WO2022195629A1 - Engineered arginase constructs, method of generation and uses thereof - Google Patents

Abstract

The present invention provides engineered arginase construct preferably engineered human arginase (EHA) polypeptide constructs comprising human arginase 1 polypeptide fused to non-arginase polypeptides via a linker peptide. The invention also provides novel polynucleotide sequences encoding EHA polypeptide constructs. The invention also provides a novel method to produce the EHA polypeptide constructs in highly pure and active form in high yield. The EHA disclosed in the invention can be used to deplete arginine and to treat arginase 1-associated diseases or conditions.

Description

“ENGINEERED ARGINASE CONSTRUCTS, METHOD OF GENERATION

AND USES THEREOF”

FIELD OF INVENTION:

The present invention relates to the broad field of Biotechnology. More specifically, the present invention relates to engineered polypeptide constructs comprising human arginase 1 fused to a non-arginase polypeptide. The present invention also relates to the method of preparing these constructs and their uses.

BACKGROUND OF THE INVENTION:

The amino acid arginine (2-amino-5-guanidinovaleric acid) is classified as a semi-essential or conditionally essential amino acid, subject to conditions such as the developmental stage and health of an individual. Arginine is a very versatile amino acid, and participates in a number of metabolic pathways. Apart from being a component of many proteins, it plays a vital role within the body in several physiological and pathophysiological processes such as in the synthesis of nitric oxide and polyamines, cell viability and proliferation, wound healing, hormone biosynthesis, ammonia disposal and immune response. Biologically available arginine may be obtained from recycling of amino acids, from dietary intake, as well as de novo synthesis. In general, it is typically required in growth and development of infants. Further, arginine requirements can be significantly elevated under conditions of stress, rapid growth or during certain pathological conditions, requiring external arginine supplementation. Moreover, cells which lack the enzymes involved in the synthesis of arginine are dependent on its import from the extracellular fluid. Due to its significant roles in various vital biological functions, arginine and its metabolism have garnered a lot of interest as a potential therapeutic target.

Three enzymes in the cells play important role in the catabolism of arginine: arginase 1, arginase 2 and nitric oxide synthase. Nitric oxide synthase catabolizes arginine into nitric oxide species, and arginase 1 and 2 catabolize arginine into urea and ornithine. Although similar in function, these 2 enzyme isoforms have considerable differences in their tissue distribution and molecular characteristics. Both enzymes share ~ 60% homology. Arginase 1, a 35 KDa protein, is present in the cytoplasm of liver cells, while arginase 2, a ~ 38.5 KDa protein, is located in the mitochondria of many cells. Arginase 1 is important to the body functions and arginase 1 knock-out animals exhibit lethal phenotype. Moreover, congenital arginase 1 deficiency in humans also leads to neuro- and metabolic-deficiencies. However, arginase 2 knock-out animals exhibit minimal physiological consequences.

Arginase I is a homotrimeric metalloenzyme and comprises of Mn²⁺ metal ion center for each subunit. Mn²⁺ participates in the catalysis by producing a metal-bound OH^" (from water molecule) which acts as a nucleophile in attacking guanidium carbon of the substrate arginine. However, arginase 1 in which Mn²⁺ is substituted with Co²⁺ exhibited improved catalytic activity, serum stability, and plasma half-life.

Many cancerous cells either lack or produce low levels of enzymes responsible for the synthesis of arginine and thus depend on the import of arginine form the extracellular fluid for their survival and maintenance. Depletion of arginine by administering arginine-hydrolyzing enzyme has been shown to inhibit the growth and progression of such arginine-auxotrophic cancers. Arginine-hydrolyzing enzymes thus have emerged as a potential candidate to selectively target and destroy such arginine-auxotrophic cancers.

Arginine-hydrolyzing enzyme mediated depletion of arginine has also emerged as a potential novel approach for treating diseases/conditions in which lowering of arginine concentration has been shown to provide therapeutic effects.

PEGylated recombinant arginine deiminase (ADI-PEG20), an enzyme from mycoplasma, is one such enzyme that lowers plasma arginine concentration efficiently and is approved for the cancer therapy. ADI-PEG20 is designated US orphan drug status by US-FDA for the treatment of hepatocellular carcinoma and invasive malignant melanomas. However, the clinical use of ADI-PEG20 suffers from several serious limitations· The enzyme hydrolyses arginine into citrulline and ammonia and due to excessive generation of ammonia, the onset of hyperammonemia is reported in many patients receiving ADI-PEG20 therapy. Also, being microbial in origin, ADI is highly immunogenic and elicit strong neutralizing antibody responses in the recipients. As an alternative to microbial ADI, recombinant human arginase 1 (rhArgl) is being developed for the treatment diseases/conditions where depletion of arginine has been shown to have a therapeutic effect. However, a major impediment of developing human arginase I for therapeutic use is its short circulating half-life (i.e., poor pharmacokinetics) due to its low molecular weight and fast renal clearance.

Different approaches have been adopted to improve the pharmacokinetic properties of human arginase 1, including its conjugation with polyethylene glycol (PEG) and its engineering by fusion with non-arginase proteins. These approaches have shown improvement in the pharmacokinetic properties of human arginase 1.

PEGylated-rhArg variants exhibit potent anticancer activity against a variety of arginine- auxotrophic cancers (US 2014/0363417 Al; US 8,440,184 B2; Yu et al, 2021). The PEGylated- rhArg variants also exhibited beneficial effect in diseases/conditions in which lowering of arginine has therapeutic effects. However, clinical use of PEGylated-proteins are known to have several severe safety and other concerns, viz., PEG toxicity, immunogenicity and hypersensitivity to PEG, problems due to heterogeneity of PEGylated proteins and coast of producing PEGylated proteins.

Another approach to improve the pharmacokinetics is to generate engineered human arginase 1 in which the enzyme is fused to a non-arginase protein. Fusion with non-arginase protein improves the properties of engineered human arginase 1 by either increasing the Fc neonatal receptor (FcRn) mediated recirculation and/or increasing the overall hydrodynamic radius/negative charge on the engineered protein resulting in its low renal clearance.

While fusion peptides for other proteins 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 require considerable human intervention and biotechnological skills.

Further, although fusion arginase 1 polypeptides have been previously attempted, these have one limitation or the other, as discussed below. A comparison of the available art and the present invention is given in Table 1. Table 1: Comparison of documents in the field with present invention.

As evident from the discussion above, although fusion/chimeric molecules for other enzymes may be known, engineered human arginase I having good specific activity is not taught or suggested by available literature. Thus, in view of the available literature and in view of the level of unpredictability and difficulty in generating fusion/chimeric molecules, there is a need to develop alternative arginase with improved properties including enhanced arginine- hydrolyzing activity. The present invention attempts to fill this gap in the art.

OBJECTS OF THE INVENTION:

One object of the present invention is to provide engineered arginase polypeptide constructs (SEQ ID No.: 33-62) in which human arginase 1 is fused to a non-arginase polypeptide.

Another object of the present invention is to provide engineered arginase polypeptide constructs that exhibit arginine-hydrolyzing activity. Another object of the present invention is to provide engineered arginase polypeptide constructs that exhibit enhanced arginine-hydrolyzing activity, compared to wild-type human arginase 1 enzyme reported in the art.

Another object of the present invention is to provide amino acid sequences of engineered arginase polypeptide constructs (SEQ ID No.: 33-62).

Yet another object of the present invention is to provide novel nucleotide sequences encoding for engineered arginase polypeptide constructs (SEQ ID No.: 2-31).

Another object of the present invention is to provide novel recombinant plasmids encoding gene for engineered arginase polypeptide constructs.

