WO2024254543A2 - Variants d'asparaginases ancestraux recombinantes et leurs utilisations dans le traitement du cancer - Google Patents

Variants d'asparaginases ancestraux recombinantes et leurs utilisations dans le traitement du cancer Download PDF

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WO2024254543A2
WO2024254543A2 PCT/US2024/033129 US2024033129W WO2024254543A2 WO 2024254543 A2 WO2024254543 A2 WO 2024254543A2 US 2024033129 W US2024033129 W US 2024033129W WO 2024254543 A2 WO2024254543 A2 WO 2024254543A2
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asparaginase
recombinant
certain embodiments
seq
variant
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WO2024254543A3 (fr
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Sunil RAIKAR
Christopher Doering
H. Trent SPENCER
Kristopher Knight
Harrison Brown
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Emory University
Childrens Healthcare of Atlanta Inc
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Emory University
Childrens Healthcare of Atlanta Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/82—Asparaginase (3.5.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01001—Asparaginase (3.5.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Asparagine amidohydrolase is a natural enzyme that catalyzes the breakdown of L-asparagine.
  • Therapeutic asparaginases also referred to as “L-asparaginase” or “L- ASNase” are commonly used in acute lymphoblastic leukemia (ALL) chemotherapy regimens and have played a significant role in improving these outcomes.
  • ALL acute lymphoblastic leukemia
  • many clinically approved asparaginases are derived from bacterial sequences. Patients often produce inactivating antibodies and are at risk of anaphylaxis among other adverse side effects such as hepatotoxicity, pancreatitis, coagulopathy, and thrombosis. Thus, there is a need to identify improvements.
  • Vrooman et al. report efficacy and toxicity of pegaspargase and calaspargase pegol in childhood acute lymphoblastic leukemia. J Clin Oncol, 2021, 39:3496-350. Fonseca et al. report strategies for circumventing the side effects of L-asparaginase. Biomed Pharmacother, 2021, 139: 111616.
  • This disclosure relates to asparaginases with ancestral variant sequences disclosed herein for uses as therapeutics.
  • the ancestral variant asparaginases have reduced immunogenicity thereby preventing or reducing the risk of host inactivation and/or anaphylaxis.
  • this disclosure relates to treating diseases associated with asparagine dependence comprising administering an effective amount of an ancestral variant asparaginase or conjugate disclosed herein to a subject in need thereof.
  • this disclosure relates to recombinant ancestral variant asparaginase comprising an amino acid sequence selected from SEQ ID NO: 1-53 or variant thereof having 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity.
  • this disclosure relates to recombinant ancestral variant asparaginase comprising an amino acid sequence selected from SEQ ID NO: 54-63 or variant thereof having 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity.
  • this disclosure relates to methods of treating cancer comprising administering an effective amount of an ancestral variant asparaginase disclosed herein to a subject in need thereof.
  • the cancer is a hematological cancer or a solid cancer.
  • the cancer is leukemia, lymphoma, acute lymphoblastic leukemia (ALL), lymphoblastic lymphoma (LBL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), chronic myelogenous leukemia, acute monocytic leukemia (AMOL), chronic myeloid leukemia (CML), B-cell acute lymphoblastic leukemia (B-ALL), myeloproliferative neoplasms (MPNs), and lymphomas, Hodgkin's lymphomas, and non-Hodgkin's lymphomas such as Burkitt lymphoma, B-cell lymphoma, or diffuse large B-cell lymphoma (DLBCL).
  • ALL acute lymphoblastic leukemia
  • LBL lymphoblastic lymphoma
  • AML acute myelogenous leukemia
  • CLL chronic lymphocytic leukemia
  • the cancer is a solid malignancy such as the cancer is a solid cancer ovarian cancer, ovarian clear cell carcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), breast cancer, metastatic breast cancer, glioblastoma, or hepatocellular carcinoma.
  • the cancer is a solid cancer ovarian cancer, ovarian clear cell carcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), breast cancer, metastatic breast cancer, glioblastoma, or hepatocellular carcinoma.
  • this disclosure relates to recombinant ancestral variant asparaginase is administered in combination with methotrexate, cytarabine, rapamycin, temozolomide, and/or 6-diazo-5-oxo-l-norleucine.
  • the recombinant ancestral variant asparaginase is conjugated to a biodegradable polymer.
  • the biodegradable polymer comprises polyethylene glycol.
  • the biodegradable polymer comprises an amide linking group connecting polyethylene glycol to the recombinant asparaginase.
  • the biodegradable polymer comprises polyethylene glycol or monomethoxy polyethylene glycol.
  • the biodegradable polymer comprises an amide linking group connecting polyethylene glycol or monomethoxy polyethylene glycol to the recombinant asparaginase through a lysine amino acid and/or to the N-terminal amino acid.
  • this disclosure relates to nucleic acids encoding a recombinant ancestral variant asparaginase disclosed herein in operable combination with a heterologous promoter. In certain embodiments, this disclosure relates to vectors encoding a nucleic acid encoding a recombinant ancestral variant asparaginase disclosed herein.
  • this disclosure relates to host cells comprising a nucleic acid or vector encoding a recombinant ancestral variant asparaginase disclosed herein.
  • the nucleic acid is naked DNA, RNA, or mRNA.
  • this disclosure relates to pharmaceutical compositions comprising an ancestral variant asparaginase or conjugated as disclosed herein.
  • this disclosure relates to the use of a recombinant ancestral variant or conjugated as disclosed herein in the production or a medicament to treat conditions and diseases disclosed herein.
  • the recombinant asparaginases disclosed herein elicit a lower immunogenic response in a patient compared to other clinically approved L-asparaginases, e.g., E. coli L-asparaginase or Erwinia chrysanthemi L-asparaginase.
  • significantly reduced immunogenicity is evidenced, e.g., by the reduction or elimination of an antibody response against the L-asparaginase preparation following administration or repeated administrations; and/or usefulness as a second-line therapy for patients who have developed sensitivity to first-line therapies using, e.g., E.
  • the recombinant asparaginases disclosed herein provide reduced immunogenicity and enhanced plasma half-life.
  • the recombinant asparaginases disclosed herein are used in a method of treating patients with relapsed ALL who were previously treated with other asparaginase preparations, in particular those who were previously treated with E. coli-derived asparaginases.
  • Figure 1 shows a sequence comparison of human L-asparaginase (subject SEQ ID NO: 64) and an ancestral recombinant ancestral construct 104 “An 104” (query SEQ ID NO: 10). There are 508 out of 573 (89%) amino acid identities.
  • Figure 2 A shows data indicating An- 104 has asparaginase activity.
  • L-asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartate.
  • Glutamic oxaloacetic transaminase subsequently catalyzes the transamination of L-aspartate and a-ketoglutarate to oxaloacetate and L-glutamate.
