inflammatory cytokines such as TNF-a, IL-23, IL-1, IL-8, IL-22, IL-17, IL-6, IL-12, etc., which worsen the inflammatory condition. Out of these cytokines, TNF-a and IL-23 are the main drivers of inflammation.

[0004] Increased level of tumor necrosis factor-alpha (TNF-a) and interleukin-23 (IL-23) plays an important role in the initiation and progression of many inflammatory diseases by activating NF-kB, INK, and STAT-3 signaling pathway. Blocking the interaction of TNF-a and IL-23 to their respective receptors inhibits signaling pathways and is an effective treatment approachagainst autoimmune and inflammatory diseases such as psoriasis, psoriatic arthritis, IBD, rheumatoid arthritis, etc.

[0005] TNF-a is a master pro-inflammatory homotrimeric cytokine secreted mainly by activated innate immune cells like macrophages, neutrophils, NK cells, dendritic cells, and other immune and non-immune cells. Blocking the interaction of TNF-a with its receptors inhibits TNF-a-mediated proinflammatory signal activation. Many TNF-blocking biologies are available for the treatment of inflammatory conditions and includes Adalimumab, Infliximab, Golimumab, Etanercept, Certolizumab pegol, and Ozoralizumab.

[0006] Interleukin-23 (IL-23) is also a potent pro-inflammatory but a heterodimeric cytokine secreted by activated dendritic cells, macrophages and other immune cells. Many IL-23 - blocking biologies are approved for clinical use as they inhibit the interaction of IL-23 ligands to its receptors. These biologies mainly include Ustekinumab, Guselkumab, Tildrakizumab, Risankizumab, Brazikumab, and Mirikizumab.

[0007] High levels of TNF-a and IL-23 are associated with a variety of inflammatory and autoimmune disorders. TNF-a and IL-23 induce multiple inflammatory pathways, including NF-kB, JAK2-STAT3, AP-1, and MAPK, in a synergistic manner in these diseases. In addition, TNF-a induces cytotoxicity via an apoptotic pathway mediated by caspase-8. These disorders include Ulcerative Colitis (UC), Crohn's Disease (CD), Psoriasis, Psoriatic Arthritis (PA), Rheumatoid Arthritis (RA), Pulmonary inflammation (Asthma, COPD, Covid), Systemic Lupus Erythematosus (SLE), and Ankylosing Spondylitis (AS), among others. These diseases are characterized by elevated TNF-a and IL-23 cytokine levels.

[0008] Thus, for their treatment many TNF-a and IL-23 blocking biologies are available for clinical use. However, these biologies are less or partial efficient, as they target only single cytokine either TNF-a or IL-23, but none of them target both pro-inflammatory cytokines for completely blocking the inflammatory pathway in these aforementioned diseases. And targeting both the main inflammatory drivers (both proinflammatory TNF-a and IL-23 cytokine) is more promising in controlling the inflammation. Thus, there is still a dire need for the development of a bispecific antibody that will target both cytokines simultaneously, inhibit their binding to their respective receptors, and ultimately block their inflammatory pathway. Bispecific antibody (anti-IL-23 / TNF-a) would be more efficacious anti-inflammatory treatment.

[0009] Bispecific antibodies are engineered antibodies that are capable of recognizing and inhibiting two different antigens (cytokines) simultaneously. They are single engineeredantibody molecules with the specificity to target two different specific epitopes either on the same antigen or different antigen. They are the next-generation antibodies and are recognized as potentially superior therapeutic molecules (over conventional monoclonal antibodies, which are monospecific). Bispecific antibody can be engineered by fusing two different antigen specific domain antibody. Domain antibody is a fragment of heavy chain antibody found in some species of camelids and sharks, which are also known as VHH (variable heavy chain domain of heavy chain antibody) and Nanobody®. They are small in size, have looped CDR3 regions make it feasible to target cryptic epitopes on antigens, highly pH and temperature stable, less immunogenic, high tissue penetration efficacy, have high nano-to-pico-molar affinity, and are easy to engineer. Thus, domain antibodies are a better option to make bispecific antibody than conventional full-length antibody. However, this bispecific protein engineering using domain antibody has a drawback of poor pharmacokinetics due to small size. Hence, bispecific domain antibody can be fused with any half-life extension partner which will enhance the pharmacokinetics of the molecule. Hence, a bispecific antibody composed of domain antibody specific to both TNF-a and IL-23 (anti-IL-23 / TNF-a bispecific domain antibody) fused with a half-life extension partner would be efficacious in the treatment of inflammatory diseases.

[0010] While few such engineered bispecific antibody variants are known in the art, it will be appreciated that each protein is different and arriving, after modification, at a functional molecule with enhanced efficacy and / or improved properties requires considerable human intervention and biotechnological skills.

[0011] Prior art of bispecific domain antibody specific to TNF-a and IL-23 are: US20210198345 Al; US2022 / 0332810 Al; WO2016156466 Al; Roberts, et al., Scientific Reports, 2021, 11: 19422 describes generation and characterization of bispecific IL 23 / TNF-a neutralizing domain antibody, in which an IL 23pl9 specific domain antibody is fused with a TNFa specific domain antibody via a central protease labile linker to create a single molecule for oral administration as therapeutic for treatment of inflammatory bowel disease. Second bispecific domain antibody specific for TNF-a and IL-23 is Wang et al, Clin Trans I Med, 2024;14(3):el636 that describe generation of two different format of a bispecific domain antibody in which one format comprises anti-TNF-a VHH fused with anti-IL-23 VHH via flexible linkers (9GS and 15GS), second format comprises anti-TNF-a VHH and anti-IL-23 VHH and fused to human IgG-Fc region, and characterized them as therapeutic for the treatment of ulcer colitis. US8258268; US9035027; US20090215992 Al disclose a DVD-Ig(Dual variable domain-immunoglobulin) bispecific antibody format for anti-TNF-a / IL-23 antibody, and US20220372128A1, US2021188963Al(Ablynx) designed a bispecific antibody comprises of at least three immunoglobulin single variable domains (ISVDs), one ISVD bind to TNF-a, at least two ISVDs bind to IL-23 (one is a blocker and one is a binder) and one binds to albumin, all ISVD linked via 9GS linkers, however, their design (sequence) and the process of development is different. Other prior art are not domain antibody, they are either the full conventional type antibody or use other antibody fragments including scFv, Fab, or etc. Prior art US10059763 B2 discloses generation and characterization of IL-23 and TNF-a targeting compound, in which recombinant VH and VL domain of TNF-a are fused with VH and VL domain of IL-23 antibody, via peptide linker. US9718884 B2 describes a bispecific antibody having an immunoglobulin G antibody (IgG) that binds TNF-a conjugated to two single chain variable fragments (scFv) that bind the pl9 subunit of IL-23 (IL-23pl9). US20190040156A1 describes IgG bispecific antibodies that bind TNF-a and the pl9 subunit of interleukin - 23 (IL-23pl9) and are characterized as having high affinity and simultaneous neutralizing properties to both TNF-a and IL - 23. CN109776684A disclosed the design of a construct that consists of full-length antibody with one Fc chain specifically terminated with CDRs for TNF-a and another Fc chain terminated with CDRs for IL-23. The designed construct showed therapeutic efficacy against rheumatoid arthritis, bone hyperplasia, cervical pain, sciatica, leg joint rehydration swelling and limb numbness. CN105294863 A disclose the development of a bifiinctional antibody by combining adalimumab (anti-TNF-a Ab) and ustekinumab (anti-IL-23 Ab) in a single construct. US20120251541 Al disclosed a CODV-Ig (cross-over dual variable- immunoglobulin) bispecific antibody format for anti-TNF-a / IL-23 antibody, but didn’t characterize its functionality. Similarly, US8258268; US9035027; US20090215992 Al disclose a DVD-Ig (Dual variable domain-immunoglobulin) bispecific antibody format for anti-TNF-a / IL-23 antibody. US20220372128A1 (Ablynx) designed a bispecific antibody comprises of at least three immunoglobulin single variable domains (ISVDs), one ISVD bind to TNF-a, at least two ISVDs bind to IL-23 (one is blocker and one is binder) and one bind to albumin, all ISVD linked via 9GS linkers.

[0012] Although several IL-23 / TNF-a blocking biologies are well-known in the art, 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.

[0013] Therefore, there is an unmet need in the art to overcome the aforementioned problems in the prior art by producing anti-IL-23 / TNF-a bispecific domain antibody that will not only provide improved therapeutic efficacy but will also provide the benefits of domain antibody.OBJECTIVES OF THE INVENTION

[0014] The main objective of the present invention is to provide a bispecific domain antibody, which is a bispecific anti-IL-23 / TNF-a domain antibody capable of inhibiting interleukin 23 (IL-23) and tumor necrosis factor-alpha (TNF-a).

[0015] Another objective of the present invention is to provide a polynucleotide sequence encoding bispecific domain antibody.

[0016] Another objective of the present invention is to provide a method for the preparation of bispecific domain antibody.