Yet another object of the present invention is to provide novel recombinant bacteria for the production of engineered arginase polypeptide constructs.

Still another object of the present invention is to provide a novel method for the production of engineered arginase polypeptide constructs in highly pure and active form in high yield.

SUMMARY OF THE INVENTION:

The present invention overcomes a major deficiency in the art by providing engineered polypeptide arginase constructs with improved arginine hydrolyzing activity. The engineered construct described herein are useful for the treatment of diseases or conditions in which arginine depletion has a therapeutic effect, such as in the treatment of cancer, viral infections, multiple sclerosis, rheumatoid arthritis, autoimmune diseases, congenital hyperargininemia, graft-versus-host disease (GvHD), inflammation, obesity, and metabolic disorders and related complications and comorbidities. The engineered construct can be used alone or in combination with at least one other agent to give a synergistic effect on disease treatment or prevention.

In a first aspect, provided herein are engineered polypeptide constructs comprising an arginase polypeptide fused to a non-arginase polypeptide via a linker peptide. The arginase employed in the construct is a human arginase 1 polypeptide. In a first embodiment of the first aspect, provided herein is the engineered polypeptide construct of the first aspect, wherein the engineered polypeptide construct comprises a polypeptide sequences as set forth in SEQ ID NO: 33-62

In a second embodiment of the first aspect, provided herein is the engineered polypeptide construct of the first aspect, wherein the engineered polypeptide construct is an Engineered Human Arginase (EHA) Polypeptide Construct.

In a third embodiment of the first aspect, provided herein is the engineered polypeptide construct of the first aspect, wherein non-arginase polypeptide can be selected from the group of polypeptides comprising: 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 transferrin (Tf) or its analogs or its fragment/portion human chorionic gonadotropic (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; 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

In a fourth embodiment of the first aspect, provided herein is the engineered polypeptide construct of the first aspect, wherein the C-terminus of the human arginase 1 polypeptide is fused to the N-terminus of the non-arginase polypeptide via a linker peptide.

In a next embodiment of the first aspect, provided herein is the engineered polypeptide construct of the first aspect, wherein the N-terminus of the human arginase 1 polypeptide is fused to the C-terminus of the non-arginase polypeptide via a linker peptide. In a fifth embodiment of the first aspect, provided herein is the engineered polypeptide construct of the first aspect, wherein the construct encodes by recombinant polynucleotides sequences as set forth in SEQ ID NO: 2-31.

In a sixth embodiment of the first aspect, provided herein is the engineered polypeptide construct of the first aspect, wherein the construct encodes by recombinant polynucleotides such that the said polynucleotide encodes at least one polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 33-62.

In a second aspect, provided herein is a method for production of the engineered construct comprises of following steps - a. designing EHA gene, b. culturing the host cells expressing EHA polypeptides, c. inducing high level of expression of EHA polypeptides, d. collecting the cell mass expressing EHA polypeptides, e. lysing the cells, f. purifying the inclusion bodies containing EHA polypeptides, g. solubilizing the inclusion bodies with the help of chaotropic agents, h. refolding the EHA polypeptides using refolding buffer, i. isolating the enzymatically active EHA polypeptides using chromatography, j. obtaining enzymatically active EHA polypeptides.

In a first embodiment of the second aspect, provided herein is a method for production of the engineered construct wherein the chaotropic agent is selected from the group comprising organic compounds, solvents, salts, detergents and is preferably guanidium; wherein the refolding buffer comprises at least one buffering agent selected from the group of Tris-HCl, CHES, EPPS, HEPES, Glycine-NaOH, Phosphate, TAPS, MOPS, and MES. wherein the refolding buffer comprises at least one buffering agent selected from the group of Tris-HCl, CHES, EPPS, HEPES, Glycine-NaOH, Phosphate, TAPS, MOPS, and MES. wherein the refolding buffer may further comprise of a mixture of at least one redox pair selected from a group of reduced glutathione/oxidized glutathione; oxidized nicotinamide dinucleotide/reduced nicotinamide dinucleotide and cy stamine/cy steamine wherein the refolding buffer further comprises at least one refolding additives selected from the group of salts, sugars, polymers, polyols, detergents and surfactants.

In a second embodiment of the second aspect, provided herein is a method for production of the engineered construct wherein the said polypeptides are derived from at least one polynucleotide sequence selected from the group comprising SEQ ID NOs: 2-31, wherein said polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 33-62, wherein said method results into high level expression of EHA polypeptides as enzymatically non-functional aggregated inclusion bodies, wherein said method further comprises the step of inducing the host cell culture with 0.1-2.0 M IPTG concentration and growing the cultures for 4-24 h at 37°C.

In a third embodiment of the second aspect, provided herein the method for production of the engineered construct wherein the purity of enzymatically active polypeptides is at least 80%.

In a third aspect, provided herein is a method of production of enzymatically active EHP polypeptides from inclusion bodies, wherein said polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 29-54, wherein said method comprises the steps of: a. purifying the inclusion bodies containing EHP polypeptides, b. solubilizing the inclusion bodies containing EHP polypeptides with the help of guanidium, c. refolding the denatured EHP polypeptides by diluting in refolding buffer, d. isolating the enzymatically active EHP polypeptides using chromatography, e. obtaining the enzymatically active EHP polypeptides.

In a first embodiment of the third aspect, provided herein is a method of production of enzymatically active EHP polypeptides from inclusion bodies, wherein the refolding buffer comprises at least one buffering agent, at least one redox pair, at least one cofactor, at least one refolding additives, or mixture thereof; the buffering agent is selected from the group of Tris- HC1, CHES, EPPS, HEPES, Glycine NaOH, Phosphate, TAPS, MOPS, and MES, the redox pair is selected from a group of reduced glutathione / oxidized glutathione; oxidized nicotinamide dinucleotide / reduced nicotinamide dinucleotide and cystamine / cysteamine, the cofactor is selected from the group of CaCI₂, C0CI2, MgCI₂, and ZnCI₂ and the refolding additives are selected from the group of salts, sugars, polymers, polyols, detergents and surfactants.

In a second embodiment of the third aspect, provided herein is a method of production of enzymatically active EHP polypeptides from inclusion bodies, wherein the purity of enzymatically active EHP polypeptides is at least 80%.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS OR FIGURES:

The accompanying drawings illustrate some of the embodiments of the invention and, together with the description, serve to explain the invention. These drawings are offered by way of illustration and not by way of limitation.

Figure 1 is a cartoon showing engineered arginase polypeptide construct of this invention and its components.

Figure 2 Panel A and B show a cartoon of gene encoding for engineered arginase polypeptide constructs of this invention and the recombinant expression plasmid containing the said gene, respectively.

Figure 3 is a picture of agarose gel electrophoresis of recombinant expression plasmids with and without restriction digestion. Double digested recombinant expression plasmids were resolved on 1% agarose gel. Legends: Lane 0: DNA marker, Lane 1: recombinant expression plasmids, Lane 2: double digested recombinant expression plasmids. Panel A - rhArg 1 ; Panel B - EHA polypeptide construct 3; Panel C - EHA polypeptide construct 4; Panel D - EHA polypeptide construct 5; Panel E - EHA polypeptide construct 6; Panel F - EHA polypeptide construct 7; Panel G - EHA polypeptide construct 8; Panel H - EHA polypeptide construct 11; Panel I - EHA polypeptide construct 12; Panel J - EHA polypeptide construct 13; Panel K - EHA polypeptide construct 14; Panel L - EHA polypeptide construct 15; Panel M - EHA polypeptide construct 16.