  • Oxaloacetate is then reduced to malate in the presence of malic dehydrogenase with the concurrent oxidation of reduced P-nicotinamide adenine dinucleotide (NADH) to P- nicotinamide adenine dinucleotide (NAD+).
  • NADH P-nicotinamide adenine dinucleotide
  • NAD+ P-nicotinamide adenine dinucleotide
  • the time-dependent change in absorbance of NADH at 340 nm is monitored on a microplate reader using the kinetic rate method.
  • the change in absorbance over time is directly proportional to the rate at which the NADH is consumed, and it is directly related to the asparaginase activity of the sample.
  • Figure 2B shows data on asparaginase activity tested at an enzyme concentration of 0.1 mg/mL and an asparagine substrate concentration of 1 pM.
  • Ancestral arginases reported herein typically have an ICso of less than 1 pM.
  • An- 104 demonstrated desirable activity, within a comparable range to the clinically relevant E. coli and Erwinia asparaginases.
  • Figure 3A shows cytotoxicity data testing the ancestral L-ASNase candidates against a T- ALL cell line.
  • CCRF-CEM ICso values calculated using calorimetric MTT assay, correlating metabolic activity with number of viable cells.
  • CCRF-CEM cells 100,000 were incubated with increasing concentrations of An-ASNase for 72 hours and ICso values determined.
  • An- 107 demonstrated the highest cytotoxicity against CCRF-CEM cells.
  • Figure 3B shows data indicating anti-leukemia properties of ancestral L-asparaginases.
  • MOLT-4 (T-ALL) cells 100,000 were incubated with increasing concentrations of An-ASNase for 72 hours and IC50 values determined using a trypan blue cell count.
  • Figure 4A shows constructs using domain swapping providing Ancestral Human Hybrids L-ASNases.
  • Non-functional C-terminal ankyrin repeat domains are replaced with human sequence to increase sequence identity to human L-ASNase.
  • Figure 4B shows a sequence identify comparison of ancestral L-asparaginase-human hybrid sequences to human L-asparaginase.
  • Figure 5 shows results of in silico immunogenicity assessment of ancestral L- asparaginases.
  • the calculations indicate that immunogenic epitope density to HLA-DRB 1*07:01 in ancestral asparaginase candidates is lower when compared to current clinical bacterial asparaginases.
  • the MHCII binding predictions were made using the IEDB analysis resource NetMHCIIpan (ver. 4.1) tool.
  • HLA-DRB 1*07:01 is associated with a higher risk of asparaginase allergies. See Fernandez et al. HLA-DRB 1*07:01 is associated with a higher risk of asparaginase allergies. Blood. 2014, 124(8): 1266-1276. See also Kaabinejadian et al. Accurate MHC Motif Deconvolution of Immunopeptidomics Data Reveals a Significant Contribution of DRB3, 4 and 5 to the Total DR Immunopeptidome. Front Immunol, 2022, 13:835454.
  • Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of immunology, medicine, organic chemistry, biochemistry, molecular biology, pharmacology, physiology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
  • the term "about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/- a percentage of the specified value. In embodiments, about includes the specified value. In certain embodiments, the term “about” can include a 5 % or 10 % difference.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • peptide having an amino acid sequence refers a peptide that may contain additional N-terminal (amine end) or C-terminal (carboxylic acid end) amino acids, i.e., the term is intended to include the amino acid sequence within a larger peptide.
  • consisting of’ in reference to a peptide having an amino acid sequence refers to a peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids expressly specified in the claim.
  • the disclosure contemplates that the “N-terminus of a peptide consists of an amino acid sequence,” which refers to the N-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a rage of amino acids specified in the claim however the C-terminus may be connected to additional amino acids, e.g., as part of a larger peptide.
  • C-terminus of a peptide consists of an amino acid sequence,” which refers to the C-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids specified in the claim however the N-terminus may be connected to additional amino acids, e.g., as part of a larger peptide.
  • a “subject” refers to any animal, preferably a human patient, livestock, or domestic pet.
  • the terms “treat” and “treating” are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments of the present disclosure also contemplate treatment that merely reduces symptoms, and/or delays disease progression.
  • the terms “prevent” and “preventing” include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.
  • the term “combination with” when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof.
  • terapéuticaally effective amount refers to the amount of a protein (e.g., asparaginase or conjugate thereof), required to produce a desired therapeutic effect.
  • nucleic acid refers to a polymer of nucleotides, or a polynucleotide, e.g., RNA, DNA, or a combination thereof. The term is used to designate a single molecule, or a collection of molecules. Nucleic acids may be single stranded or double stranded and may include coding regions and regions of various control elements.
  • encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • polypeptide polypeptide
  • peptide protein
  • polymers of amino acids of any length can comprise modified amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids such as homocysteine, ornithine, p- acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art.
  • a "heterologous" nucleic acid sequence or peptide sequence refers to a nucleic acid sequence or a peptide sequence that does not naturally occur, e.g., because the whole sequence contains a segment from other plants, bacteria, viruses, other organisms, or joinder of two sequences that occur the same organism but are joined together in a manner that does not naturally occur in the same organism or any natural state.
  • nucleic acid molecule when made in reference to a nucleic acid molecule refers to a nucleic acid molecule which is comprised of segments of nucleic acid joined together by means of molecular biological techniques provided that the entire nucleic acid sequence does not occurring in nature, i.e., there is at least one mutation in the overall sequence such that the entire sequence is not naturally occurring even though separately segments may occur in nature. The segments may be joined in an altered arrangement such that the entire nucleic acid sequence from start to finish does not naturally occur.
  • recombinant when made in reference to a protein or a peptide refers to a protein molecule that is expressed using a recombinant nucleic acid molecule.
  • vector refers to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free expression system.
  • Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences.
  • Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
  • this disclosure contemplates a vector encoding a peptide disclosed herein in operable combination with a heterologous promoter.
  • Protein "expression systems” refer to in vivo and in vitro (cell free) systems. Systems for recombinant protein expression typically utilize somatic cells transfected with a DNA expression vector that contains the template. The cells are cultured under conditions such that they translate the desired protein. Expressed proteins are extracted for subsequent purification. In vivo protein expression systems using prokaryotic and eukaryotic cells are well known. Proteins may be recovered using denaturants and protein-refolding procedures.
  • In vitro (cell-free) protein expression systems typically use translation-compatible extracts of whole cells or compositions that contain components sufficient for transcription, translation, and optionally post-translational modifications such as RNA polymerase, regulatory protein factors, transcription factors, ribosomes, tRNA cofactors, amino acids, and nucleotides. In the presence of an expression vector, these extracts and components can synthesize proteins of interest.
  • Cell-free systems typically do not contain proteases and enable labeling of the protein with modified amino acids. See, e.g., Shimizu et al., Cell-free translation reconstituted with purified components, 2001, Nat. Biotechnol., 19, 751-755 and Asahara & Chong, Nucleic Acids Research, 2010, 38(13): el41 , both hereby incorporated by reference in their entirety.