[0017] Another objective of the present invention is to provide a bispecific domain antibody that is useful in the treatment of various inflammatory and autoimmune diseases like Inflammatory Bowel Disease (IBD) including both Ulcer Colitis (UC) and Crohn’s Disease (CD), Lung inflammation (Asthma, SARS-Covid), Psoriasis, Psoriatic Arthritis (PA), Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE) and Ankylosing Spondylitis (AS).SUMMARY OF THE INVENTION

[0018] This summary is provided to introduce a selection of concepts in a simplified form that is further described below in the detailed description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0019] The present invention relates to the field of biotechnology. Particularly, the present invention relates to a bispecific domain antibody, which is a bispecific anti-IL-23 / TNF-a domain antibody capable of inhibiting interleukin 23 (IL-23) and tumor necrosis factor-alpha (TNF-a). More particularly, the present invention relates to a method for the preparation of bispecific domain antibody. Further, the antibody of the present invention is useful in the treatment of various inflammatory and autoimmune diseases like Inflammatory Bowel Disease (IBD) including both Ulcer Colitis (UC) and Crohn’s Disease (CD), Lung inflammation (Asthma, SARS-Covid), Psoriasis, Psoriatic Arthritis (PA), Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE) and Ankylosing Spondylitis (AS).

[0020] In one aspect, the present invention relates to a bispecific domain antibody consisting of polypeptide sequence as set forth in SEQ ID NO: 1.

[0021] In another aspect of the present invention, the said bispecific domain antibody is a IL- 23 and TNF-a blocking domain antibody.

[0022] In another aspect of the present invention, the polypeptide sequence comprises an anti- IL-23 domain antibody polypeptide, anti-TNF-a domain antibody polypeptide, a non-antibody polypeptide, a linker polypeptide and (His)e-tag.

[0023] In another aspect of the present invention, a polynucleotide sequence encoding bispecific domain antibody construct, as set forth in SEQ ID NO: 2 comprising: a) a polynucleotide sequence comprising a nucleic acid encoding an anti-IL-23 domain antibody polypeptide and anti-TNF-a domain antibody polypeptide; b) a polynucleotide sequence comprising a nucleic acid encoding a non-antibody polypeptide selected from the group consisting of human serum albumin (HSA) or its analogs or its fragment / portion, human serum albumin targeting polypeptide (HSATP) or its analogs or its fragment / portion, human chorionic gonadotropin (hCG) hormone or its fragment / portion, elastin or its analogs or its fragment / portion, gelatin or its analogs or its fragment / portion, al -antitrypsin or its analogs or its fragment / portion, immunoglobulin (Ig) or its analogs or its fragment / portion, and non-natural polymeric amino acid sequences (NNPAAS); c) a polynucleotide sequence comprising a nucleic acid encoding a linker polypeptide d) a polynucleotide sequence comprising a nucleic acid encoding a (His)e-tag

[0024] In another aspect of the present invention, the nucleic acid construct encodes a bispecific domain antibody with (His)e-tag at either C-terminal or N-terminal, and wherein the bispecific domain antibody is having SEQ ID NO 1.

[0025] In another aspect of the present invention, the non-natural polymeric amino acid sequences (NNPAAS) are selected from but not limited to alanine, glutamic acid, glycine, proline, serine and threonine or proline, alanine, and serine or glycine and serine,

[0026] In another aspect of the present invention, the non-antibody polypeptide is an analog or fragment / portion of human serum albumin.

[0027] In another aspect of the present invention, a recombinant plasmid for cloning and expression of bispecific domain antibody of SEQ ID NO: 1, comprising the polynucleotide sequence of SEQ ID NO: 2.

[0028] In another aspect, the present invention provides the recombinant plasmid comprising the construct of SEQ ID NO. 2 for secretory expression of bispecific domain antibody of SEQ ID NO. 1 in host cells, wherein the construct comprises of an arrangement having: a) a first polynucleotide comprising a nucleic acid encoding an anti-IL-23 domain antibody polypeptide; b) a second polynucleotide sequence encoding a linker; c) a third polynucleotide sequence comprising a nucleic acid encoding a non-antibody polypeptide; d) a fourth polynucleotide sequence encoding a linker; e) a fifth polynucleotide sequence comprising a nucleic acid encoding an anti-TNF-a domain antibody; and f) a polynucleotide comprising a nucleic acid encoding a (His)6-tag at either C- or N-terminus.

[0029] In another aspect of the present invention, the host cells are selected from the group consisting of bacterial cells, yeast cells, mammalian cells and insect cells.

[0030] In another aspect of the present invention, the host cells are yeast cells.

[0031] In yet another aspect, present invention provides a method for the production of bispecific domain antibody of SEQ ID NO: 1, wherein the method comprising the steps of: i. preparing recombinant plasmid containing polynucleotide sequence of SEQ ID NO: 2 for the secretory expression of bispecific domain antibody of SEQ ID NO: 1; ii. transforming the host cells with recombinant plasmids of step (i) to obtain recombinant host cells; iii. culturing the recombinant host cells of step (ii) in culture media for the growth of recombinant host cells; iv. inducing the secretary expression of bispecific domain antibody of SEQ ID NO: 1 by treating the culturing host cells of step (iii); v. isolating and purifying the bispecific domain antibody of SEQ ID NO: 1 from the culture media of step (iv) by using affinity chromatography, vi. removing endotoxin from the purified bispecific domain antibody, by using endotoxin removal columns; and vii. reconstituting the purified bispecific domain antibody of step (vi) for long-term storage and followed by In-vitro characterization of bispecific domain antibody.

[0032] In another aspect of the present invention, the host cells are selected from the group consisting of bacterial cells, yeast cells, mammalian cells and insect cells.

[0033] In another aspect of the present invention, the recombinant host cells are recombinant P. pastoris SMD 1168 cells.

[0034] In another aspect of the present invention, the culture media is selected from a group consisting of YPD (Yeast extract, Peptone, Dextrose compositions), BMGY (Medium comprises peptone, yeast extract, yeast nitrogen base, glycerol and a phosphate buffer), and BMMY (Medium comprises peptone, yeast extract, yeast nitrogen base, methanol and a phosphate buffer).

[0035] In another aspect, the present invention provides treating the culturing host cells of step (iv) with 1-5% methanol followed by further culturing of host cells at 29°C for 3-5 days.

[0036] In another aspect of the present invention, the long-term storage of bispecific domain antibody in step (vii) is done at -80°C by adding 10% glycerol.

[0037] In another aspect of the present invention, the bispecific domain antibody produced using the method binds to IL-23 and TNF-a with high affinities and inhibits IL-23 and TNF-a activities.

[0038] In another aspect, the present invention provides a pharmaceutical composition comprising a bispecific domain antibody of SEQ ID NO: 1 and a pharmaceutically acceptable carrier, excipient, or diluent.

[0039] In another aspect of the present invention, the pharmaceutical composition comprises a bispecific domain antibody of SEQ ID NO: 1, wherein the pharmaceutical composition is in the form of a tablet, a capsule, a solution, a gel, a suspension or a powder.

[0040] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The invention has other advantages and features which will be more readily apparent from the following detailed description of the invention, when taken in conjunction with the accompanying drawings, in which:

[0042] Figure 1: depicts the designing, construction, production and purification of bispecific domain antibody. (A) The designed bispecific domain antibody (BiSpekDAb) construct of the present invention and its components. (B) The designed gene and (C) The recombinant vector containing the designed gene for cloning and secretory expression of BiSpekDAb polypeptide construct in P pastoris. (D) The chromatogram showing the resolution of BiSpekDAb polypeptide on the Ni-NTA affinity chromatography column. Red dots denote the absorbanceat 595 nm (protein content by Bradford’s assay) of the eluted fractions from the column. Fractions containing pure protein were pooled, dialyzed (against PBS, pH 5.8), subjected to endotoxin removal and concentrated using a centrifugal concentrator. (E) The image of Coomassie-stained SDS-PAGE gel of final BiSpekDAb preparation. (F) The image of the BiSpekDAb prototype. Legends: Lane M: protein molecular weight markers; Lane 1-3: 5, 10 and 15 pg of BiSpekDAb protein, respectively.

[0043] Figure 2: depicts the in-vitro characterization of bispecific domain antibody. (A) The graphical representation of binding of BiSpekDAb to surface coated TNF-a or IL-23 as determined by Solid-Phase ELISA. (B) The graphical representation of TNF-a blocking efficacy of BiSpekDAb and Adalimumab (positive control) as determined by L929 cell cytotoxicity assay determined by MTT assay. Legends: 1- Control; 2- TNF-a 1.5 nM; 3- 0.006 nM; 4- 0.06 nM; 5- 0.6 nM; 6- 6.0 nM; 7- 60 nM of BiSpekDAb or Adalimumab. (C) The graphical representation of inhibition of IL-23 induced proliferation by BiSpekDAb and Ustekinumab (positive control) in Caco-2 cells as determined by MTT assay. Legends: 1- Control; 2- IL-23 100 ng / ml; 3- 0.6 nM; 4- 6 nM of BiSpekDAb or Ustekinumab (Positive control). Data were analyzed by GraphPad Prism 9.0 and significance statistics done by oneway anova. (* represent comparison vs TNF-a or IL-23, # represent comparison vs healthy control, *p<0.05, **p<0.00I, ***p<0.0001, ****p<0.0001).