Figure 4 shows images of SDS-PAGE (Panels A) and western blot analysis (Panels B) of insoluble debris fractions of recombinant E. coli cells expressing EHA polypeptide constructs. The western blots were developed using monoclonal mouse anti-human arginase 1 antibodies as primary antibodies. Legends: Lane 0: Protein molecular weight markers; Lane 3 - EHA polypeptide construct 3; Lane 4 - EHA polypeptide construct 4; Lane 5 - EHA polypeptide construct 5; Lane 7 - EHA polypeptide construct 7; Lane 12 - EHA polypeptide construct 12; Lane 13 - EHA polypeptide construct 13; Lane 15 - EHA polypeptide construct 15; Lane 16 - EHA polypeptide construct 16.

Figure 5 Panel A shows a scheme followed for the production of EHA polypeptide constructs. Panels B-E are the images of Coomassie stained SDS-PAGE gel (Left) and western blot (Right) of purified EHA polypeptide constructs. Legends: Lane 0: Protein molecular weight markers; Lane 3 - EHA polypeptide construct 3; Lane 4 - EHA polypeptide construct 4; Lane 11 - EHA polypeptide construct 11; Lane 12 - EHA polypeptide construct 12.

DETAILED DESCRIPTION OF THE INVENTION:

In the detailed description of the present invention, numerous specific details are described to provide a thorough understanding of the various embodiments of the present invention. However, a person skilled in the relevant art will recognize that an embodiment of the present invention can be practiced without one or more of the specific details, or with other apparatus, systems, assemblies, methods, components, materials, parts, and/or the like. In other instances, well-known structures, materials, or operations are not specifically shown or described in detail to avoid obscuring aspects of embodiments of the present invention. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of person skill in the art. Some of the terms are defined briefly here below; the definitions should not be construed in a limiting sense.

The term ‘heterologous proteins’ refers to those proteins that are foreign to the host cells used, such as human proteins produced in E. coli.

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).

The term ‘optimized polynucleotide sequence’ refers to a synthetically synthesized nucleic acid (gene) optimized for high level expression of recombinant protein in host cells (e.g., E. coli).

The term ‘isolated’ and ‘biologically pure’ refer to a material which is substantially or essentially free from components which normally accompany it as found in its native state.

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.

The term ‘ recombinant E. colV refers to host cells that contain recombinant expression plasmid and that can express the target proteins into them.

The term ‘enzymatically active’ or ‘functionally active’ refer the ability of polypeptide / enzyme to exert one or more activities known to be associated with human arginase 1 enzyme, such as ability to hydrolyze argininie.

The term ‘chaotropic agent’ refers to a compound that in a suitable concentration in aqueous solution is capable of changing the spatial conformation of proteins so as to render the proteins soluble in an aqueous solution.

The term ‘inclusion bodies (IBs)’ refers to cytoplasmic aggregates of over-expressed, misfolded recombinant proteins expressed in recombinant E. coli cells and which may or may not be biologically active. The term ‘refolding’ used in this invention refers to a process of reintroducing secondary and tertiary structure to a protein that has had some or all of its native secondary or tertiary structure removed, either in vitro or in vivo , e.g., as a result of expression conditions or intentional denaturation and/or reduction. The refolded protein is functionally active and may possess three-dimensional conformation similar to native protein (enzyme).

The term ‘refolding additives’ refers to various chemicals that are known to help in refolding of the denatured proteins.

The term ‘refolding buffer’ refers to a buffered solution containing various refolding additives that assist in refolding of the denatured proteins.

The term ‘transformed cells’ refers to host E. coli cells containing plasmid containing target gene encoding for recombinant protein.

The term ‘unfolded or denatured protein’ refers to the biologically inactive form of the recombinant protein obtained after dissolving the inclusion bodies with aqueous solution of chao tropic agents.

The term ‘arginase 1 - associated diseases or conditions’ refer to the diseases or conditions in which arginine depletion has a therapeutic effect. This can be achieved by increasing the activity of arginase 1 enzyme in a subject by administering recombinant arginase polypeptide. Examples of such diseases or conditions include, but are not limited to cancer, bone related conditions, viral infections, multiple sclerosis, rheumatoid arthritis, autoimmune diseases, congenital hyperargininemia, ocular disease, graft-versus-host disease (GVHD), inflammation, immune diseases or conditions obesity, and metabolic disorders and related complications and comorbidities.

Arginase 1 - associated diseases or conditions can be treated by administering recombinant arginase polypeptide either alone or in combination with other agents. For example, in the treatment of certain cancers recombinant arginase can be given with chemotherapeutic agent(s). For example, in the treatment of viral infection, recombinant arginase can be given with anti-viral chemical drug(s). The term ‘arginase protein’ or ‘arginase polypeptide’ or ‘arginase enzyme’ refers to naturally occurring (EC 3.5.3.1) or recombinant enzyme that hydrolyze arginine substrate. Arginase polypeptide include, but are not limited to, native or recombinant human arginase enzyme. Non-human arginase polypeptide include, but are not limited to, arginase polypeptide from rabbit, mouse, rat, pig, cow, chicken, turkey and dog. Recombinant arginase 1 enzyme refers to a recombinantly produced arginase 1 produced in a foreign host cell like mammalian cell, bacterial cell, insect cell, plant cell, etc. Exemplary arginase include, but are not limited to, human arginase 1.

The term ‘wild-type human arginase 1’ used in this invention refers to a recombinantly produced wild-type human arginase 1 enzyme which is (100 %) identical to naturally occurring human liver arginase 1 in terms of its amino acid sequence and enzymatic properties (SEQ ID NO. 32).

The term ‘non-arginase polypeptides’ used in this invention refers to polypeptides that are different from arginase polypeptide in terms of their amino acid sequences and biological properties and that cannot hydrolyze arginine substrate. The non-arginase polypeptides when fused with arginase polypeptide, the resulting engineered molecules may possess arginine- hydrolyzing activity and increased hydrodynamic radius, increased FcRn-mediated recycling, increased negative charge, decreased renal clearance, etc, compared to arginase polypeptide or non-arginase polypeptide alone.

The term ‘engineered human arginase (EHA) polypeptide constructs’ refer to polypeptides of this invention (SEQ ID NO. 33-62) which include a human arginase 1 polypeptide whose amino acid sequence is set forth in (SEQ ID No. 32), a linker peptide and a non-arginase polypeptide comprising at least one of the following: 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 transferrin (Tf) or its analogs or its fragment/portion: human chorionic gonadotropic (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. The EHA polypeptide constructs used in this invention refers to polypeptides or proteins created through the in-frame joining of two or more polynucleotide sequences which originally encoded human arginase 1 polypeptide whose amino acid sequence is set forth in (SEQ ID No.: 32), any one of a linker peptide (SEQ ID No.: 63-64) and any one of the above mentioned non- arginase polypeptide. Translation of the EHA polynucleotide sequences (SEQ ID No.: 2-31) will result in a single polypeptide sequence which may have functional properties derived from each of the original peptides. Polynucleotide sequences encoding EHA polypeptide constructs may be created artificially by standard molecular biology methods. The resulting polynucleotide sequence may be inserted into an appropriate expression vector that supports the expression of EHA polypeptide construct in a standard host organism.

The EHA polypeptide constructs of the present invention comprise human arginase 1 polypeptide fused to any of the above-mentioned non-arginase polypeptide. The C-terminus of the human arginase 1 polypeptide may be fused directly, or fused via a linker peptide to the N- terminus of any of the above mentioned non-arginase polypeptide. Conversely, the N-terminus of the human arginase 1 polypeptide can be fused directly, or fused via a linker peptide, to the C-terminus of any of the above-mentioned non-arginase polypeptide. These EHA polypeptide constructs are biologically active and exhibit arginine-hydrolyzing activity.