  • a “variant” refers to a chemically similar peptide sequence because of amino acid changes.
  • a variant contains one or two, or more amino acid substitutions, deletions, or insertions.
  • the substitutions are conserved substitutions.
  • a variant contains one, two, or ten or more, or ten or less amino acid additions.
  • the additions may be to the N-terminus or the C-terminus.
  • the variant may be substituted with one or more chemical substituents.
  • a conservative amino acid substitution refers to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
  • a variant may have "non-conservative" changes (e.g., replacement of a glycine with a tryptophan).
  • Similar minor variations may also include amino acid deletions or insertions (in other words, additions), or both.
  • Guidance in determining which and how many amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art. Variants can be tested in functional assays.
  • variants have less than 10%, and preferably less than 5%, and still more preferably less than 2% changes (whether substitutions, deletions, and so on). Variants can be prepared for testing by mutating a vector to produce appropriate codon alternatives for peptide translation.
  • sequence “identity” refers to the number of exactly matching amino acids (expressed as a percentage) in a sequence alignment between two sequences of the alignment calculated using the number of identical positions divided by the greater of the shortest sequence or the number of equivalent positions excluding overhangs wherein internal gaps are counted as an equivalent position.
  • any recitation of sequence identity expressed herein may be substituted for sequence similarity.
  • Percent “similarity” is used to quantify the similarity between two sequences of the alignment. This method is identical to determining the identity except that certain amino acids do not have to be identical to have a match.
  • Amino acids are classified as matches if they are among a group with similar properties according to the following amino acid groups: Aromatic - F Y W; hydrophobic-A V I L; Charged positive: R K H; Charged negative - D E; Polar - S T N Q.
  • the amino acid groups are also considered conserved substitutions.
  • conjugation refers to linking molecular entities through covalent bonds, or by other specific binding interactions, such as due to hydrogen bonding or other van der Walls forces.
  • the force to break a covalent bond is high, e.g., about 1500 pN for a carbon-to- carbon bond.
  • the force to break a combination of strong protein interactions is typically a magnitude less, e.g., biotin to streptavidin is about 150 pN.
  • conjugation must be strong enough to bind molecular entities in order to implement the intended results.
  • a "linking group” refers to any variety of molecular arrangements that can be used to bridge to molecular moieties together.
  • linking groups include bridging amide groups, alkyl groups, alkoxy groups, alkoxyalkyl groups, and combinations thereof.
  • nucleic acid molecule when made in reference to a nucleic acid molecule refers to a nucleic acid molecule which is comprised of segments of nucleic acid joined together by means of molecular biological techniques.
  • recombinant when made in reference to a protein or a polypeptide refers to a protein molecule which is expressed using a recombinant nucleic acid molecule.
  • vector or “ expression vector” refer to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free.
  • Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences.
  • Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
  • Protein “expression systems” refer to in vivo and in vitro (cell free) systems.
  • Systems for recombinant protein expression typically utilize cells, e.g., somatic cells, transfected with a DNA expression vector that contains the template. The cells are cultured under conditions such that they translate the desired protein. Expressed proteins are extracted for subsequent purification.
  • In vivo protein expression systems using prokaryotic and eukaryotic cells are well known. Also, some proteins are recovered using denaturants and protein-refolding procedures.
  • In vitro (cell-free) protein expression systems typically use translation-compatible extracts of whole cells or compositions that contain components sufficient for transcription, translation, and optionally post- translational modifications such as RNA polymerase, regulatory protein factors, transcription factors, ribosomes, tRNA cofactors, amino acids, and nucleotides. In the presence of an expression vectors, these extracts and components can synthesize proteins of interest. Cell-free systems typically do not contain proteases and enable labeling of the protein with modified amino acids. Some cell free systems incorporated encoded components for translation into the expression vector.
  • a “selectable marker” is a nucleic acid introduced into a recombinant vector that encodes a polypeptide that confers a trait suitable for artificial selection or identification (report gene), e.g., beta-lactamase confers antibiotic resistance, which allows an organism expressing beta-lactamase to survive in the presence antibiotic in a growth medium.
  • a trait suitable for artificial selection or identification e.g., beta-lactamase confers antibiotic resistance, which allows an organism expressing beta-lactamase to survive in the presence antibiotic in a growth medium.
  • Another example is thymidine kinase, which makes the host sensitive to ganciclovir selection. It may be a screenable marker that allows one to distinguish between wanted and unwanted cells based on the presence or absence of an expected color.
  • the lac-z-gene produces a beta-galactosidase enzyme which confers a blue color in the presence of X-gal (5-bromo-4-chloro-3-indolyl-P-D-galactoside). If recombinant insertion inactivates the lac-z-gene, then the resulting colonies are colorless.
  • selectable markers e.g., an enzyme that can complement to the inability of an expression organism to synthesize a particular compound required for its growth (auxotrophic) and one able to convert a compound to another that is toxic for growth.
  • URA3 an orotidine-5 1 phosphate decarboxylase, is necessary for uracil biosynthesis and can complement ura3 mutants that are auxotrophic for uracil. URA3 also converts 5-fluoroorotic acid into the toxic compound 5 -fluorouracil. Additional contemplated selectable markers include any genes that impart antibacterial resistance or express a fluorescent protein.
  • Examples include, but are not limited to, the following genes: ampr, camr, tetr, blasticidinr, neor, hygr, abxr, neomycin phosphotransferase type II gene (nptll), p-glucuronidase (gus), green fluorescent protein (gfp), egfp, yfp, mCherry, p- galactosidase (lacZ), lacZa, lacZAM15, chloramphenicol acetyltransferase (cat), alkaline phosphatase (phoA), bacterial luciferase (luxAB), bialaphos resistance gene (bar), phosphomannose isomerase (pmi), xylose isomerase (xylA), arabitol dehydrogenase (atlD), UDP- glucose:galactose-l -phosphate uridyltransferase (galT), feedback-insensitive a sub
  • GSA-AT glutamate 1 -semialdehyde aminotransferase
  • DAAO D-amino acidoxidase
  • rstB ferredoxin-like protein
  • pflp ferredoxin-like protein
  • AtTPSl trehalose-6-P synthase gene
  • lyr lysine racemase
  • dapA dihydrodipicolinate synthase
  • AtTSBl tryptophan synthase beta 1
  • dehalogenase dhlA
  • M6PR mannose-6-phosphate reductase gene
  • HPT hygromycin phosphotransferase
  • dsdA D-serine ammonialyase
  • this disclosure relates to recombinant ancestral variant asparaginases comprising an amino acid sequence selected from SEQ ID NO: 1-53 or variant thereof having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity or similarity.
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution.