[0044] Figure 3: depicts the beneficial effect of bispecific domain antibody in TNF-a and IL- 23 related diseases. (A) The scheme of asthmatic rat model developed by novel combination of Ovalbumin (OVA) and Lipopolysaccharide (LPS). (B) The graphical representation of the airway hyperresponsiveness (AHR) in response to different concentrations of Methacholine (Meh challenge) showed as PenH (enhanced pause) value. (C) The improvement in Lung Functional Parameters (LFP) such as respiratory rate (BPM), Inspiratory time (sec), Mid tidal expiratory flow rate (ml / sec). (D) The effect of BiSpekDAb on blood cell counts (TLC, Neutrophils, Eosinophil). (E) The effect of BiSpekDAb on BALF (Bronchoalveolar lavage fluid) cell counts (TLC, Neutrophils, Eosinophil). (F) The graphical representation of lung tissue biochemicals (Nitrite and MPO (Myeloperoxidase) activity). (G) The graphical representation of the effect of BiSpekDAb on the body weight of animals during the OVA + LPS challenging phase. Legends: 1; Healthy control, 2; Asthmatic, 3; BiSpekDab (1 mg / kg), 4; DXM (2 mg / kg). Data represents the mean ± SEM (n=9-12 per group) and analyzed by oneway ANOVA or two-way ANOVA followed by multiple comparisons tests. (* representcomparison vs disease group, # represent comparison vs healthy control, *p<0.05, **p<0.001, ***p<0.0001, ****p<0.0001).DETAILED DESCRIPTION OF THE INVENTION

[0045] The following is a detailed description of embodiments of the disclosure. The embodiments are in such details as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention.

[0046] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0047] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0048] In some embodiments, numbers have been used for quantifying weight percentages, ratios, and so forth, to describe and claim certain embodiments of the invention and are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be constructed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the inventionmay contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0049] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0050] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise. Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”

[0051] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0052] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0053] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.

[0054] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present invention. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.

[0055] It should also be appreciated that the present invention can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.

[0056] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0057] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements a, b, and c, and a second embodiment comprises elements b and d, then the inventive subject matter is also considered to include other remaining combinations of a, b, c, or d, even if not explicitly disclosed.Definitions

[0058] 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.

[0059] 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.

[0060] 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.

[0061] The term “polypeptide” as used herein refers to two or more polymers of natural or unnatural amino acids.

[0062] The term “heterologous proteins” refers to those proteins that are foreign to the host cells used, such as human proteins produced in P. pastoris.

[0063] The term “engineered proteins” refers to those proteins that are modified with recombinant DNA technology to get a desirable function for better use in medicine and industry.

[0064] 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).

[0065] The term “optimized polynucleotide sequence” refers to a synthetically synthesized nucleic acid (gene) optimized for high level expression of recombinant protein in host cells.

[0066] The term ‘recombinant’ as used herein means that a heterologous protein is derived from recombinant (e.g., microbial or mammalian) expression systems. ‘Microbial’ refers to bacterial or fungal (e.g., yeast) expression systems. ‘Recombinant microbial product’ defines a protein produced in a recombinant microbial expression system, which is essentially free of native endogenous substances.

[0067] 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.

[0068] The term “recombinant host cell” refers to host cells that contain a 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. pasioris. animal cells, plant cells, mammal cells, or insect cells.

[0069] 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.

[0070] 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) and interleukin 23 (IL-23), preferably human TNF-a and IL-23.

[0071] The term ‘linker’ is meant to refer to a compound or moiety that acts as a molecular bridge to operably link each monomeric antibody. The linker peptide may facilitate correct folding of the individual protein or peptide parts and may make it more likely for the individual protein or peptide parts to retain their unique functional properties.

[0072] The term “immunoglobulin” is used herein to refer to a protein consisting 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. Also, it contains different domains among these chains, such as, variable domain and constant domain. The variable region of the heavy chain is often called the “VH domain” and the variable region of the light chain is often called the VL domain. Same like this, for the constant region of heavy chain often called as CH domain and the constant region of light chain often called as CL domain.

[0073] The term “conventional antibody” is used herein to refer to one form of immunoglobulin which is derived from a single cell and it is tetrameric in nature and comprises 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.

[0074] The term “single domain antibody (sdAb)” or “domain antibody” or “VHH” is used herein to refer to antigen binding fragments or portions 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.

[0075] 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), and lacks the light chain.

[0076] The term ‘isolated’ or ‘purified’, as used in the context of this specification to define the purity of BiSpekDAb polypeptide construct, means that the polypeptide is substantially free of other proteins of natural or endogenous origin and contains less than about 1% by mass of protein contaminants residual of production processes.

[0077] The term ‘biologically pure’ refers to a material that is substantially or essentially free from components that normally accompany it as found in its native state.

[0078] The term "recombinant P. pasioris' refers to host cells expressing target heterologous protein.

[0079] The term ‘functionally active’ refers to the ability of the polypeptide to exert one or more activities known to be associated with engineered antibody- 1, such as the ability to bind to human TNF-a and human IL-23 and inhibit their binding to their respective receptor.

[0080] The term “Affinity” refers to a binding strength between an antigen-binding site at the antibody and an antigen. It is the net result of attractive and repulsive forces between an antigen-antibody interaction. It is the degree of association between an antigen epitope and a paratope or antigen-binding site on an antibody.

[0081] The term “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.

[0082] The term “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.

[0083] The term ‘autoimmune disease’ refers to the physiological condition in which the body’s immune cell itself starts activating against self-proteins. A person’s immune cells fail to differentiate between self and non-self-antigens and hyperactivate against self-proteins and exert immune inflammation. Such autoimmune diseases include multiple sclerosis (MS), systemic lupus erythematosus (lupus), inflammatory bowel disease (IBD), rheumatoid arthritis (RA).

[0084] The term “antigen” refers to a substance that is capable of interacting with an antibody and in the context of the present invention is Interleukin-23 (IL-23) and tumor necrosis factor alpha (TNF alpha), preferably Interleukin-23 (IL-23) and human TNF alpha or any Interleukin-23 (IL-23) and TNF alpha representing an antigen for engineered antibody.

[0085] In an embodiment, the non-antibody polypeptide used to create BiSpekDAb polypeptide construct / engineered antibody- 1 of the present disclosure is a polypeptide that isdifferent from engineered antibody polypeptide in terms of their amino acid sequences and biological properties. The non-antibody polypeptides when fused with VHH of TNF-a and IL- 23 polypeptide, the resulting engineered molecules may possess binding and inhibiting property against both IL-23 and TNF-a, simultaneously and increased circulatory half-life, compared to antibody polypeptide or non-antibody polypeptide alone. The non-antibody polypeptide used to create polypeptide constructs of the present disclosure comprises a fragment of human serum albumin (HSA). The linker peptides used to fuse IL-23 and TNF-a VHH domain polypeptide to a non-antibody polypeptide are short stretches of amino acids.

[0086] The term ‘non-antibody polypeptides’ used in the present disclosure refers to polypeptides that are different from antibody polypeptides used in the present invention in terms of their amino acid sequences and biological properties and that cannot neutralize TNF-a and IL-23 antigens.

[0087] The term ‘linker’ is meant to refer to a compound or moiety that acts as a molecular bridge to operably link two different molecules, wherein one portion of the linker is linked to an arginase polypeptide, and wherein another portion of the linker is linked to a nonantibody polypeptide.

[0088] The term Tinker peptide’ used in the present disclosure refers to a peptide sequence that separates the two polypeptide components of BiSpekDAb polypeptide construct of engineered antibody of the present disclosure. The linker peptide may facilitate correct folding of the individual protein or peptide parts and may make it more likely for the individual protein or peptide parts to retain their unique functional properties.

[0089] The term ‘half-life extending partner (HLEP)’ refers to a non-antibody polypeptide which when genetically fused with antibody polypeptides can increase in-vivo circulatory half- life of fusion protein. Example of HLEP polypeptide suitable for genetic fusion to arginase polypeptide in connection with the present disclosure includes: human serum albumin (HSA) or its analogs or its fragment / portion; human serum albumin targeting polypeptide (HSATP) or its analogs or its fragment / portion; human chorionic gonadotropin (hCG) hormone or its fragment / portion; elastin or its analogs or its fragment / portion; gelatin or its analogs or its fragment / portion; al -antitrypsin or its analogs or its fragment / portion; immunoglobulin (Ig) or its analogs or its fragment / portion; and non-natural polymeric amino acid sequences (NNPAAS) containing either alanine, glutamic acid, glycine, proline, serine and threonine or proline, alanine, and serine or glycine and serine, preferably analog or fragment or portion of human serum albumin, and any combination thereof.

[0090] The term “immunoglobulin1is used herein to refer to a protein consisting of one or more polypeptides Substantially 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 chain and one heavy chain. In each pair the heavy and light chain variable regions together provide the binding surface capable of interacting with the antigen.