The terms ‘analogs’ or ‘fragments’ or ‘portion’ used in this invention 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.

The term ‘polypeptide or protein or enzyme’ used in this invention collectively refers to human arginase 1 polypeptide and EHA polypeptide constructs of this invention.

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 2).

Table 2: Naturally occurring amino acids

Human serum albumin (HSA) is ~ 65.5 kDa 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 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 this invention refer to either HSA or its analogs or its fragment/portion (e.g., Ill domain of HAS; 3dHSA). The term ‘human serum albumin targeting polypeptide (HSATP) or its analogs or its fragment/portion’ refers to any polypeptide that can specifically bind to HSA.

Transferrin (Tf) is ~ 76 kDa glycoproteins involved in the binding and transport of iron in blood plasma. It is produced in the liver and contains binding sites for two Fe³⁺ atoms. It is made up of two domains (N- and C-terminal domains) and has a serum half-life of approximately 10-12 days. The term ‘human transferrin (Tf) or its analogs or its fragment/portion’ used in this invention refer to either Tf or its analogs or its fragment/portion (e.g., N-terminal domain of Tf; nTf).

Human chorionic gonadotropin (HCG) is ~ 36.7 KDa glycoprotein composed of two subunits: 14.5 KDa a-hCG and 22.2 KDa b-hCG. It is produced by trophoblast cells that surround a growing embryo. The C-terminal peptide (CTP) of the b-hCG is responsible for the longer serum half-life of hCG. The term ‘human chorionic gonadotropic (hCG) hormone or its analogs or its fragment/portion’ used in this present invention refer to either hCG or its analogs or its fragment/portion (e.g., its C-terminal peptide; CTP).

Elastin is a key protein of the extracellular matrix and is involved in the maintenance of many tissues in the body. Elastin is rich in hydrophobic amino acids. The term ‘elastin or its analogs or its fragment/portion’ used in this invention refer to either elastin or its fragment/portion or its analog (e.g. elastin-like polypeptides; ELP - a polymeric repeats of peptide motifs identical to the hydrophobic domain of human elastin).

Gelatin is a peptide produced by hydrolysis of collagen and consists of repeating G-X-Y triplets (where, G is glycine and X and Y are often proline). It is hydrophilic in nature and displays an increased hydrodynamic diameter that is attributable to its open, unfolded conformation and its hydrophilic nature. The term ‘gelatin or its fragment/portion or its analog’ used in this invention refer to either gelatin or its fragment/portion or its analogs (e.g., gelatin-like polypeptides; GLP - which is composed of short polymeric repeats of peptide motifs identical to the ones present in human gelatin). al -antitrypsin is ~ 52 KDa glycoprotein belonging to serpin superfamily and present in blood where it inhibits various protease and enzymes. In humans, the concentration of a-1 antitrypsin in blood is ~ 2 mg/mL. The term ‘al -antitrypsin or its analogs or its fragment/portion’ used in this invention refer to al -antitrypsin or its analogs or its fragment/portion.

Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells. They act as a critical part of the immune response by specifically recognizing and binding to particular antigens, such as bacteria or viruses, and aiding in their destruction. The five primary classes of immunoglobulins are IgG, IgM, IgA, IgD and IgE. Igs are composed of one or more units, each containing four polypeptide chains: two identical heavy chains (H) and two identical light chains (L). The amino terminal ends of the polypeptide chains show considerable variation in amino acid composition and are referred to as the variable (V) regions to distinguish them from the relatively constant (C) regions. Each L chain consists of one variable domain (VL), and one constant domain (CL). The H chains consist of a variable domain (VH) and three constant domains (CHI, CH2 and CH3). The fragment crystallizable region (Fc region) is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system. The term ‘immunoglobulin (Ig) or its analogs or its fragment/portion’ used in this invention refer to immunoglobulin or its analogs or its fragment/portion (e.g., Fc region of IgG2; G2Fc, and Fc region of IgG4; G4Fc).

The term ‘non-natural polymeric amino acid sequences’ (NNPAAS) used in this invention refer to non-natural polymeric amino acid sequences containing either alanine, glutamic acid, glycine, proline, serine and threonine (NNPAAS- 1) or proline, alanine, and serine (NNPAAS- 2) or glycine and serine (NNPAAS -3). NNPAAS are highly hydrophilic, unstructured polypeptides designed to prolong the serum half-life of pharmaceuticals by introducing a bulking effect similar to that of poly (ethylene glycol) and retarding the renal filtration of the pharmaceuticals to which they are fused with.

The term Tinker peptide’ used in this invention refers to a peptide sequence that separates the two polypeptides components of EHA polypeptide constructs of this invention. 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 individual functional properties.

The most preferred linker peptide is designated as LI and comprises the sequence Gly-Gly- Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 63). Additional preferred linker peptide includes a linker specified as L2 and comprises the sequence Leu-Glu- Ala-Glu-Ala-Ala-Ala-Lys-Glu-Ala-Ala-Ala-Lys-Glu-Ala-Ala-Ala-Lys-Glu-Ala-Ala-Ala- Lys-Ala-Leu-Glu-Ala-Glu-Ala-Ala-Ala-Lys-Glu-Ala-Ala-Ala-Lys-Glu-Ala-Ala-Ala-Lys- Glu-Ala-Ala-Ala-Lys-Ala-Leu-Glu (SEQ ID NO: 64). The present disclosure with reference to the accompanying examples describes the present invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. It is understood that the examples are provided for the purpose of illustrating the invention only, and are not intended to limit the scope of the invention in any way.

Example 1: Designing of EHA polypeptide constructs.

The EHA polypeptide constructs described in this invention consist of three components: human arginase 1 polypeptide fused to a non-arginase polypeptide via a linker peptide (FIG. 1).

Amino acid sequences of the components of EHA polypeptide constructs (i.e., human arginase 1 polypeptide, linker peptide and non-arginase polypeptides) were used to design polynucleotides (genes) coding for EHA polypeptide constructs of the inventions (SEQ ID No.: 2-31). The details of EHA of this invention are provided in Table 3. The human arginase 1 polypeptide is similar to naturally occurring human liver arginase 1 in terms of its amino acid sequence and enzymatic properties and is capable of hydrolyzing arginine into ornithine and urea. The non-arginase polypeptides used to create EHA polypeptide constructs of this invention are polypeptides that are different from arginase polypeptide in terms of their amino acid sequences and biological properties and that cannot hydrolyze arginine substrate. The non- arginase polypeptides when fused with arginase polypeptide, the resulting engineered molecules may possess arginine-hydrolyzing activity and increased hydrodynamic radius, increased FcRn-mediated recycling, increased negative charge, decreased renal clearance, etc, compared to arginase polypeptide or non-arginase polypeptide alone.

The non-arginase polypeptide used to create EHA polypeptide constructs of this invention comprises of at least one of the following: 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 transferrin (Tf) or its analogs or its fragment/portion: human chorionic gonadotropic (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; 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.

The linker peptides used to fuse human arginase 1 polypeptide to a non-arginase polypeptide are short stretch of amino acids.

Table 3. EHA polypeptide constructs of the invention.

Example 2: Generation of recombinant expression plasmid.