  • the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase is a truncated version of SEQ ID NO: 1-53, e g., comprising an amino acid sequence selected from amino acids 1 to 359 of SEQ ID NO: 1-53 or variant thereof having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the truncated version, wherein the N-terminal methionine (M) is position 1.
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase is a truncated version of SEQ ID NO: 1-53, e g., comprising an amino acid sequence selected from amino acids 1 to 396 of SEQ ID NO: 1-53 or variant thereof having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the truncated version.
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution.
  • the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase is a truncated versions of SEQ ID NO: 1-53, e.g., wherein the N-terminal methionine (M) is position 1, comprising an amino acid sequence selected from amino acids 10 to 573 of SEQ ID NO: 1-53, amino acids 1 to 563 of SEQ ID NO: 1-53, amino acids 20 to 573 of SEQ ID NO: 1-53, amino acids 1 to 553 of SEQ ID NO: 1-53, amino acids 30 to 573 of SEQ ID NO: 1-53, amino acids 1 to 543 of SEQ ID NO: 1-53, amino acids 40 to 573 of SEQ ID NO: 1-53, amino acids 1 to 533 of SEQ ID NO: 1-53, amino acids 50 to 573 of SEQ ID NO: 1-53, amino acids 1 to 523 of SEQ ID NO: 1-53, amino acids 60 to 573 of SEQ ID NO: 1-53, amino acids 1 to 513 of SEQ ID NO: 1-53, amino acids 70 to 573 of SEQ ID NO:
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO: 10 (An 104) or a variant having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity.
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution.
  • the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO: 2 (An 69) or a variant having 98%, 99%, or greater sequence identity. In certain embodiments, the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO:
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO: 3 (An 70) or a variant having 99%, or greater sequence identity. In certain embodiments, the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO:
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO:
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO:
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO:
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO: 12 (An 107) or a variant having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity.
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution.
  • the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO: 53 (An 108) or a variant having 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity.
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution.
  • the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • this disclosure relates to recombinant ancestral variant asparaginases comprising an amino acid sequence selected from SEQ ID NO: 54-63 or variant thereof having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity or similarity.
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution.
  • the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase is a truncated version of SEQ ID NO: 54-63, e.g., comprising an amino acid sequence selected from amino acids 1 to 359 of SEQ ID NO: 54-63 or variant thereof having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the truncated version, wherein the N-terminal methionine (M) is position 1.
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution.
  • the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase is a truncated version of SEQ ID NO: 54-63, e.g., comprising an amino acid sequence selected from amino acids 1 to 396 of SEQ ID NO: 54-63 or variant thereof having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the truncated version.
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution.
  • the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase is a truncated versions of SEQ ID NO: 54-63, e.g., wherein the N-terminal methionine (M) is position 1, comprising an amino acid sequence selected from amino acids 10 to 573 of SEQ ID NO: 54-63, amino acids 1 to 563 of SEQ ID NO: 54-63, amino acids 20 to 573 of SEQ ID NO: 54-63, amino acids 1 to 553 of SEQ ID NO: 54-63, amino acids 30 to 573 of SEQ ID NO: 54-63, amino acids 1 to 543 of SEQ ID NO: 54-63, amino acids 40 to 573 of SEQ ID NO: 54-63, amino acids 1 to 533 of SEQ ID NO: 54-63, amino acids 50 to 573 of SEQ ID NO: 54-63, amino acids 1 to 523 of SEQ ID NO: 54-63, amino acids 60 to 573 of SEQ ID NO: 54-63, amino acids 1 to 513 of SEQ ID NO
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO:
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO:
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO:
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO:
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO:
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO:
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO:
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO:
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has SEQ ID NO:
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the recombinant ancestral variant asparaginase has SEQ ID NO:
  • the recombinant ancestral variant asparaginase variant has 1 amino acid substitution. In certain embodiments, the recombinant ancestral variant asparaginase has 2 or 3 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 4 or 5 amino acid substitutions. In certain embodiments, the recombinant ancestral variant asparaginase has 6 or 7 amino acid substitutions.
  • the ancestral variant asparaginases as disclosed herein can be produced by any commonly used method. Typical examples include the recombinant expression in suitable host systems, e.g., cell, mammalian cell, bacteria, or yeast.
  • suitable host systems e.g., cell, mammalian cell, bacteria, or yeast.
  • the ancestral variant asparaginases may be produced by living host cells that have been genetically engineered to produce the polypeptide. Methods of genetically engineering cells to produce proteins are well known in the art. See e.g., Ausubel et al., eds. (1990), Current Protocols in Molecular Biology (Wiley, New York). Such methods include introducing nucleic acids that encode and allow expression of the polypeptide into host cells. These host cells can be bacterial cells, fungal cells, or animal cells grown in culture.
  • ancestral variant asparaginases are produced in mammalian cells.
  • Typical mammalian host cells for expressing the asparaginase include Chinese Hamster Ovary (CHO cells), lymphocytic cell lines, e.g., NSO myeloma cells, SP2 cells, COS cells.
  • the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e ., U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017).
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced.
  • Standard molecular biology techniques can be used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the ancestral variant asparaginases or cells coated with the peptide from the culture medium.
  • the ancestral variant asparaginases or cells can be isolated by affinity chromatography.
  • this disclosure relates to nucleotide sequences or nucleic acids that encode ancestral variant asparaginases as disclosed herein, genetic constructs that include nucleotide sequences or nucleic acids and one or more elements for genetic constructs known per se. In certain embodiments, this disclosure relates to hosts or host cells that contain such nucleotide sequences or nucleic acids, and/or that express (or are capable of expressing) ancestral variant asparaginases disclosed herein.
  • this disclosure relates to methods for preparing ancestral variant asparaginases or cells expressing the asparaginases using constructs disclosed herein, which method comprises cultivating or maintaining a host cell under conditions such that said host cell produces or expresses the ancestral variant asparaginases as disclosed herein.
  • the disclosure relates to recombinant ancestral variant asparaginases comprising sequences disclosed herein or variants or fusions thereof wherein the interior amino acid sequence, the amino terminal end, or the carbon terminal end of the amino acid sequence are optionally attached to a heterologous amino acid sequence, label, or reporter molecule.
  • the disclosure relates to the recombinant vectors comprising a nucleic acid encoding an ancestral variant asparaginase as disclosed herein.
  • the recombinant vector optionally comprises a mammalian, human, insect, viral, bacterial, bacterial plasmid, yeast associated origin of replication or gene such as a gene or retroviral gene or lentiviral LTR, TAR, RRE, PE, SLIP, CRS, and INS nucleotide segment or gene selected from tat, rev, nef, vif, vpr, vpu, and vpx or structural genes selected from gag, pol, and env.