[0091] 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.

[0092] The term “conventional antibody” is used herein to refer to one form of immunoglobulin which is tetrameric in nature and comprises 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.

[0093] 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-VL 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-VL 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.

[0094] 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 traditional antibody have been joined to form one chain.

[0095] 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).

[0096] The term “domain antibody (DAb)” or “domain antibody” or “VHH” is used herein to refer to the variable heavy chain fragment or variable domain 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.

[0097] The term “Affinity” refers to a binding strength between an antigen-binding site at the antibody and an antigen. It is the net result of attractive and repulsive forces between antigen-antibody interactions. It is the degree of association between an antigen epitope and a paratope or antigen-binding site on an antibody.

[0098] The terms ‘analogs’ or ‘fragments’ used in the present disclosure refer to an amino acid sequence comprising at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of a naturally occurring said protein or mutant thereof.

[0099] The term ‘polypeptide’ used in the present disclosure collectively refers to neutralizing BiSpekDAb 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

[0100] In an embodiment, the Human serum albumin (HSA) is ~65.5 kDa, the most abundant glycoprotein present in human blood plasma (35-50 g / L). It is produced in the liver and is involved in the transport of hormones, fatty acids, and other compounds in the body. It is a helicoidal protein, consisting of three structurally similar domains (I, II, III domain). It has a serum half-life of approximately 21 days. The term ‘human serum albumin (HSA) or its analogs or its fragment / portion’ used in the present disclosure refer to either HSA or its analogs or its fragment / portion (e.g., Ill domain of HSA; 3dHSA).

[0101] The term “binding property” refers to the ability of an antibody to preferentially bind to a particular analyte that is present in a homogeneous mixture of different analytes. In certain embodiments, a specific binding interaction will discriminate between desirable and undesirable analytes in a sample, in some embodiments more than about 10 to 100-fold or more (e.g., more than about 1000- or 10,000-fold).

[0102] The term "TNF alpha" is tumor necrosis factor alpha and is a cytokine involved in systemic inflammation. TNF alpha 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 alpha and its receptors (CD 120a andCD120b) have been studied in great detail. TNF alpha in its bioactive form is a trimer. Several strategies to antagonize the action of TNF-a using anti-TNF-a antibodies or binders have been developed and are currently commercially available, such as Remicade* and Humira*.

[0103] The term “Interleukin-23” or “IL-23” is a cytokine produced by dendritic cells and the like, and is a heterodimeric cytokine consisting of two subunits, i.e., a pl9 subunit which is a component specific for IL-23 and the p40 subunit which is also a component of IL- 12 (nonpatent document 1). IL-23 binds to the IL-23 receptor (also referred to as IL-23R) to transduce signals into cells (non-patent document 2). IL-23R is a heterodimeric receptor consisting of an IL-23R subunit and the IL-12Rpi subunit which is also a component of the IL-12 receptor. Also, it is known that the IL 12 receptor is a complex of an IL-12Rpi subunit and an IL-12RP2 subunit, and that IL-23 does not bind to the IL- 12 receptor.

[0104] The term “anti-IL-23 VHH” or “binding fragment thereof’ encompasses a fragment or a derivative of an heavy chain antibody that still substantially retains its biological activity of inhibiting IL-23 activity. Therefore, the term “antibody fragment” or IL-23 binding fragment refers to a portion of a full-length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; and multispecific antibodies formed from antibody fragments. Typically, a binding fragment or derivative retains at least 10% of its IL-23 inhibitory activity. Preferably, a binding fragment or derivative retains at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (or more) of its IL-23 inhibitory activity, although any binding fragment with sufficient affinity to exert the desired biological effect will be useful. It is also intended that a IL-23 binding fragment can include conservative amino acid substitutions that do not substantially alter its biologic activity.

[0105] The term “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.

[0106] The term “neutralizing engineered antibody”, “antibody that neutralizes antigen activity” or any grammatical equivalent thereof, is intended to refer to an antibody whose binding to antigen results in inhibition of a biological activity of the same. This inhibition of the biological activity of antigen can be assessed by measuring one or more indicators of antigen biological activity, such as antigen-induced cytotoxicity (either in vitro or in vivo), antigen-induced cellular activation or inflammation or antigen binding its receptor.

[0107] The term “TNF-a and IL-23 related diseases or conditions” refers to the diseases or conditions in which TNF-a and IL-23 inhibition has therapeutic effects. This can be achieved by administering certain drugs and anti-TNF-a and anti-IL-23 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, pulmonary inflammation such as asthma, COPD (Chronic Obstructive Pulmonary Disease), 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 a structural lesions of the cerebellum; spinocerebellar degenerations (spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations, multiple systems degenerations (Mencel, Dejerine-Thomas, Shy-Drager, and Machado-Joseph); and systemic disorders (Refsum’s disease, abetalipoproteinemia, 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 and IL-23-secreting tumors or other malignancies involving TNF-a and IL-23, such as, but not limited to leukemias (acute, chronic myelocytic, chronic lymphocytic and / or myelodysplastic syndrome);lymphomas (Hodgkin’s and non-Hodgkin’s lymphomas, such as malignant lymphomas (Burkitt’s lymphoma or Mycosis fungoides); andF. alcohol-induced hepatitis.

[0108] In a specific embodiment, the TNF-a and IL-23 related diseases or conditions refer to psoriasis, IBD-intestinal bowel disease (Ulcer colitis, Crohn’s disease), pulmonary inflammation (asthma), multiple sclerosis, viral infections, rheumatoid arthritis, autoimmune diseases, graft-versus-host disease (GVHD), inflammation and related complications and comorbidities.

[0109] The term “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.

[0110] 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.[oni] 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 (Remington ’s Pharmaceutical Sciences 16thEdition, 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 dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butylbenzyl alcohol, alkyl parabenssuch as methyl or propyl paraben, catechol, resorcinol, cycloHexanol, 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, com and other starches; Binder; additive; coloring agent; Salt-forming counter ions 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.

[0112] The term “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 skills 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.

[0113] The term "bispecific antibody" refers to a molecule comprising a single antibody or fragment thereof with binding affinity to two different epitopes or antigens. As referred to herein, a bispecific antibody of the present invention comprises one domain antibody covalently linked to each other via a flexible linker. Carboxy terminal of anti-IL-23 domain antibody bind to the amino terminal of the amino terminal of domain antibody polypeptide part via flexible linker and the amino terminal of anti-TNF-a domain antibody bind to the carboxy terminal of the domain antibody polypeptide via flexible linker. This bispecific antibody herein used to have binding affinity to two different antigens (TNF-a and the IL-23).

[0114] 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 cellis 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.

[0115] The term “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.

[0116] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skills 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.

[0117] In a general embodiment, the present invention relates to engineered antibodies: bispecific domain antibody (BiSpekDAb) that can bind IL-23 and TNF-a simultaneously and can inhibits their biological activity, wherein the amino acid sequences of the engineered bispecific domain antibody (BiSpekDAb) are set forth in SEQ ID NO: 1 given below.SEQ ID NO: 1:Amino acid sequence of bispecific domain antibody (BiSpekDAb):> BiSpekDAb (465 aa)DVQLLESGGGVVQPGGSLRLSCAASGRIFSLPASGNIFNLLTIAWYRQAPGKGRELVA TINSGSRTYYADSVKGRFTISRDNSKKTVYLQMNSLRPEDTALYYCQTSGSGSPNFWG QGTLVTVSSGGGGSGGGGSVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVK HKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLVAASQAALGLGGGGS GGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEI NTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSGFNRGQGT LVTVSS