Amino acid sequence of human arginase 1 polypeptide (Seq ID: 32), linker peptide (Seq ID: 63-64) and a non-arginase polypeptide were used to design polynucleotide (gene) (Seq ID: 2-

31) encoding for EHA polypeptide constructs (Seq ID: 33-62). The designed genes were codon-optimized for high level expression in E. coli and custom synthesized from Gene Script, NJ. In the designed gene, the 5'end of the open reading frame (ORF) is flanked by a Nde 1 restriction site. The 3'end of the ORF is flanked by nucleotides encoding for a stop codon and a EcoR 1 restriction site (FIG. 2A). The designed genes were sub-cloned in pET23a(+) expression plasmid, between Nde 1 and EcoR 1 restriction sites, to generate recombinant expression plasmids encoding for particular EHA polypeptide construct of this invention (FIG. 2B). Example 3: Confirmation of recombinant expression plasmids.

Confirmation by double digestion of recombinant expression plasmids.

By following standard molecular biology techniques known in the art, the recombinant expression plasmids were individually transformed into E. coli BL21 (DE3) to generate recombinant E. coli cells for the production of EHA polypeptide constructs. The recombinant plasmids were then isolated from the recombinant E. coli cells and the presence of desired genes encoding for particular EHA polypeptide constructs in the recombinant expression plasmids was confirmed by digesting the purified plasmids with restriction enzymes Nde 1 and EcoR 1 followed by agarose gel electrophoresis Representative results of agarose gel electrophoresis of digested recombinant expression plasmids are given in FIG. 3. Observation of drop-out (fragment of DNA) of desired sizes in the digested plasmid samples confirm the presence of desired gene encoding for particular EHA polypeptide constructs in the recombinant expression plasmids.

Confirmation by expression of EHA polypeptide constructs

To confirm the expression of EHA polypeptide constructs in recombinant cells, glycerol stock of recombinant E. coli BL21 (DE3) cells was streaked on Luria Bertani (LB)-agar plate containing carbenicillin and the plates were incubated overnight at 37°C. A single colony from the plate was used to initiate a seed culture in LB-broth supplemented with carbenicillin and the seed culture was grown at 37°C overnight at 200 rpm. One percent of this seed culture was then inoculated into fresh LB-broth supplemented with carbenicillin and the main culture was grown at 37°C till ODeoo reached 0.6-0.8. The culture was then induced with 0.5 mM isopropyl b-D-l-thiogalactopyranoside (IPTG) and was allowed to grow further at 20°C. The wet cell mass (WCM) of E. coli was then harvested by centrifugation (10,000xg, 10 min, 4°C) and re- suspended in ice-cold lysis buffer (50 mM Tris-HCl, pH 8.0 containing 150 mM NaCl, 1 mM b-ME, 0.1 mM of protease inhibitor cocktail and 10 μg/ml lysozyme). The cell suspensions were gently stirred at room temperature for 1 h, passed through syringe and the cells were subjected to sonication (60 % amplitude, 10 pulses of 1 min each with 1 min break after each pulse on ice) using Ultrasonics, Inc. model W830 ultrasonic processor with a macro-probe tip. The sonicated cell suspensions were then centrifuged (16,000xg, 30 min, 4°C) to separate supernatant cell lysate fractions from insoluble cell debris fractions. These fractions were then analyzed for the presence of EHA polypeptide constructs by SDS-PAGE and western blot analysis.

Results

No or very faint bands of recombinant proteins were observed in western blot when supernatant cell lysate fraction of the recombinant E. coli cells expressing EHA polypeptide constructs were analyzed (data not shown). Prominent target protien bands were observed in SDS-PAGE and western blot analysis when insoluble cell debris fractions of recombinant E. coli cells expressing EHA polypeptide constructs were analyzed. Representative results of this expression studies are given in FIG. 4. In western blot analysis, when anti-arginasel antibody was used as a primary antibody, a prominent protein band corresponding to expected molecular weight was observed in the insoluble cell debris fraction, indicating expression of EHA proteins in recombinant E. coli cells. The results also suggest that major amount of overexpressed EHA propteins were aggregating and forming inclusion bodies (IBs) in E. coli, despite growing the bacterial cells at lower temperature and induction of recombinant proteins with lower concentration of IPTG.

Example 4: Production of EHA-1 polypeptide constructs.

Scheme followed for the production of EHA polypeptide constructs are given in FIG 5A.

A. Cultivation of recombinant E. coli cells containing gene for EHA polypeptide constructs and isolation of inclusion bodies:

Glycerol stocks of recombinant E. coli BL21 (DE3) cells containing gene for EHA polypeptide constructs were streaked on Luria Bertani (LB)-agar plate containing carbenicillin and incubated overnight at 37°C. A single colony from the plates was used to initiate the seed cultures in LB-broth supplemented with carbenicillin and the seed culture was grown at 37°C overnight at 200 rpm. One percent of this seed culture was then inoculated into fresh LB-broth supplemented with carbenicillin and the main culture was grown at 37°C till ODeoo reached 0.6-0.8. The culture was then induced with IPTG and was allowed to grow further at 37°C. The wet cell mass of bacteria was then harvested by centrifugation (10,000xg, 10 min, 4°C) and the cell pellet was used to purify the inclusion bodies. The cells were re-suspended in ice-cold lysis buffer. The cell suspensions were gently stirred at room temperature for 1 h, passed through syringe with a needle and the cells were then disrupted by sonication. The sonicated cell suspensions were immediately cooled on ice and treated with DNase for 1 h. To the sample was then added 2V of buffer to make total volume 3V. The samples were vortexed and incubated at 4°C for 30 min with gentle shaking. The samples were then centrifuged to separate clear supernatant cell lysate fraction from insoluble fraction containing EHA-enriched IBs. The IB was then washed with washing buffer to remove the contaminants present and stored at - 80°C till further use.

B. In vitro refolding of recombinant EHA polypeptide construct:

Different refolding additives that are known to facilitate in vitro refolding of recombinant proteins were selected and include: a buffering agent selected from the group of Tris-HCl, CHES, EPPS, HEPES, Glycine-NaOH, phosphate, TAPS, MOPS, and MES, a salt selected from the group of NaCl, KC1 and NH4CI, a detergent selected from the group of tween-20, tween-20, NP-10, NP-40, triton X-100, CHAPS and Brij 35, an amino acid is selected from the group of lysine, histidine, glutamic acid, aspartic acid, glycine, alanine, proline, serine, threonine, tryptophan, phenylalanine, cysteine, methionine, valine, leucine, isoleucine, tyrosine, asparagine and glutamine, a sugar selected from a group of maltose, glucose, mannose, trehalose, sucrose, dextrose, lactose, glycerol, sorbitol, mannitol, myo-inositol, xylitol and ethylene glycol, a polymer selected from a group of cyclodextrins and polyethylene glycols, a surfactant selected from a group of NDSB201 and NDSB256, and a reducing agent selected from TCEP and DTT. EHA polypeptide construct (1 mg/ml) was diluted to 100 μg/ml in different refolding buffers and allowed to incubate at room temperature at 150 rpm for desired period of time.

C. Purification of EHA polypeptide constructs from refolding mixture:

EHA polypeptide constructs were purified from the refolding mixture by using hydrophobic interaction chromatography. The phenyl-agarose resin column was pre-equilibrated with buffer containing 50 mM Tris HC1, pH 8.0, and 0.5 M sodium sulphate. After equilibration, the refolding reaction mixture (containing 0.5 M sodium sulphate) was loaded onto column and the column was washed with buffer containing 50 mM Tris HC1, pH 8.0, and 0.5 M sodium sulphate. Elution of bounded protein was done in water and the eluted fractions were analyzed for both protein content as well as arginine-hydrolyzing activity. Qualitative analysis of these fractions was done by performing SDS-PAGE electrophoresis and western blot by using monoclonal mouse anti-human arginase 1 antibody as primary antibody. The fractions containing enzymatically active protein were pooled and concentrated and used further.