  • the recombinant vector optionally comprises a gene vector element (nucleic acid) such as a selectable marker region, lac operon, a CMV promoter, a hybrid chicken B-actin/CMV enhancer (CAG) promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter, internal ribosome entry site (IRES) sequence, cis-acting woodchuck post regulatory element (WPRE), scaffold-attachment region (SAR), inverted terminal repeats (ITR), c-myc tag coding region, metal affinity tag coding region, streptavidin binding peptide tag coding region, polyHis tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, polyadenylation coding region, SV40 polyadenylation signal, SV40 origin of replication, Col El origin of replication, fl origin, pBR322 origin, or pUC origin, TEV prote
  • the recombinant ancestral variant asparaginase is conjugated, e.g., through a linking group, to a biodegradable polymer such as polyethylene glycol, mono-methoxypolyethylene glycol, a fatty acid, or combinations thereof.
  • a biodegradable polymer such as polyethylene glycol, mono-methoxypolyethylene glycol, a fatty acid, or combinations thereof.
  • this disclosure relates to a conjugate of an ancestral variant asparaginase as disclosed herein having substantial L-asparagine amidohydrolase activity and polyethylene glycol, wherein the polyethylene glycol has a molecular weight less than or equal to or about 100, 500, 1000, 2000, 3000, 4000, or 5000 Da.
  • the ancestral variant asparaginase conjugate has a sequence as reported herein and has an in vitro activity of at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the ancestral variant asparaginase when not conjugated to a biodegradable polymer, e.g., polyethylene glycol.
  • a biodegradable polymer e.g., polyethylene glycol
  • the recombinant asparaginases disclosed herein elicit a lower immunogenic response in a patient compared to other clinically approved L-asparaginases, e.g., E. coli L-asparaginase or Erwinia chrysanthemi L-asparaginase.
  • significantly reduced immunogenicity is evidenced, e.g., by the reduction or elimination of an antibody response against the L-asparaginase preparation following administration or repeated administrations; and/or usefulness as a second-line therapy for patients who have developed sensitivity to first-line therapies using, e.g., E.
  • the recombinant asparaginases disclosed herein provide reduced immunogenicity and enhanced plasma half-life. In certain embodiments, the recombinant asparaginases disclosed herein are used in methods of treating patients with relapsed ALL who were previously treated with other asparaginase preparations, in particular those who were previously treated with E. coli-derived asparaginases.
  • the conjugate has a longer in vivo circulating half-life compared to the ancestral variant asparaginase when not conjugated to a biodegradable polymer, e.g., polyethylene glycol.
  • a biodegradable polymer e.g., polyethylene glycol
  • the biodegradable polymer e.g., polyethylene glycol
  • the biodegradable polymer, e.g., polyethylene glycol is covalently linked to the one or more amino groups by an amide bond.
  • the biodegradable polymer e.g., polyethylene glycol
  • the biodegradable polymer is covalently linked to at least from about 40% to about 100% of the accessible amino groups (e.g., lysine residues and/or the N-terminus of the protein) or at least from about 40% to about 90% of total amino groups (e.g., lysine residues and/or the N-terminus of the protein).
  • the ancestral variant asparaginase or conjugate is administered at a dose of 4000 IU/m 2 intravenously. In certain embodiments, the ancestral variant asparaginase or conjugate is administered at a dose of 3000 IU/m 2 intravenously. In certain embodiments, the ancestral variant asparaginase or conjugate is administered at a dose of 2500 IU/m 2 intravenously. In certain embodiments, the ancestral variant asparaginase or conjugate is administered at a dose of 2000 IU/m 2 intravenously. In certain embodiments, the ancestral variant asparaginase or conjugate is administered at a dose of 1500 IU/m 2 intravenously. In certain embodiments, the ancestral variant asparaginase or conjugate is administered at a dose of 1000 IU/m 2 intravenously.
  • the ancestral variant asparaginase or conjugate depletes blood or plasma asparagine levels to between or less than 0.05-0.4 lU/mL, or less than 0.02 lU/mL, or an undetectable level for at least about 12, 24, 48, 96, 108, or 120 hours.
  • this disclosure relates to recombinant ancestral variant asparaginases or conjugate that provide a reduced risk of anaphylaxis, urticaria, itching, bronchospasm, hepatotoxicity, pancreatitis, coagulopathy, hyperammonemia, neurotoxicity, hemorrhage, thrombosis, and or high titers of serum immunoglobulin G (IgG) antibodies, e.g., when compared to pegaspargase (SS-PEG asparaginase), calaspargase pegol (SC-PEG asparaginase, E.
  • IgG serum immunoglobulin G
  • this disclosure relates to recombinant ancestral variant asparaginases or conjugate that provide a reduced glutaminase activity, e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, e.g., when compared to pegaspargase (SS-PEG asparaginase), calaspargase pegol (SC-PEG asparaginase, E. coli L-asparaginase), asparaginase Erwinia chrysanthemi, or crisantaspase (recombinant asparaginase Erwinia chrysanthemi).
  • L-asparaginase is a component of the chemotherapy armamentarium used to treat acute lymphoblastic leukemia (ALL). L-ASNase plays a significant role in improving ALL survival outcomes. Preclinical data indicated that L-ASNase is beneficial in several solid malignancies such as pancreatic cancer, colorectal cancer, and metastatic breast cancer. However, because existing clinical L-ASNases are bacterial in origin, e.g., derived from either Escherichia coli or Erwinia chrysanthemi. Thus, they are sometimes highly immunogenic, with reactions ranging from silent inactivation to severe anaphylaxis, mediated by the development of anti-L- ASNase IgG and IgE antibodies.
  • liver and pancreatic toxicity is seen in adults treated with L-ASNase, thus limiting its widespread use beyond ALL, especially in adult solid malignancies. Emerging data indicates that this toxicity is related to the concurrent glutaminase activity seen in bacterial L-ASNases. Thus, experiments were performed to identify less immunogenic L-ASNase with reduced glutaminase activity.
  • L-ASNase catalyzes the production of free L-aspartic acid from the amino acid L-asparagine, thereby depleting the circulating pool of L-asparagine.
  • ALL and other tumors that lack asparagine synthase activity are dependent on the extrinsic supply of asparagine for protein synthesis and are therefore extremely sensitive to L-ASNase.
  • the anti-tumor properties of L-ASNase were reported to be first discovered when mice treated with guinea pig serum demonstrated regression in subcutaneous lymphomas. This response was later confirmed to be because of guinea pig L-ASNase.