[0118] In an embodiment, the present invention provides a polynucleotide sequence encoding bispecific domain antibody (BiSpekDAb) and the polynucleotide sequences are set forth in SEQ ID NO: 2 given below.SEQ ID NO: 2:Nucleotide sequence of bispecific domain antibody (BiSpekDAb):> BiSpekDAb (1395 bp)GATGTTCAATTGTTGGAATCTGGTGGTGGTGTTGTTCAACCAGGTGGTTCTTTGAG ATTGTCTTGTGCTGCTTCTGGTAGAATTTTCTCTTTGCCAGCTTCTGGTAACATTTT CAATTTGTTGACTATCGCTTGGTACAGACAAGCTCCAGGTAAAGGTAGAGAATTG GTTGCTACTATTAACTCTGGTTCCAGAACTTACTATGCTGATTCTGTTAAGGGTAG ATTCACTATTTCCAGAGATAACTCTA AGAAAACTGT TTATTTGCAA ATGAACTCTC TTAGACCAGAGGATACTGCTTTGTACTATTGTCAAACTTCTGGTTCTGGTTCTCCTA ATTTCTGGGGTCAAGGTACTTTGGTTACTGTTTCTTCTGGAGGTGGTGGTTCTGGA GGAGGTGGTTCTGTTGAAGAGCCACAAAACTTGATTAAACAAAACTGTGAATTGT TTGAGCAATTGGGTGAATACAAGTTCCAAAACGCTTTGTTGGTTAGATACACTAAG AAAGTTCCACAAGTTTCTACTCCTAC TTTGGTTGAA GTTTCCAGAA ATTTGGGTAAAGTTGGTTCTAAATGTTGTAAGCATCCAGAAGCTAAGAGAATGCCTTGTGCTGAG GATTACTTGTCTGTTGTTTTGAACCAATTGTGTGTTTTGCACGAAAAGACTCCTGTT TCTGATAGAGTTACTAAGTGTTGTACTGAGTCTTTGGTTAACCGTAGACCATGTTTT TCTGCTTTGGAAGTTGATGAGACTTATGTTCCTAAAGAGTTTAACGCTGAGACTTT TACTTTCCATGCTGATATTTGTACTTTGTCTGAAAAGGAGAGACAAATTAAGAAA CAAACTGCTTTGGTTGAATTGGTTAAGCACAAACCTAAGGCTACTAAAGAGCAAT TGAAG GCTGTTATGG ATGATTTTGCTGCTTTCGTTGAAAAATGTT GTAAGGCTGA TGATAAAGAGACTTGTTTTGCTGAAGAGGGTCCAAAGTTGGTTGCTGCTTCTCAAG CTGCTTTGGGTTTGGGTGGTGGTGGTTCTGGTGGAGGAGGTTCTGAAGTTCAATTG GTTGAGTCTGGTGGTGGTTTGGTTCAACCTGGTGGTTCTTTGAGATTGTCTTGCGCTGCTTCTGGTTTTACTTTCTCTGATTACTGGATGTATTGGGTTAGACAAGCTCCTGGT AAAGGTTTGGAATGGGTTTCTGAGATTAACACTAACGGTTTGATCACTAAGTACCC AGACTCCGTCAAAGGAAGATTCACTATTTCCAGAGATAATGCTAAGAATACTTTGT ATTTGCAAATGAACTCTCTTAGACCAGAAGATACTGCTGTTTACTATTGTGCTAGA TCCCCATCTGGTTTCAATAGAGGACAGGGTACTTTG GTTACTGTTT CTTCT

[0119] In an embodiment, the present invention provides a recombinant plasmid comprising the polynucleotide sequences (SEQ ID NO: 2), wherein the plasmid is a cloning and expression vector suitable for cloning and expression of BiSpekDAb in host cells selected from the group consisting of bacterial cells, yeast cells, mammalian cells and insect cells.

[0120] In an embodiment, the present invention provides a host cell comprising the polynucleotide sequences (SEQ ID NO: 2), wherein the host cell is the yeast cell.

[0121] In an embodiment, the present invention provides a polynucleotide sequence encoding a bispecific domain antibody (BiSpekDAb) construct as set forth in the SEQ ID NO: 2 comprising: a) a polynucleotide sequence comprising a nucleic acid encoding an anti-IL-23 domain antibody and anti-TNF-a domain antibody polypeptide; wherein anti-IL-23 domain antibody is a nanobody or domain antibody specific for human IL-23, anti-TNF-a domain antibody is a nanobody or domain antibody specific for human TNF-a. b) a polynucleotide sequence encoding a non-antibody polypeptide selected from the group consisting of human serum albumin (HSA) or its analogs or its fragment / portion, human serum albumin targeting polypeptide (HSATP) or its analogs or its fragment / portion, human chorionic gonadotropin (hCG) hormone or its fragment / portion, elastin or its analogs or its fragment / portion, gelatin or its analogs or its fragment / portion, al -antitrypsin or its analogs or its fragment / portion, immunoglobulin (Ig) or its analogs or its fragment / portion, and non-natural polymeric amino acid sequences (NNPAAS) containing either alanine, glutamic acid, glycine, proline, serine and threonine or proline, alanine, and serine or glycine and serine, preferably analog or fragment or portion of human serum albumin; c) a polynucleotide sequence comprising a nucleic acid encoding a linker polypeptide; and d) a polynucleotide sequence comprising a nucleic acid encoding a (His)e-tag, wherein the nucleic acid construct encodes a bispecific domain antibody (BiSpekDAb), construct with (His)e-tag either at C- or N- terminus; and wherein the bispecific domain antibody (BiSpekDAb), is having SEQ ID NO 1.

[0122] In yet another embodiment, the present invention provides a method for the production of bispecific domain antibody (BiSpekDAb), of SEQ ID NO: 1 and the method consists of following steps: i. Preparing recombinant plasmid containing polynucleotide sequence of SEQ ID NO: 2, and is suitable for the secretory expression of bispecific domain antibody (BiSpekDAb), of SEQ ID NO: 1; ii. Preparing recombinant host cells containing bispecific domain antibody (BiSpekDAb), sequence of SEQ ID NO: 2, by transforming the yeast cells with recombinant plasmids of step (i); iii. Culturing the recombinant host cells of step (ii) in suitable media for the growth of recombinant cells; iv. Inducing the secretary expression of bispecific domain antibody (BiSpekDAb), of SEQ ID NO: 1 by treating the culturing cells of step (iii) with 1-5% methanol followed by further culturing of host cells at 29°C for 3-5 days; v. Isolating and purifying the bispecific domain antibody (BiSpekDAb), from the culture media of step (iv) by using affinity chromatography; vi. Removal of endotoxin from the purified bispecific domain antibody (BiSpekDAb), by using endotoxin removal columns; vii. Reconstituting the purified bispecific domain antibody (BiSpekDAb), of step (vi) for long-term storage at -80°C by adding 10% glycerol; and viii. In-vitro characterization of bispecific domain antibody (BiSpekDAb).

[0123] In another embodiment, the present invention provides BiSpekDAb suitable for end application without being processed for removal of (His)6-tag.

[0124] In another embodiment, the present invention provides a culture media for the culturing of recombinant host cells and selected from the group consisting of series of culturing media selected on the basis of expression of the targeted protein wherein the culturing media selected from the group consisting of YPD (Y east extract, Peptone, Dextrose compositions); BMGY (Medium comprises peptone, yeast extract, yeast nitrogen base, glycerol and a phosphate buffer); and BMMY (Medium comprises peptone, yeast extract, yeast nitrogen base, methanol and a phosphate buffer).

[0125] In another embodiment, the present invention provides a method of producing BiSpekDAb of SEQ ID NO. 1, wherein the recombinant plasmid is pPIC9K- BiSpekDAb and the yeast cells are Pichia pastoris SMD 1168.

[0126] In another embodiment, the present invention provides a method of producing BiSpekDAb of SEQ ID NO. 1, wherein production steps also contain removal of endotoxin by using High-Capacity Endotoxin Removal Spin Columns.

[0127] In another embodiment, the present invention provides BiSpekDAb which binds to IL-23 and TNF-a with good affinities.

[0128] In another embodiment, the present invention provides BiSpekDAb which inhibits TNF-a induced cytotoxicity of L929 cells (TNF-a activity), and inhibits IL-23 induced proliferation of Caco-2 cells (IL-23 activity).

[0129] In another embodiment, the present invention provides a method of producing BiSpekDAb, wherein the BiSpekDAb of SEQ ID NO. 1 can bind IL-23 and TNF-a simultaneously and inhibits their activity.

[0130] In another embodiment, the present invention provides a method of getting beneficial activity of BiSpekDAb in OVA + LPS (ovalbumin + lipopolysaccharides) induced asthma in a rodent model.

[0131] In another embodiment, the present invention provides a pharmaceutical composition comprising the BiSpekDAb, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0132] In another embodiment of the present invention, the BiSpekDAb can be used for the treatment of TNF-a and IL-23 related diseases or conditions, wherein the diseases is selected from the list comprising of psoriasis, IBD-intestinal bowel disease (Ulcer colitis, Crohn’s disease), pulmonary inflammation (asthma), multiple sclerosis, viral infections, rheumatoid arthritis, autoimmune diseases, graft-versus-host disease (GVHD), inflammation and related complications and comorbidities.

[0133] While the foregoing description discloses various embodiments of the disclosure, other and further embodiments of the invention may be devised without departing from the basic scope of the disclosure. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.EXAMPLES

[0134] The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are byway of example and for the purpose of illustrative discussion of preferred embodiments of the invention.