Representative results of SDS-PAGE and western blot analysis of purified EHA polypeptide constructs are given in FIG 5B. Single protein bands corresponding to desired molecular weights were observed in SDS-PAGE and western blot.

Example 5: Enzymatic characterization of recombinant EHA polypeptide constructs.

A coupled spectrophotometric assay was used to determine the arginine-hydrolysing activity of EHA polypeptide constructs. Purified EHA proteins were activated by incubating them with either MnS0₄ or C0CI2 (final concentration 10 mM) at 50 °C for 20 min. After activation, the arginine-hydrolysing activity of EHA was determined by incubating activated EHA with 50 mM Arginine in 100 mM HEPES buffer of pH 8.4 or pH 7.6 (for 10 minutes at 30°C). The urea generated was detected by using urea detection kit (AutoZyme Urea; Accurex Biomedical Pvt. Ltd.). One Unit (U) of arginase activity was defined as the amount of enzyme that can produce 1 pmol urea/min at 30°C at pH 8.5 or pH 7.6.

Specific activity of representative EHA proteins of this invention is given in Table 4.

Table 4. Arginine-hydrolyzing activity of EHA polypeptide constructs.

EHA polypeptides exhibited considerably different arginine-hydrolyzing potency. Some EHA polypeptide constructs (e.g. EHA polypeptide construct 3 and 12) exhibited considerably enhanced arginine-hydrolyzing activity. It is important to note here that specific activity (arginine-hydrolyzing activity) of 389-518 has been reported for wild-type human arginase 1 previously (US 2015/0010522 Al; US 8,679,810 B2; Ikemoto et al., 1989, Ann. Clin. Biochem. 26, 547-553; Ikemoto et al., 1990, Biochem. J. 270, 697-703; Tsui, et al., 2009. Cancer Cell Int, 2009. 9:p. 9). The results suggest that due to engineering, some of the EHA variants acquired enhanced arginine-hydrolyzing activity, compared to wild-type human arginase 1.

It will be appreciated by those of skill in the art that the success of production of any structurally stable and functionally active fusion polypeptide depends on a number of factors, including, inter alia, the particular protein in question, choice of the linker, particular fusion partner polypeptide, particular configuration of these three components which may or may not result in structurally stable and functionally active fusion polypeptide.

EMBODIMENTS OF THE INVENTION:

The above examples and the technical descriptions are illustrative of the invention and any variations to the same should be regarded as within the scope of the invention. Some of the embodiments are as described herein:

One embodiment of the present invention relates to EHA polypeptide constructs, whose amino acid sequences are set forth as SEQ ID No.: 33-62 and include a human arginase 1 polypeptide whose amino acid sequence is set forth as SEQ ID No.: 32 , a linker peptide and a non-arginase polypeptide comprising of at least one of the following: 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 transferrin (Tf) or its analogs or its fragment/portion: human chorionic gonadotropic (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; 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.

The EHA polypeptide constructs according to the present invention are proteins created through the in-frame joining of two or more polynucleotide sequences which originally encoded human arginase 1 polypeptide, a linker peptide and a non-arginase polypeptide. Translation of EHA polynucleotide sequence will result in a single polypeptide sequence which may have functional properties derived from each of the component (original) polypeptides. Polynucleotide sequences encoding EHA polypeptide constructs may be created artificially by standard molecular biology methods. The resulting polynucleotide sequence may be inserted into an appropriate expression vector that supports the heterologous EHA polypeptide construct expression in a standard host organism.

EHA polypeptide constructs may contain a linker peptide that separates the two component polypeptides of EHA polypeptide constructs. The linker peptide may facilitate the 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 individual functional properties.

Another embodiment of the present invention relates to EHA polypeptide constructs comprising human arginase 1 polypeptide covalently joined to a non-arginase polypeptide via a linker peptide. In one aspect, the EHA polypeptide constructs comprise human arginase 1 covalently joined to human serum albumin (HSA) or its analogs or its fragment/portion via a linker peptide. In another aspect, the EHA polypeptide constructs comprise human arginase 1 covalently joined to human serum albumin targeting polypeptide (HSATP) or its analogs or its fragment/portion via a linker peptide. In another aspect, the EHA polypeptide constructs comprise human arginase 1 covalently joined to human transferrin (Tf) or its analogs or its fragment/portion via a linker peptide. In another aspect, the EHA polypeptide constructs comprise human arginase 1 covalently joined to human chorionic gonadotropic (hCG) hormone or its fragment/portion via a linker peptide. In another aspect, the EHA polypeptide constructs comprises human arginase 1 covalently joined to elastin or its analogs or its fragment/portion via a linker peptide. In another aspect, the EHA polypeptide constructs comprises human arginase 1 covalently joined to gelatin or its analogs or its fragment/portion via a linker peptide. In another aspect, the EHA polypeptide constructs comprises human arginase 1 covalently joined to al -antitrypsin or its analogs or its fragment/portion via a linker peptide. In another aspect, the EHA polypeptide constructs comprises human arginase 1 covalently joined to immunoglobulin (Ig) or its analogs or its fragment/portion via a linker peptide. In another aspect, the EHA polypeptide constructs comprises human arginase 1 covalently joined to non- natural polymeric amino acid sequences (NNPAAS) containing alanine, glutamic acid, glycine, proline, serine and threonine or proline, alanine, and serine or glycine and serine via a linker peptide.

In one embodiment, in the said EHA polypeptide constructs the said human arginase 1 polypeptide comprises an amino acid sequence that is at least 90 % identical to the amino acid sequence denoted by SEQ ID NO: 32 and wherein said human arginase 1 polypeptide component retains the functional activity of human arginase 1.

In another embodiment of the present invention is provided a EHA polypeptide constructs whose nucleotide sequences are set forth as SEQ ID No.: 2-31.

In one embodiment, non-arginase polypeptide component of EHA polypeptide construct comprise human serum albumin (HSA) or its analogs or its fragment/portion.

In one embodiment, non-arginase polypeptide component of EHA polypeptide construct comprise human serum albumin targeting polypeptide (HSATP) or its analogs or its fragment/portion.

In one embodiment, non-arginase polypeptide component of EHA polypeptide construct comprise human transferrin (Tf) or its analogs or its fragment/portion

In one embodiment, non-arginase polypeptide component of EHA polypeptide construct comprise human chorionic gonadotropic (hCG) hormone or its fragment/portion.

In one embodiment, non-arginase polypeptide component of EHA polypeptide construct comprise elastin or its analogs or its fragment/portion.

In one embodiment, non-arginase polypeptide component of EHA polypeptide construct comprise gelatin or its analogs or its fragment/portion. In one embodiment, non-arginase polypeptide component of EHA polypeptide construct comprise al -antitrypsin or its analogs or its fragment/portion.

In one embodiment, non-arginase polypeptide component of EHA polypeptide construct comprise immunoglobulin (Ig) or its analogs or its fragment/portion.

In one embodiment, non-arginase polypeptide component of EHA polypeptide construct comprise 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.

In one embodiment, the C-terminus of human arginase 1 polypeptide is covalently linked to the N-terminus of the non-arginase polypeptide via a linker peptide.

In another embodiment, the N-terminus of the human arginase 1 polypeptide is covalently linked to the C-terminus of the non-arginase polypeptide a linker peptide.

In other embodiments, said EHA polypeptide constructs further comprises a linker peptide covalently linking human arginase 1 polypeptide component and the non-arginase polypeptide component.