  • ASR Ancestral sequence reconstruction
  • guinea pig L-ASNase has now confirmed the significantly favorable anti-leukemic and enzymatic properties of guinea pig L-ASNase compared to human L-ASNase. Furthermore, guinea pig L- ASNase shares approximately 70% sequence identity with human L-ASNase compared to the approximately 30% identity shared by bacterial L-ASNases. Additionally, guinea pig L-ASNase has reduced glutaminase activity which may result in lower toxicity in adults. It is contemplated that ancestral sequence reconstruction is a viable platform to bioengineer variant recombinant L- asparaginases with reduced toxicides and improved enzymatic specificity. Experiments were performed to identify and characterize ancient DNA sequences along the human and guinea pig lineage to allow for the mapping of functional residues, with the goal of developing an enhanced, less immunogenic, therapeutic L-ASNase candidate.
  • a phylogenic tree was constructed utilizing extant L-ASNase sequences and ancestral sequences along the human and guinea pig lineage were identify.
  • cDNA sequences for ancestral L-ASNase (An-ASNase) variants are ligated into a His6-SUMO-pET14b vector and transformed into E. coli BL21(DE3) cells for protein expression.
  • kinetic properties of An-ASNase variants are determined and compared to bacterial, guinea pig and human L-ASNase controls. Crystal structures of lead candidates are evaluated. Combining the functional and structural data gained, variant bioengineered human L-ASNase variants are designed and tested.
  • this disclosure relates to methods of treating a disease or condition such as cancer comprising administering a recombinant ancestral variant asparaginase or conjugate as disclosed herein to a subject in need thereof.
  • the recombinant ancestral variant asparaginase or conjugate comprises an amino acid sequence selected from SEQ ID NO: 1-53 or variant thereof having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity.
  • this disclosure relates to methods of treating a disease or condition such as cancer comprising administering a recombinant ancestral variant asparaginase or conjugate as disclosed herein to a subject in need thereof.
  • the recombinant ancestral variant asparaginase or conjugate comprises an amino acid sequence selected from SEQ ID NO: 54-63 or variant thereof having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity.
  • this disclosure relates to a method for treating acute lymphoblastic leukemia (ALL) comprising administering to a patient in need of a therapeutically effective amount of an ancestral variant asparaginase or conjugate as disclosed herein.
  • treatment with an ancestral variant asparaginase or conjugate is administered as a first line therapy or second line therapy in patients, particularly patients with ALL, where objective signs of allergy or hypersensitivity, including “silent hypersensitivity,” have developed to other asparaginase preparations, e.g., subject is “antibody positive” for an asparaginase enzyme.
  • administration is about twice a week to about once a month, typically once per week or once every other week, as a single agent (e.g., monotherapy) or as part of a combination of chemotherapy drugs, including, but not limited to glucocorticoids, corticosteroids, anticancer compounds or other agents, including, but not limited to methotrexate, dexamethasone, prednisone, prednisolone, vincristine, cyclophosphamide, and anthracycline.
  • patients with ALL will be administered the ancestral variant asparaginase or conjugate as disclosed herein as a component of multi-agent chemotherapy during 1, 2, or 3 chemotherapy phases including induction, consolidation or intensification, and maintenance.
  • the patient has relapsed ALL.
  • the patient was previously treated with other asparaginase preparations, e.g., bacterial or E. coli derived asparaginases.
  • the recombinant asparaginases disclosed herein elicit a lower immunogenic response in a patient compared to other clinically approved L-asparaginases, e.g., E. coli L-asparaginase or Erwinia chrysanthemi L-asparaginase.
  • significantly reduced immunogenicity is evidenced, e.g., by the reduction or elimination of an antibody response against the L-asparaginase preparation following administration or repeated administrations; and/or usefulness as a second-line therapy for patients who have developed sensitivity to first-line therapies using, e.g., E.
  • the recombinant asparaginases disclosed herein provide reduced immunogenicity and enhanced plasma half-life. In certain embodiments, the recombinant asparaginases disclosed herein are used in methods of treating patients with relapsed ALL who were previously treated with other asparaginase preparations, in particular those who were previously treated with E. coli-derived asparaginases.
  • the ancestral variant asparaginase or conjugate is administered at a dose of 4000 IU/m 2 intravenously. In certain embodiments, the ancestral variant asparaginase or conjugate is administered at a dose of 3000 IU/m 2 intravenously. In certain embodiments, the ancestral variant asparaginase or conjugate is administered at a dose of 2500 IU/m 2 intravenously. In certain embodiments, the ancestral variant asparaginase or conjugate is administered at a dose of 2000 IU/m 2 intravenously. In certain embodiments, the ancestral variant asparaginase or conjugate is administered at a dose of 1500 IU/m 2 intravenously. In certain embodiments, the ancestral variant asparaginase or conjugate is administered at a dose of 1000 IU/m 2 intravenously.
  • the ancestral variant asparaginase or conjugate depletes plasma asparagine levels to between 0.05-0.4 lU/mL, or less than 0.05 lU/mL, or less than 0.04 lU/mL, or less than 0.03 lU/mL, or less than 0.02 lU/mL, or an undetectable level for at least about 12, 24, 48, 96, 108, or 120 hours.
  • Cancer refers to any of various cellular diseases with malignant neoplasms characterized by the proliferation of cells. It is not intended that the diseased cells must actually invade surrounding tissue and metastasize to new body sites. Cancer can involve any tissue of the body and has many different forms in each body area. Within the context of certain embodiments, whether “cancer is reduced” may be identified by a variety of diagnostic manners known to one skill in the art including, but not limited to, observation the reduction in size or number of tumor masses or if an increase of apoptosis of cancer cells observed, e.g., if more than a 5 % increase in apoptosis of cancer cells is observed for a sample compound compared to a control without the compound. It may also be identified by a change in relevant biomarker or gene expression profile, such as PSA for prostate cancer, HER2 for breast cancer, or others.
  • the cancer is a hematological cancer or a solid cancer.
  • the cancer is leukemia, lymphoma, acute lymphoblastic leukemia (ALL) lymphoblastic lymphoma (LBL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), chronic myelogenous leukemia, acute monocytic leukemia (AMOL), chronic myeloid leukemia (CML), B-cell acute lymphoblastic leukemia (B-ALL), myeloproliferative neoplasms (MPNs), and lymphomas, Hodgkin's lymphomas, and non-Hodgkin's lymphomas such as Burkitt lymphoma, B-cell lymphoma, or diffuse large B-cell lymphoma (DLBCL).
  • ALL lymphoblastic leukemia
  • LBL acute myelogenous leukemia
  • AML acute myelogenous le
  • treatment is done in combination with chemotherapy in combination with radiation therapy or a stem cell transplant, e.g., hematopoietic stem cell transplant.
  • a stem cell transplant e.g., hematopoietic stem cell transplant.
  • the recombinant ancestral variant asparaginase or conjugate is administered in combination with another anticancer agent.
  • a “chemotherapy agent,” “chemotherapeutic,” “anti-cancer agent,” or the like, refer to molecules that are recognized to aid in the treatment of a cancer.