[0135] Example 1: Designing, cloning, expression and purification of BiSpekDAb polypeptide constructs. a) Designing of BiSpekDAb polypeptide and polynucleotide constructs'. A polypeptide construct of BiSpekDAb of SEQ ID NO. 1 or 2 is designed by fusing the amino acid sequence of anti-IL-23 domain antibody (PCT / EP 2016 / 076076) with amino acid sequence of anti-TNF- a domain antibody (PCT / EP2016 / 077595) via a non-antibody polypeptide (fragment of human serum albumin, HSA) and linker peptides as depicted in Figure 1A. A gene encoding for BiSpekDAb was then designed. In the designed gene, the 5' end of the open reading frame (ORF) is preceded by two codons (AAA and AGG), which codes for two positively charged amino acids (proteolytic cleavage site for the secretion of mature BiSpekDAb polypeptide in the culturing medium) and an Xhol restriction site. The 3' end of the open reading frame (ORF) is preceded by Notl restriction site. The designed gene also contains a polynucleotide sequence that codes for a (His)e-tag (either at the N-terminal or C-terminal) of BiSpekDAb polypeptide. b) Generation of recombinant expression plasmid (vector)'. The designed genes were codon- optimized for high-level expression in Pichia pastoris and cloned in pPIC9K plasmid to create recombinant pPIC9K plasmids (pPIC9K-BiSpekDAb) (Figure IB) (Figure 1C), and custom synthesized from GenScript Biotech (Singapore) Pte. Ltd. This unique design of recombinant pPIC9K-BiSpekDAb permits to clone the gene in P. pastoris genome and express BiSpekDAb polypeptides into the culturing medium (which can be purified easily). Presence of designed genes in the recombinant plasmids was confirmed by digesting the individual plasmid with Xhol and Notl (NEB, USA) and monitoring the digestion with agarose gel electrophoresis. Presences of digested products of desired sizes confirm the presence of designed genes in the recombinant plasmids. c) Generation of recombinant host cell'. The recombinant plasmids (pPIC9K-BiSpekDAb) were amplified in A'. coli DH5a cells and purified by following standard molecular biology techniques. The purified plasmids were subjected to restriction digestion with Bglll . For this, 10-12 pg of pPIC9K-BiSpekDAb DNA was treated with 10 U of Bglll enzyme (NEB, USA) in a specific buffer (CutSmart buffer compatible with the enzyme) and the reaction was incubated at 37 C for 12 h in a water bath. After this, the digested reaction mixture was subjected to 1% agarose gel electrophoresis and a linearized pPIC9K-BiSpekDAb fragment was sliced from thegel using a UV transilluminator and gel purified using a gel extraction kit. The amount of purified linearized pPIC9K-BiSpekDAb fragment was quantitated using a Nano photometer (at 260 nm) and used for transfection of competent P. pastoris cells. For this, the linearized pPIC9K-BiSpekDAb fragment (10-20 pl containing 10-15 pg DNA) was added to 100 pl of competent P. pastoris cells SMD1168 cells (Cat # Cl 7500, Invitrogen, USA) and the mixture was kept in electroporation cuvette (Gene Pulser® cuvette, Cat # 165-2086, Bio-Rad, USA) (5 min on ice) and electroporated at 1700 V for ~10 msec. Then, 1 ml of IM sorbitol was added immediately to the cuvette and the cuvette was incubated at 29°C for 45 min. After incubation, the mixture was centrifuged (3000 xg for 10 min), the majority of supernatant was discarded and the remaining cells were plated on minimal dextrose (MD) plates (1.34 % yeast nitrogen base without amino acids and without ammonium sulphate, 2% dextrose, 2 % agar) and the plates were incubated at 29°C for 72-96 h. Colonies obtained on the MD plates were resuspended in autoclaved ddH2O, centrifuged at 3000 xg for 10 minutes and after discarding supernatant, remaining cells were plated on YPD (1% yeast extract, 2% peptone, and 2% dextrose, 1.5 % agar) plates containing increasing concentration of Geneticin sulphate (G418) antibiotic. The plates were allowed to grow for 4-5 days and the colonies present in the plate containing the highest concentration of G418 were screened for the expression of BiSpekDAb. For screening, selected clone (obtained above) was inoculated in 10 ml of BMGY (Buffered complex glycerol medium containing 1% yeast extract, 2% peptone, 1% glycerol, 1.34 % yeast nitrogen base without amino acids, without ammonium sulphate), 0.1 M phosphate buffer of pH 5.8 media containing 10 pg / ml kanamycin and 100 pg / ml ampicillin and the cultures were grown for 24 h at 29°C, 250 rpm. Subsequently, 1% culture was transferred to the 500 ml of BMGY media and grown further at 29 C for 48 hours. The cultures were then centrifuged at 5000 rpm for 5 minutes to remove BMGY media and the cell mass was washed with BMMY (Buffered methanol -complex medium containing 1% yeast extract, 2% peptone, 1.34 % yeast nitrogen base without amino acids without ammonium sulphate, 0.1 M phosphate buffer pH 5.8 and 1% methanol) and resuspended in fresh BMMY media. The cultures were grown further at 29 C and induced with 1% methanol every 24 hours for 5 days. After this, the cultures were centrifuged (3500 rpm for 10 minutes) and the cell mass was separated from the cultivating media. The presence of BiSpekDAb in cultivating media was determined by western blot analysis by using mouse anti-His antibody (Cat. No. AE003, Abclonal, USA; 1:5000 diluted in TBST) as a primary antibody. Clones showing maximum expression of targetrecombinant protein were then selected and their glycerol stocks were made for storage at - 80°C. These stored recombinant P. pastoris cells were used further. d) Production of BiSpekDAb'. For production of BiSpekDAb polypeptides, glycerol stocks of recombinant P. pastoris cells (obtained above) were streaked on YPD plate containing kanamycin and ampicillin for 24 hours at 29 C, and further single colony was selected from the streaked plate and cultured for expression of BiSpekDAb by following the procedure essentially described above. After 5 days of induction of the culture with methanol, the cultivating media was collected and subjected to affinity chromatography using of Ni2+-NTA resin (50 ml). Elution of the bounded protein was done in a 100 mM phosphate buffer containing 300 mM imidazole and 250 mM NaCl and the eluted fractions were collected and analyzed for protein content using Bradford assay.Results: Representative chromatogram of purification of BiSpekDAb polypeptide is shown in Figure ID. Fractions containing protein were pooled and dialyzed against a 20 mM PBS buffer containing 150 mM NaCl, pH 7.4. Dialyzed sample was then subjected to endotoxin removal step by using High-Capacity Endotoxin Removal Spin Columns (Cat # 88274, Invitrogen, USA) and concentrated using centrifugal filter units. A concentrated protein sample was then made 10% glycerol (final concentration) and stored at -80 C for further use. Purity of this sample was checked by SDS-PAGE analysis and a representative result is given in Figure IE. Single protein band corresponding to desired molecular weight confirms the purity of BiSpekDAb. BiSpekDAb polypeptide produced using this procedure remains biologically active and stable for a long period of time, and the final prototype is being developed (Figure IF).