In one specific embodiment, the said linker peptide comprises the amino acid sequences set forth in SEQ ID NO: 63-64.

In another embodiment of the present invention is provided optimized polynucleotide sequences encoding for EHA polypeptide constructs of this invention and comprising at least one polynucleotide sequence selected from the group comprising SEQ ID NOs: 2-31.

In another embodiment of the present invention is provided a optimized polynucleotide sequences, wherein said optimized polynucleotide sequences encode at least one polypeptide chain, wherein the polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 33-62. In another embodiment of the present invention is provided a EHA polynucleotides and EHA polypeptide constructs thereof comprising at least one polynucleotide sequence selected from the group comprising SEQ ID NOs: 2-31, wherein the polynucleotide encodes at least one polypeptide chain comprising at least one amino acid sequence selected from the group comprising SEQ ID NOs: 33-62, wherein at least one polypeptide chain has increased arginine- hydrolyzing activity.

In a further related aspect, the invention provides methods of making all above mentioned EHA polypeptide constructs comprising the step of culturing the host cell under suitable conditions for expression of recombinant polypeptides. In certain embodiments, the method further comprises the step of obtaining the recombinant polypeptides from the cell culture. The recombinant polypeptides can be obtained from the cell culture by any methods of protein isolation or purification known in the art, including without limitation, collecting recombinant cells, freezing/thawing, centrifugation, cell lysis, inclusion bodies isolation, in vitro refolding and hydrophobic interaction column chromatography. In certain embodiments, the host cell is an E. coli cell.

In another embodiment of the present invention is provided a method for production of EHA polypeptide constructs comprising amino acid sequence of recombinant polypeptides as set forth in SEQ ID No.: 33-62 by refolding inclusion bodies comprising the steps of- designing gene encoding for recombinant polypeptide constructs, culturing the host cells expressing recombinant polypeptide constructs, inducing high level of expression of recombinant polypeptide constructs, collecting the cell mass expressing recombinant polypeptide constructs, lysing the cells, purifying the inclusion bodies containing recombinant polypeptide constructs, solubilizing the inclusion bodies with the help of chaotropic agents, refolding the recombinant polypeptide constructs using refolding buffer, isolating the enzymatically active recombinant polypeptide constructs using chromatography , obtaining enzymatically active recombinant polypeptide constructs.

In another embodiment of the present invention is provided a method for production of EHA polypeptide constructs as set forth in SEQ ID No.: 33-62 by refolding inclusion bodies, wherein the chaotropic agent is selected from the group comprising organic compounds, solvents, salts, detergents and is preferably guanidium.

In another embodiment of the present invention is provided a method for production of EHA polypeptide constructs as set forth in SEQ ID No.: 33-62 by refolding inclusion bodies, wherein the refolding buffer comprises at least one buffering agent selected from the group of Tris-HCl, CHES, EPPS, HEPES, Glycine-NaOH, Phosphate, TAPS, MOPS, and MES.

In another embodiment of the present invention is provided a method for production of EHA polypeptide constructs as set forth in SEQ ID No.: 33-62 by refolding inclusion bodies, wherein the refolding buffer further comprises a mixture of at least one redox pair selected from a group of reduced glutathione/oxidized glutathione; oxidized nicotinamide dinucleotide/reduced nicotinamide dinucleotide and cystamine/cysteamine.

In another embodiment of the present invention is provided a method for production of EHA polypeptide construct comprising amino acid sequence of recombinant polypeptides as set forth in SEQ ID No.: 33-62 by refolding inclusion bodies, wherein the refolding buffer further comprises at least one refolding additives selected from the group of salts, sugars, polymers, polyols, detergents and surfactants.

In another embodiment of the present invention is provided a method for production of EHA polypeptide constructs, wherein said polypeptides are derived from at least one polynucleotide sequence selected from the group comprising SEQ ID NOs: 2-31, wherein said polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 33-62, wherein the method results into high level expression of said polypeptides as enzymatically non-functional aggregated inclusion bodies, wherein said method comprises the step of inducing the host cell culture with 0.1-2.0 M IPTG concentration and growing the cultures for 4-24 h at 37°C.

In another embodiment of the present invention is provided a method of production of enzymatically active recombinant polypeptides from inclusion bodies, wherein said polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 33-62, wherein said method comprises the steps of: isolating the inclusion bodies containing EHA polypeptide constructs, solubilizing the inclusion bodies containing EHA polypeptide constructs with the help of guanidium, refolding the denatured recombinant polypeptide constructs by diluting in refolding buffer, isolating the enzymatically active recombinant polypeptide constructs using chromatography , obtaining the enzymatically active recombinant polypeptide constructs.

In another embodiment of the present invention is provided a method of production of enzymatically active EHA polypeptide constructs from inclusion bodies, wherein said polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 33-62, wherein the refolding buffer comprises at least one buffering agent, at least one redox pair, at least one refolding additives, or mixture thereof; the buffering agent is selected from the group of Tris-HCl, CHES, EPPS, HEPES, Glycine-NaOH, Phosphate, TAPS, MOPS, and MES, the redox pair is selected from a group of reduced glutathione/oxidized glutathione; oxidized nicotinamide dinucleotide/reduced nicotinamide dinucleotide and cystamine/cysteamine, and the refolding additives are selected from the group of salts, sugars, polymers, polyols, detergents and surfactants.

In another embodiment of the present invention is provided a method of production of enzymatically active EHA polypeptide constructs from inclusion bodies, wherein said polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 33-62, wherein the purity of enzymatically active EHA polypeptide constructs is at least 80%.

In another embodiment of the present invention is provided EHA polypeptide constructs wherein the purified recombinant polypeptides are expressed and purified from living cell not limiting to a bacterium, a yeast, a mammalian cell, an insect cell, and a plant cell.

In another embodiment of the present invention are provided recombinant expression plasmid or vector comprising the isolated polynucleotides of the present invention. In another embodiment of the present invention are provided recombinant expression plasmid comprising the isolated polynucleotides encoding recombinant polypeptides of the present invention.

In another embodiment of the present invention is provided an isolated host cell comprising the recombinant expression plasmid of the present invention.

In another embodiment of the present invention the host cell is selected from the group of living cells but not limiting to bacterial cells, yeast cells, animal cells, plant cells, insect cells, and may be cell free expression system. Preferably the host cell is E. coli.

In another embodiment of the present invention are provided polynucleotides which are optimized polynucleotide sequences for high level expression in E. coli, as set forth in SEQ ID NOs: 2-31.

Yet another embodiment of the present invention provides a method for producing recombinant polypeptides comprising of the following steps: i) expressing the human arginase 1 enzymes as inclusion bodies in host cell, ii) purification of the inclusion bodies, iii) denaturation of the inclusion bodies with the help of chaotropic agent, iv) refolding of denatured proteins with the help of various refolding additives, v) separation of enzymatically active recombinant polypeptides from inactive polypeptides, and vi) characterizing the hydrolytic activities of the purified recombinant polypeptides.

According to yet another embodiment of the present invention the recombinant E. coli cells are grown under the conditions favorable for high level expression of heterologous recombinant polypeptides. One or more favorable experiment conditions may include effective media, temperature, pH, oxygen condition, shaking time and speed, and like.

According to yet another embodiment of the present invention various chemical or physical agents, such as IPTG, lactose, low or high temperature change, and like, are used to induce high level expression of heterologous recombinant polypeptides in E. coli cells. According to yet another embodiment of the present invention the E. coli cells were lysed by using either physical or chemical methods such as sonication, French press, lysozyme- and detergent-treatment, and like, to release the recombinant polypeptides.