  • Contemplated examples include the following molecules or derivatives such as abemaciclib, abiraterone acetate, methotrexate, paclitaxel, adriamycin, acalabrutinib, brentuximab vedotin, ado-trastuzumab emtansine, aflibercept, afatinib, netupitant, palonosetron, imiquimod, aldesleukin, alectinib, alemtuzumab, pemetrexed disodium, copanlisib, melphalan, brigatinib, chlorambucil, amifostine, aminolevulinic acid, anastrozole, apalutamide, aprepitant, pamidronate disodium, exemestane, nelarabine, arsenic trioxide, ofatumumab, atezolizumab, bevacizumab, ave
  • the anticancer agent is cytarabine (ara-C) and an anthracycline drug.
  • the anthracycline drug is daunorubicin or idarubicin.
  • the anticancer agent is cladribine, fludarabine, or etoposide.
  • the anticancer agent is prednisone, dexamethasone, vincristine, daunorubicin, doxorubicin, cyclophosphamide, methotrexate, cytarabine, 6-mercaptopurine, 6- thioguanine or nelarabine.
  • the subject is diagnosed with leukemia cells having an FLT3 gene mutation and the ancestral variant asparaginase or conjugate is administered in combination with administering midostaurin.
  • the subject is diagnosed with leukemia cells having a high expression of CD33 and the ancestral variant asparaginase or conjugate is administered in combination with administering gemtuzumab ozogamicin.
  • the chemotherapy agent is an anti-PD-1, anti-PD-Ll anti-CTLA4 antibody or combinations thereof, such as an anti-CTLA4 (e.g., ipilimumab, tremelimumab) and anti-PDl (e.g., nivolumab, pembrolizumab, cemiplimab) and anti-PD-Ll (e.g., atezolizumab, avelumab, durvalumab).
  • an anti-CTLA4 e.g., ipilimumab, tremelimumab
  • anti-PDl e.g., nivolumab, pembrolizumab, cemiplimab
  • anti-PD-Ll e.g., atez
  • the ancestral variant asparaginase or conjugate is administered before or after receiving bone marrow transplant or blood stem cell transplant and optionally radiation therapy.
  • the method of administration is in a subject with a lymphodepleted environment due to prior or concurrent administration of lymphodepl eting agents.
  • lymphodepleting agents e.g., cyclophosphamide and fludarabine.
  • the subject is a human patient.
  • the cancer is selected from bladder cancer, lung cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, pancreatic cancer, kidney cancer, prostate cancer, thyroid cancer, brain cancer, multiple myeloma, lymphoma, or leukemia.
  • the disclosure also provides for the use of an ancestral variant asparaginase or conjugate disclosed herein for the preparation of a medicament.
  • Such preparations are contemplated for the treatment of a cancer or neoplasm in a human patient or other mammal.
  • this disclosure relates to methods for the treatment a subject at risk of, exhibiting symptoms of, suspected of, or diagnosed with a cancer or neoplasm selected from skin cancer, melanoma, Barret's adenocarcinoma; biliary tract carcinomas; breast cancer; cervical cancer; cholangiocarcinoma; central nervous system tumors including primary CNS tumors such as glioblastomas, astrocytomas (including glioblastoma multiforme) and ependymomas, and secondary CNS tumors (i.e., metastases to the central nervous system of tumors originating outside of the central nervous system), colorectal cancer, including large intestinal colon carcinoma; gastric cancer; carcinoma of the head and neck including squamous cell carcinoma of the head and neck; hematologic cancers including leukemias and lymphomas such as acute lymphoblastic leukemia, acute myelogenous leukemia (AML), myelodysplastic syndromes, chronic mye
  • this disclosure relates to treating diseases associated with asparagine dependence comprising administering an effective amount of an ancestral variant asparaginase or conjugate disclosed herein to a subject in need thereof.
  • the disease is a non-malignant hematologic disease which respond to asparagine depletion include immune system-mediated blood diseases, e.g., infectious diseases such as those caused by HIV infection (i.e., AIDS).
  • immune system-mediated blood diseases e.g., infectious diseases such as those caused by HIV infection (i.e., AIDS).
  • non-hematologic diseases associated with asparagine dependence include autoimmune diseases, for example rheumatoid arthritis, SLE, autoimmune, collagen vascular diseases, AIDS, etc.
  • the disease is an autoimmune disease such as osteoarthritis, psoriasis, insulin dependent diabetes mellitus, multiple sclerosis, sclerosing panencephalitis, systemic lupus erythematosus, rheumatic fever, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), chronic active hepatitis, glomerulonephritis, myasthenia gravis, pemphigus vulgaris, and Graves' disease.
  • autoimmune disease such as osteoarthritis, psoriasis, insulin dependent diabetes mellitus, multiple sclerosis, sclerosing panencephalitis, systemic lupus erythematosus, rheumatic fever, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), chronic active hepatitis, glomerulonephritis, myasthenia gravis, pemphigus vulgaris, and Graves' disease.
  • the ancestral variant asparaginase or conjugate disclosed herein can be used alone in the treatment of each of the foregoing conditions or can be used to provide additive or potentially synergistic effects with certain existing chemotherapies, radiation, biological or immunotherapeutics (including monoclonal antibodies) and vaccines.
  • the ancestral variant recombinant asparaginase or conjugate disclosed herein may be useful for restoring effectiveness of certain existing chemotherapies and radiation and or increasing sensitivity to certain existing therapies, chemotherapies, and/or radiation.
  • the subject is a human subject is 2, 12, or 16 years old or older. In certain embodiments, the subject is a human subject is 2, 12, or 15 years old or less than 2, 12, or 16 years old. In certain embodiments, the subject is a human subject is 55 or 65 years old or older.
  • the subject is a human subject is an infant, e.g., from one month to two years of age. In certain embodiments, the subject is a human subject is a child, e.g., from one two to twelve years of age. In certain embodiments, the subject is a human subject is an adolescent, e.g., from twelve to sixteen years of age. In certain embodiments, the subject is a human subject sixteen years of age or older.
  • this disclosure relates to pharmaceutical compositions comprising ancestral variant asparaginase or conjugate disclosed herein and optionally a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises a recombinant ancestral variant or conjugated as disclosed herein which is in a vial in lyophilized form.
  • the pharmaceutical composition comprises a recombinant ancestral variant or conjugated as disclosed herein which is in an aqueous pH buffered isotonic solution.
  • this disclosure relates to pharmaceutical compositions comprising ancestral variant asparaginase or conjugate disclosed herein contained in a vial as a lyophilized powder to be reconstituted with a solvent.
  • the pharmaceutical composition is a “ready to use” solution enabling, further to an appropriate handling, an administration through, e.g., intramuscular, intravenous (infusion and/or bolus), intra-cerebro-ventricular (icv), or subcutaneous routes.