[0136] Example 2: Determination of antigen binding affinity and blocking activity of BiSpekDAb by in-vitro methods.BiSpekDAb has bispecific binding affinity to both human TNF-a and IL-23. Binding affinity to human TNF-a and human IL-23 performed by solid-phase ELISA, surface plasmon resonance, and cellular assay. Commercially available human TNF-a and IL-23 without HISe tag was used to confirm the binding of BiSpekDAb. a) Binding affinity of BiSpekDAb to surface coated TNF-a as determined by Solid-Phase ELISA: BiSpekDAb was produced as described above and solid-phase ELISA was carried out to determine its binding to TNF-a. Briefly, wells of 96-well polystyrene plates were coated overnight at 25 °C with lOOpL of 20 ng / ml of hTNF-a (Elab Sciences Biotechnology Inc, Cat #PKSH033490) in phosphate buffer (50 mM, pH 7.4). After washing the wells with PBST(phosphate buffered saline containing 0.05% (v / v) Tween-20), the wells were blocked with PBS containing 3% (w / v) bovine serum albumin (BSA) for 2 h at 25 °C. After PBST washing, wells were incubated with desired concentrations of purified BiSpekDAb (diluted in PBS buffer) 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 mouse anti-His antibody (Cat. No. M1001020, G-Biosciences, USA; 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 thrice with PBST and then developed by incubating with substrate, 3.3',5,5'-tetramethylbenzidine (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.Results: Using the above mentioned procedure, manually human TNF-a was successfully coated on polystyrene 96-well plate and BiSpekDAb binds to surface coated TNF-a in a dose dependent manner, as represented in Figure 2A. BiSpekDAb produced by the selected clones is active and has good binding affinity to the human TNF-a. b) Binding affinity of BiSpekDAb to surface coated IL-23 as determined by Solid-Phase ELISA: BiSpekDAb was produced as described above and solid-phase EUISA was carried out to determine its binding to hIU-23. Briefly, wells of 96-well plates were coated overnight at 25 °C with lOOpU of 20 ng / ml of IU-23 (ProSpecBio, Cat #cyt-050) 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 PBST washing, wells were incubated with desired concentrations of purified BiSpekDAb (diluted in PBS buffer) 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 mouse anti-His antibody (Cat. No. Ml 001020, G-Biosciences, USA; 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 thrice with PBST and then developed by incubating with substrate, 3.3', 5,5'- tetramethylbenzidine (Cat. No. Abl71523, Abeam, UK) for 30 min. Color development wasstopped by adding 100 pl / well of stop solution (0.2 M H2SO4) and the absorbance was measured at 450 nm using a microplate reader.Results: Using the above mentioned procedure, manually human IL-23 was successfully coated on a polystyrene 96-well plate and BiSpekDAb binds to surface coated IL-23 in a dose dependent manner, as represented in Figure 2A. BiSpekDAb produced by the selected clones is active and has good binding affinity to the human IL-23. c) Determination of inhibition ofTNF-a induced cytotoxicity in L929 cells'.Inhibition of TNF-a activity by BiSpekDAb was studied by monitoring inhibition of TNF-a induced cytotoxicity of L929 cells. L929 cells are mouse fibroblast cells that naturally express TNF-a receptors and are highly sensitive to human TNF-a treatment. Incubation of L292 cells with TNF-a results in binding of TNF-a to its receptors and induction of cytotoxicity in L929 cells. Cell death can be experimentally measured by using standard MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay.For assay, L929 cells (obtained from National Centre for Cell Science, Pune, India) were grown in DMEM medium (Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum, 1% glutamine and 1% penicillin / streptomycin solution) in a humidified environment having 5% CO2. Approximately 10k cells were seeded in each well of 96-well plate and incubated for 24 h at 37 °C (under a humidified atmosphere and 5% CO2). Stock solution of BiSpekDAb or Adalimumab (Exemptia®; Cadila Healthcare Ltd, Gujarat, India; Batch # B100040) was diluted in DMEM media containing 1.5 nM hTNF-a, to obtain desired concentrations. This mixture was then added to the wells containing L929 cells and the plates were incubated for further 24h. DMEM media containing 1.5 nM hTNF-a only was considered as the control group. After incubation, 0.5 mg / mL of MTT (final concentration) was added to the wells and the plates were incubated for further 4h. After this, the supernatant in the wells were discarded and 100 ul of dimethyl sulfoxide (87.5%) was added to the wells and the plates were further incubated for 40 min at 25°C with shaking. The absorbance was measured at 570 nm and the data was used to calculate percentage inhibition ofTNF-a mediated cytotoxicity by BiSpekDAb or adalimumab. Four independent experiments were done in triplicates. The statistical significance was determined by one-way AN OVA using the SigmaPlot program (version 14.0, SPSS Inc.).Results: Compared to untreated cells, addition of hTNF-a resulted in cytotoxicity of L929 cells. Increasing concentration of adalimumab (which was used as a positive control in this experiment) resulted in inhibition of TNF-a mediated cytotoxicity. Under similar conditions,increasing concentration of BiSpekDAb also resulted in a dose-dependent inhibition of TNF-a mediated cytotoxicity of L929 cells (Figure 2B). c) Determination of inhibition of IL-23 induced proliferation of Caco-2 cells'. Inhibition of IL- 23 activity by BiSpekDAb was studied by monitoring inhibition of IL-23 induced proliferation of Caco-2 cells. Caco-2 cells are human epithelial cells from colon tissue that are highly proliferative in response to human IL-23 treatment. Incubation of Caco-2 cells with IL-23 results in binding of IL-23 to its receptors and induction of proliferation of Caco-2 cells. Cell growth can be experimentally measured by using standard MTT (3-(4,5-Dimethylthiazol-2-yl)- 2,5-Diphenyltetrazolium Bromide) assay.For assay, Caco-2 cells (obtained from National Centre for Cell Science, Pune, India) were grown in DMEM medium (Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum, 1% glutamine and 1% penicillin / streptomycin solution) in a humidified environment having 5% CO2. Approximately 50000 cells were seeded in each well of 96-well plate and incubated for 24 h at 37° C (under a humidified atmosphere and 5% CO2). Stock solution of BiSpekDAb or ustekinumab (ProteoGenix #PX-TA1011) was diluted in DMEM media containing 100 ng / ml hIL-23, to obtain desired concentrations. This mixture was then added to the wells containing Caco-2 cells and the plates were incubated for further 24h. DMEM media containing 100 ng / ml hIL-23 only was considered as the control group. After incubation, 0.5 mg / mL of MTT (final concentration) was added to the wells and the plates were incubated for further 4h. After this, the supernatant in the wells were discarded and 100 ul of dimethyl sulfoxide (87.5%) was added to the wells and the plates were further incubated for 40 min at 25 °C with shaking. The absorbance was measured at 570 nm and the data was used to calculate percentage inhibition of TNF-a mediated cytotoxicity by BiSpekDAb or ustekinumab. Three independent experiments were done in triplicates. The statistical significance was determined by one-way ANOVA using the SigmaPlot program (version 14.0, SPSS Inc.).Results: Compared to untreated cells, addition of hIL-23 resulted in an increase in the proliferation of IL-23 cells. Increasing concentration of ustekinumab (which was used as a positive control in this experiment) resulted in inhibition of IL-23 mediated proliferation. Under similar conditions, increasing concentration of BiSpekDAb also resulted in a dosedependent inhibition of IL-23 mediated proliferation of Caco-2 cells (Figure 2C).

[0137] Example 3: Beneficial effect of BiSpekDAb in IL23 and TNF-a related diseases (OVA + LPS induced asthma in rat)AnimalsAll the experiments were performed on female adult Wistar rats (200-260g; 8-9 weeks old). The animals were procured from the Central Animal Facility, National Institute of Pharmaceutical Education and Research (NIPER), S.A.S. Nagar, India. The housing of the animals was done as three animals per cage and was acclimated for 7 days before experimentation to standard laboratory conditions in a room with controlled ambient temperature (22 ± 2°C), humidity 45 ± 10 %, and light / dark cycle (12 h light- 12 h dark and had free access to food and water. All experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC no: 23 / 36) and were performed in accordance with the Committee for the Purpose of Control and Supervision of Experimentation on Animals (CPCSEA) guidelines. a) Scheme for the development of OVA + LPS induced asthmatic rat model and determination of therapeutic efficacy of BiSpekDAh in this model: Severe asthma in rats were induced by sensitization and challenge with the OVA (Ovalbumin) and LPS (lipopolysaccharides) (Figure 3A).Sensitization by ovalbumin and LPS: Brief, rats were first sensitized with an intraperitoneal injection of ovalbumin (1 mg / mL) (Cas No. 9006-59-1) in combination with aluminum hydroxide (Cas No. 21645-51-2) as an adjuvant to enhance the sensitivity and immune response and followed by LPS (10 ug / kg) (Cas No. 93572-42-0) intraperitoneal injection after 1 hr on days 1, 7, and 14.Intranasal challenge with ovalbumin and LPS: Onward to the sensitization phase, rats were put on the challenge phase where animals were challenged with 1% (w / v) OVA by placing them inside a closed glass chamber (70cm dia. * 40 cm height) and nebulizing the prepared suspension by inhalation route for 30 minutes every day from day 15 to 25. After 1 hr of OVA inhalation, rats were intraperitoneally injected with LPS (10 ug / kg) every day with OVA challenge. Commercially available Dexamethasone (DXM) (Cadila Pharmaceuticals) (2 mg / kg, s.c) administered subcutaneously every day from the 15thto the 25thday of the experiment as a first-line drug for asthma treatment so it is a positive control of the study. Animals administered with BiSpekDAb (1 mg / kg, s.c) subcutaneously on alternate days (15th, 17th, 19th, 21st, 23rd, 25th) of the challenging phase.On the 26thday of the model, various Lung Functional Parameters (LFPs) of each animal were recorded using Whole Body Plethysmograph (Buxco Fine Pointe, DSI). On the 27thday, a methacholine challenge test was performed which is the confirmatory test for asthma. Thestudy was terminated on the 28thday with the sample collection (Blood, BALF, Lung tissue), and euthanasia with urethane (0.6 g / ml) (Cas No. 51-79-6). b) BiSpekDAb enhance lung functions and improve OVA + LPS induced airway hyperresponsiveness (AHR) in female Wistar rats: To investigate the beneficial effect of BiSpekDAb in asthmatic rats, lung functions were measured recorded on day 26thof model. Respiratory rate, and mid-expiratory flow rate were significantly high and the inspiratory time was decreased in the asthmatic group, which shows the establishment of poor lung function in OVA + LPS treated rats, representing an asthmatic condition. BiSpekDAb treated groups normalize the lung function, significantly decrease the respiratory rate and mid-tidal expiratory flow rate up to the basal level similar to the healthy control group, which is also the same in the DXM group (positive control). BiSpekDAb also significantly improved the inspiratory flow rate which was reduced in the asthmatic disease group. Results show that BiSpekDAb (1 mg / kg) improves lung function parameters (Figure 3C). To check the airway hyper responsiveness (AHR) in response to methacholine chloride, rats were challenged with different concentrations of Meh, as a potent bronchoconstrictor. Asthmatic group animals show higher Penh values with increasing doses of Meh, and show higher bronchoconstriction, confirming the airway hyperresponsiveness of the lung, while the BiSpekDAb (1 mg / kg) treated animals have significantly lower Penh values similar to a healthy group, and the DXM treated groups, represents BiSpekDAb normalizes the lung responsiveness (Figure 3B). Collectively, these results showed that BiSpekDAb enhances the lung overall health or lung functionality. c) BiSpekDAb reduce OVA + LPS induced airway inflammation in asthmatic rats: OVA + LPS is known to induce both eosinophilic and neutrophilic asthma in animals characterized by the increase in the level of total leukocytes (TLC) (cells / cu.mm), and also results in increase in level of DLC (differential cell count, % of eosinophils and % of neutrophils) in both blood circulation and in the BALF. In our experimental model, blood parameters as TLC, as well as DLC, were measured and the level of both DLC and TLC were significantly increased in response to asthma inducers OVA + LPS due to recruitment and infiltration of various immune cells that shows the establishment of ideal asthmatic disease conditions. BiSpekDAb (1 mg / kg) group greatly reduced the counts as well as the percentage of particular immune cells to the basal level similar to healthy control group animals which also reflected in DXM; positive control group of animals in blood (Figure 3D) and BALF (Figure 3E). Nitrite level and MPO activity was biochemically measured in the lung tissue lysates and the level of NO and MPOactivity was significantly high in the OVA + LPS induced asthmatic group because of the lung inflammation while the level of NO and MPO activity was reduced to basal level in BiSpekDAb animals similar to the DXM group (Figure 3F). Increase in the systemic circulatory immune cells (Neutrophils and Eosinophils) and particularly infiltration of eosinophils and neutrophils in the lung BALF, and the increase NO and MPO activity in lung tissue confirms the inflammatory condition of the lungs in OVA + LPS induced inflammatory asthma while this level significantly reduced to the basal level in response to BiSpekDAb, collectively confirms that BiSpekDAb greatly reduce systemic as well as the airway inflammation. d) BiSpekDAb prevent the side effects of the commercially available anti-inflammatory Dexamethasone (DXM) drug in OVA + LPS induced asthmatic rats: Body weight of each animal was measured and the change in body weight was observed to be decreased due to exposure to OVA + LPS in the disease group but not significantly, but the change in weight of DXM group; positive control group was observed to be significantly decreased in comparison to healthy control as well as BiSpekDAb (1 mg / kg) treated animals (Figure 3G). Dexamethasone (DXM), a glucocorticoid that is a first-line drug for asthma, has a side effect of inducing metabolic disturbance which also represented the weight reduction in the DXM- treated groups. However, BiSpekDab is a novel antibody that blocks proinflammatory TNF-a, and IL-23 cytokines simultaneously and improves the lung physiological conditions as well as there is no change in the body weight of the BiSpekDab treated animals. Thus, BiSpekDAb could be a novel biologic for the treatment of asthma with lesser side effects unlike chemical drugs such as dexamethasone.