In yet another embodiment of the present invention the purity of isolated inclusion bodies varies from at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% to 100%.

According to still another embodiment of the present invention, the recombinant polypeptides are refolded with the help of chemical assisted dilution refolding selected from a group of refolding methods such as simple batch dilution, continuous dilution, dialysis, chromatography-column based refolding, chaperone assisted refolding, and like.

According to still another embodiment of the present invention the refolding additives used for the chemical assisted dilution refolding are used in various combinations and may include but are not limited to compounds selected from the group of various buffers, ionic detergents, non- ionic detergents, non-detergent sulfobetains (NDSBs), redox pair, salts, denaturing agents, chelating agents, cofactors, sugars, amino acids, proteins, polyols, polymers, reducing agents, and like. A buffer system for refolding of the protein in question may easily be designed by the person skilled in the art.

According to yet another embodiment of the present invention, the optimum refolding conditions can be same or different for recombinant polypeptides.

According to still another embodiment of the present invention, the final concentrations of recombinant polypeptides used in the refolding reaction varies from less than 1 mg/ml including less than 300 μg/ml, such as less than 100 μg/ml.

According to yet another feature of the present invention the optimum time of refolding varies from 2 to 24 hours depending upon the refolding condition and recombinant polypeptides.

According to yet another embodiment of this invention the yield of refolding varies from at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% to 100% depending on different refolding conditions, purity of inclusion bodies and recombinant polypeptides. According to yet another embodiment of the present invention the yield of enzymatically active recombinant polypeptides is at least 10 mg, 100 mg, 1 g, 10 g, 100 g, 0.1 kg, 10 kg, or 100 kg.

Yet another embodiment of the present invention provides an isolated polynucleotide and polypeptide sequences encoding EHA polypeptide constructs exhibiting enhanced Arginine- hydrolyzing activity, and are set forth in SEQ ID NOs: 2-31 and 33-62, respectively, wherein at least one polypeptide chain has increased arginine-hydrolyzing activity.

For example, EHA polypeptide constructs of this embodiment have enhanced arginine- hydrolyzing activity and are set forth in SEQ ID NOs: 35, 44.

According to yet another embodiment of the present invention, the recombinant polypeptides of the invention can be used to treat arginase 1-associated diseases or conditions in the subjects. Although the present invention has been described in detail for specific embodiments thereof, it is apparent to those skilled in the art that various alterations and modifications are conductible without departing from the spirit and scope of the present invention. Acknowledgement: The inventors thank the Department of Biotechnology (New Delhi, Government of India; grant # BT/PR23283/MED/30/1953/2018) and NIPER SAS Nagar for providing support through research grants.

Claims

The Claims:

1. Engineered polypeptide construct comprising of arginase polypeptide fused to a non- arginase polypeptide via a linker peptide.

2. The construct as claimed in claim 1, wherein said arginase polypeptide is a human arginase 1 polypeptide.

3. The construct as claimed in claim 2, wherein said constructs are set forth in SEQ ID NO: 33-62.

4. The construct as claimed in claim 1 and 2, wherein said non-arginase polypeptide is selected from the group comprising of following: i. human serum albumin (HSA) or its analogs or its fragment/portion; ii. human serum albumin targeting polypeptide (HSATP) or its analogs or its fragment/portion ; iii. human transferrin (Tf) or its analogs or its fragment/portion iv. human chorionic gonadotropic (hCG) hormone or its fragment/portion v. elastin or its analogs or its fragment/portion; vi. gelatin or its analogs or its fragment/portion; vii. al -antitrypsin or its analogs or its fragment/portion; viii. immunoglobulin (Ig) or its analogs or its fragment/portion; ix. 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.

5. The construct as claimed in claim 2, wherein the C-terminus of the human arginase 1 polypeptide is fused to the N-terminus of the non-human arginase polypeptide via a linker peptide.

6. The construct as claimed in claim 2, wherein the N-terminus of the human arginase 1 polypeptide is fused to the C-terminus of the non-human arginase polypeptide via a linker peptide.

7. The construct as claimed in claim 2, encoded by recombinant polynucleotides sequences as set forth in SEQ ID NO: 2-31.

8. The recombinant polynucleotide as claimed in claim 7, wherein said polynucleotide encodes at least one polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 33-62.

9. A method for production of the engineered construct as claimed in claim 1 and 2, said method comprising of following steps - a. designing EHA gene, b. culturing the host cells expressing EHA polypeptides, c. inducing high level of expression of EHA polypeptides, d. collecting the cell mass expressing EHA polypeptides, e. lysing the cells, f. purifying the inclusion bodies containing EHA polypeptides, g. solubilizing the inclusion bodies with the help of chaotropic agents, h. refolding the EHA polypeptides using refolding buffer, i. isolating the enzymatically active EHA polypeptides using chromatography, j. obtaining enzymatically active EHA polypeptides.

10. The method as claimed in claim 9, wherein said chaotropic agent is selected from the group comprising organic compounds, solvents, salts, detergents and is preferably guanidium; wherein the said refolding buffer comprises at least one buffering agent selected from the group of Tris-HCl, CHES, EPPS, HEPES, Glycine-NaOH, Phosphate, TAPS, MOPS, and MES; wherein the said refolding buffer comprises at least one buffering agent selected from the group of Tris-HCl, CHES, EPPS, HEPES, Glycine-NaOH, Phosphate, TAPS, MOPS, and MES; wherein the said refolding buffer may further comprise of a mixture of at least one redox pair selected from a group of reduced glutathione/oxidized glutathione; oxidized nicotinamide dinucleotide/reduced nicotinamide dinucleotide and cystamine/cysteamine; wherein the said refolding buffer further comprises at least one refolding additives selected from the group of salts, sugars, polymers, polyols, detergents and surfactants.

11. The method as claimed in claim 9, wherein the said polypeptides are derived from at least one polynucleotide sequence selected from the group comprising SEQ ID NOs: 2-31, wherein the said polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 33-62, wherein the said method results into high level expression of EHA polypeptides as enzymatically non-functional aggregated inclusion bodies, wherein the said method further comprises the step of inducing the host cell culture with 0.1-2.0 M IPTG concentration and growing the cultures for 4-24 h at 37°C.

12. A method of production of enzymatically active EHP polypeptides from inclusion bodies, wherein said polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 29-54, wherein said method comprises the steps of: a. purifying the inclusion bodies containing EHP polypeptides, b. solubilizing the inclusion bodies containing EHP polypeptides with the help of guanidium, c. refolding the denatured EHP polypeptides by diluting in refolding buffer, d. isolating the enzymatically active EHP polypeptides using chromatography, e. obtaining the enzymatically active EHP polypeptides.

13. The method as claimed in claim 12, wherein the refolding buffer comprises at least one buffering agent, at least one redox pair, at least one cofactor, at least one refolding additives, or mixture thereof; the buffering agent is selected from the group of Tris-HCl, CHES, EPPS, HEPES, Glycine NaOH, Phosphate, TAPS, MOPS, and MES, the redox pair is selected from a group of reduced glutathione / oxidized glutathione; oxidized nicotinamide dinucleotide / reduced nicotinamide dinucleotide and cystamine / cysteamine, the cofactor is selected from the group of CaCI₂, CoCI₂, MgCI₂, and ZnCI₂ and the refolding additives are selected from the group of salts, sugars, polymers, polyols, detergents and surfactants.

14. The method as claimed in claim 12 wherein the purity of enzymatically active EHP polypeptides is at least 80%.

15. The method as claimed in 9 wherein the purity of enzymatically active EHA polypeptides is at least 80%.