  • compositions comprising ancestral variant asparaginase or conjugate disclosed herein can be administered to a patient using standard techniques. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa., 1990 (herein incorporated by reference). Suitable dosage forms, in part, depend upon the use or the route of entry, for example, oral, transdermal, transmucosal, or by injection (parenteral). Such dosage forms should allow the therapeutic agent to reach a target cell or otherwise have the desired therapeutic effect. For example, pharmaceutical compositions injected into the blood stream preferably are soluble.
  • ancestral variant asparaginase or conjugate disclosed herein can be formulated as pharmaceutically acceptable salts and complexes thereof.
  • Pharmaceutically acceptable carriers and/or excipients can also be incorporated into a pharmaceutical composition to facilitate administration of the particular ancestral variant asparaginase, or conjugate disclosed herein.
  • carriers include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and physiologically compatible solvents.
  • physiologically compatible solvents include sterile solutions of water for injection (WFI), saline solution and dextrose.
  • the ancestral variant asparaginase or conjugate disclosed herein may be formulated into conventional oral dosage forms such as capsules, tablets, pills, microparticles, and liquid preparations such as syrups, elixirs, and concentrated drops.
  • Pharmaceutical compositions according to the invention can be administered by different routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical (transdermal), or transmucosal administration.
  • injection parenteral administration
  • pharmaceutical compositions are formulated in liquid solutions, preferably in physiologically compatible buffers or solutions, such as saline solution, Hank's solution, or Ringer's solution.
  • the ancestral variant asparaginase or conjugate disclosed herein may be formulated in solid form and redissolved or suspended immediately prior to use. For example, lyophilized forms of the conjugate can be produced.
  • the conjugate is administered intramuscularly. In certain embodiments, the conjugate is administered intravenously.
  • Systemic administration can also be accomplished by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are well known in the art, and include, for example, transmucosal administration, bile salts, and fusidic acid derivatives.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration for example, may be through nasal sprays, inhalers (for pulmonary delivery), or rectal suppositories.
  • compounds can be formulated into ointments, salves, gels, or creams.
  • the amounts of the conjugate to be delivered will depend on many factors, for example, the biological half-life of the compound, the age, size, weight, and physical condition of the patient, and the disease or disorder to be treated. The importance of these and other factors to be considered are well known to those of ordinary skill in the art.
  • the amount of the conjugate to be administered will range from about 10 International Units per square meter of the surface area of the patient's body (IU/m 2 ) to 50,000 IU/m 2 , with a dosage range of about 1,000 IU/m 2 to about 15,000 IU/m 2 , and a range of about 6,000 IU/m 2 to about 15,000 IU/m 2 , and a range of about 10,000 to about 15,000 IU/m 2 (about 20-30 mg protein/m 2 ) to treat a malignant hematologic disease, e.g., leukemia.
  • a malignant hematologic disease e.g., leukemia.
  • these dosages are administered via intramuscular or intravenous injection at an interval of about once per week or once every other week during the course of therapy, or 3 times weekly, to about once per month.
  • Other dosages and/or treatment regimens may be employed as determined by the attending physician.
  • ASR is an in-silico method for studying the predicted molecular evolution of a specific protein. The process involves comparing DNA and/or protein sequences of orthologs and inferring evolutionary ancestors of extant species using a bioinformatics pipeline. By custom synthesizing the ancestral cDNA sequences and expressing them in cell culture, recombinant versions of the predicted ancient proteins can be isolated and studied in the laboratory with the goal of identifying amino acid substitutions that contribute to enhanced functionality.
  • ASR will be performed utilizing publicly available extant mammalian L-ASNase sequences including the human and guinea pig LASNase sequence.
  • Guinea pig L-ASNase demonstrating improved enzyme kinetic properties despite a 69.8% sequence identity and 88.6% homology with human L-ASNase suggest one can build a phylogenic tree common to ancestors of both human and guinea pig.
  • L-ASNase sequences were aligned using MUSCLE and an evolutionary phylogeny tree.
  • Ancestral L-ASNase sequences were identified for analysis which have an identity ranging from about 70%-99% when compared to human L-ASNase.
  • Table 1 shows a comparison of amino acid identities of ancient L-Asparaginase to human L- Asparaginase.
  • cDNA Complementary DNA sequences were generated using an E. coli codon optimization algorithm and synthesized de novo to contain 5' Ndel and 3' BamHI restriction sites for subcloning into a His6-SUMO-pET14b expression vector. Sequences along the lineage where human and guinea pig converge are the nodes designated for resurrection. Plasmids with correctly inserted genes are transformed into E. coli BL21(DE3) cells for protein expression. Cells are grown at 37 °C in Terrific Broth (TB) until optical density of 0.6-0.8 is reached.
  • TB Terrific Broth
  • Target gene is induced for expression by 0.3 mM isopropyl P-D-l- thiogalactopyranoside (IPTG), followed by incubation at 18 °C overnight growth. After lysis by sonification, the An-ASNase candidates are purified on a nickel column and N-terminal His6 tags are cleaved. Sequence comparison of An 104 and a human consensus sequence is shown in figure 1.
  • LGQPGYDGRSALHVAEAAGNLEVVTLLQSLQGGAGAQAPGPEVLPGV SEQ ID NO: 51
  • Ancestral Human Chimeras (An h c ) - Utilizing 365-573 of human L-ASNase protein as the C terminal consisting of ankyrin repeat.
  • VLPPASPDQRVIYT VLECQPLFD S SDMTITEW VQIAQTIERHYEQ YHGF VVIHGTDT MAFAASVLSFVLENLQKPVILTGAQVPIHALWNDGRENLLGALLMAGQYIIPEVCLFFQ NQLFRGNRTTKVDARRFAAFCSPNLPPLATVGADVTINRELVRKASGKDRLVVHSSME RDVGLLRLYPGIPASLVRAFLQPPLKGVVMETFGSGNGPTKPDLLQELRAAAERGLVIV NCTHCLQGAVTSDYASGMAMAGAGIVSGFDMTSEAALAKLSYVLGQPGLSLDDRKEL

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Abstract

Sont divulguées des asparaginases recombinantes avec des séquences de variants ancestraux destinées à des utilisations en tant qu'agents thérapeutiques. Dans certains modes de réalisation, il est envisagé que les variants d'asparaginases ancestraux présentent une immunogénicité réduite, ce qui permet de prévenir ou de réduire le risque d'inactivation et/ou d'anaphylaxie de l'hôte. Dans certains modes de réalisation, la présente divulgation concerne le traitement de maladies associées à la dépendance à l'asparagine comprenant l'administration d'une quantité efficace d'une asparaginase ou d'un conjugué variant ancestral divulgué ici à un sujet en ayant besoin.
PCT/US2024/033129 2023-06-09 2024-06-07 Variants d'asparaginases ancestraux recombinantes et leurs utilisations dans le traitement du cancer Ceased WO2024254543A2 (fr)

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