[0138] A skilled artisan will appreciate that the quantity and type of each ingredient can be used in different combinations or singly. All such variations and combinations would be falling within the scope of present disclosure.

[0139] The foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.ADVANTAGES OF THE INVENTION• The present invention provides an engineered BiSpekDAb which is a bispecific anti- IL-23 / TNF-a domain antibody, capable of inhibiting interleukin 23 (IL-23) and tumor necrosis factor-alpha (TNF-a).• The present invention provides double benefit by inhibiting two cytokines TNF-a and IL-23 simultaneously.• The present invention provides a domain antibody which are most stable in diverse range of pH and temperature conditions, less immunogenic, high nano-to-pico molar affinity, and very small in size as compare to conventional full-length antibody, and it is fused with a half-life extension partner that will improve the pharmacokinetics.• The present invention is useful in the treatment of various inflammatory and autoimmune diseases like Inflammatory Bowel Disease (IBD) including both Ulcer Colitis (UC) and Crohn’s Disease (CD), Lung inflammation (Asthma, SARS-Covid), Psoriasis, Psoriatic Arthritis (PA), Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), Ankylosing Spondylitis (AS), etc.

Claims

We Claim:

1. A bispecific domain antibody comprising of polypeptide sequence as set forth in SEQ ID NO: 1, wherein the said bispecific domain antibody is an IL-23 and a TNF-a blocking domain antibody.

2. The bispecific domain antibody as claimed in claim 1, wherein the polypeptide sequence comprises an anti-IL-23 domain antibody polypeptide, an anti-TNF-a domain antibody polypeptide, a non-antibody polypeptide, a linker polypeptide and (His)6-tag.

3. A polynucleotide sequence encoding bispecific domain antibody of SEQ ID NO: 1 as claimed in claim 1, as set forth in SEQ ID NO: 2 comprising: a. a polynucleotide sequence comprising a nucleic acid encoding an anti-IL-23 domain antibody polypeptide and anti-TNF-a domain antibody polypeptide; b. a polynucleotide sequence comprising a nucleic acid encoding a non-antibody polypeptide selected from the group consisting of human serum albumin (HSA) or its analogs or its fragment / portion, human serum albumin targeting polypeptide (HSATP) or its analogs or its fragment / portion, human chorionic gonadotropin (hCG) hormone or its fragment / portion, elastin or its analogs or its fragment / portion, gelatin or its analogs or its fragment / portion, al- antitrypsin or its analogs or its fragment / portion, immunoglobulin (Ig) or its analogs or its fragment / portion, and non-natural polymeric amino acid sequences (NNPAAS); c. a polynucleotide sequence comprising a nucleic acid encoding a linker polypeptide; and d. a polynucleotide sequence comprising a nucleic acid encoding a (His)e-tag, wherein, the nucleic acid construct encodes a bispecific domain antibody of SEQ ID NO: 1 with (His)e-tag at either C-terminal or N-terminal.

4. The polynucleotide sequence as claimed in claim 3, wherein the non-antibody polypeptide is an analog or fragment / portion of human serum albumin.

5. The polynucleotide sequence as claimed in claim 3, wherein the non-natural polymeric amino acid sequences (NNPAAS) are selected from but not limited to alanine, glutamic acid, glycine, proline, serine and threonine or proline, alanine, and serine or glycine and serine.

6. A recombinant plasmid for cloning and expression of bispecific domain antibody of SEQ ID NO: 1 as claimed in claim 1, comprising a polynucleotide sequence of SEQ ID NO: 2 as claimed in claim 3.

7. The recombinant plasmid as claimed in claim 6, wherein the recombinant plasmid comprising polynucleotide sequence construct of SEQ ID NO. 2 for secretory expression of bispecific domain antibody of SEQ ID NO. 1 in host cells, wherein the construct comprises of an arrangement having: a) a first polynucleotide comprising a nucleic acid encoding an anti-IL-23 domain antibody polypeptide; b) a second polynucleotide sequence encoding a linker; c) a third polynucleotide sequence comprising a nucleic acid encoding a nonantibody polypeptide; d) a fourth polynucleotide sequence encoding a linker; e) a fifth polynucleotide sequence comprising a nucleic acid encoding an anti- TNF-a domain antibody; and f) a polynucleotide comprising a nucleic acid encoding a (His)6-tag at either C- or N-terminus.

8. The recombinant plasmid as claimed in claim 6, wherein the host cells are selected from the group consisting of bacterial cells, yeast cells, mammalian cells and insect cells.

9. The recombinant plasmid as claimed in claim 6, wherein the host cells are yeast cells.

10. A method for the production of bispecific domain antibody of SEQ ID NO: 1 as claimed in claim 1, wherein the method comprising the steps of: i. preparing recombinant plasmid as claimed in claim 6, containing polynucleotide sequence of SEQ ID NO: 2 as claimed in claim 3 for the secretory expression of bispecific domain antibody of SEQ ID NO: 1; ii. transforming the host cells as claimed in claim 8, with recombinant plasmids of step (i) to obtain recombinant host cells; iii. culturing the recombinant host cells of step (ii) in culture media for the growth of recombinant host cells; iv. inducing the secretary expression of bispecific domain antibody of SEQ ID NO: 1 by treating the culturing recombinant host cells of step (iii);v. isolating and purifying the bispecific domain antibody of SEQ ID NO: 1 from the culture media of step (iv) by using affinity chromatography; vi. removing endotoxin from the purified bispecific domain antibody of SEQ ID NO: 1, by using endotoxin removal columns; and vii. reconstituting the purified bispecific domain antibody of step (vi) for longterm storage and followed by in-vitro characterization of bispecific domain antibody of SEQ ID NO: 1.

11. The method as claimed in claim 10, wherein the recombinant host cells are recombinant Pichia pastoris SMD 1168 cells.

12. The method as claimed in claim 10, wherein the culture media is selected from a group consisting of YPD (Y east extract, Peptone, Dextrose compositions), BMGY (Medium comprises peptone, yeast extract, yeast nitrogen base, glycerol and a phosphate buffer), and BMMY (Medium comprises peptone, yeast extract, yeast nitrogen base, methanol and a phosphate buffer).

13. The method as claimed in claim 10, wherein treating the culturing host cells of step (iv) with 1-5% methanol followed by further culturing of host cells at 29°C for 3-5 days.

14. The method as claimed in claim 10, wherein the long-term storage of bispecific domain antibody of SEQ ID NO: 1 in step (vii) is done at -80°C by adding 10% glycerol.

15. The method as claimed in claim 10, wherein the bispecific domain antibody of SEQ ID NO: 1 binds to IL-23 and TNF-a with high affinities and inhibits IL-23 and TNF-a activities.

16. A pharmaceutical composition comprising a bispecific domain antibody of SEQ ID NO: 1 as claimed in claim 1 and a pharmaceutically acceptable carrier, excipient, or diluent.

17. The pharmaceutical composition as claimed in claim 16, wherein the pharmaceutical composition is in the form of a tablet, a capsule, a solution, a gel, a suspension or a powder.

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