L-asparaginase variants and fusion proteins with reduced L-glutaminase activity and enhanced stability
Inventors
Lavie, Arnon • NGUYEN, Hien-Anh
Assignees
US Department of Veterans Affairs • University of Illinois System
Publication Number
US-10821160-B2
Publication Date
2020-11-03
Expiration Date
2037-03-01
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Abstract
Variant Erwinia chrysanthemi L-asparaginases with reduced L-glutaminase activity and enhanced in vivo circulation are described as are fusion proteins containing an L-asparaginase and three tandem soluble domains of TRAIL for use in the treatment of cancers such as acute lymphoblastic leukemia and acute myeloid leukemia.
Core Innovation
This invention provides variant Erwinia chrysanthemi L-asparaginases (ErA variants) that have reduced L-glutaminase activity and enhanced in vivo circulation time, and fusion proteins composed of an L-asparaginase linked to three tandem soluble domains of TRAIL. The amino acid substitutions are at one or more of positions 31, 63, and 254 of SEQ ID NO:1, aiming to maintain high L-asparaginase activity with low L-glutaminase activity. The fusion proteins are linked via peptide linking groups and may include tags such as histidine, SUMO, and albumin-binding domains to increase stability and half-life.
The problem addressed arises from the toxicity associated with the L-glutaminase side activity of L-asparaginases used in cancer treatment, particularly in acute lymphoblastic leukemia (ALL). While L-asparaginase activity is important for depleting circulating asparagine to kill cancer cells, L-glutaminase activity contributes to side effects that limit therapeutic use. There is conflicting data about L-glutaminase's role in killing leukemic cells, but its toxicity is established. Therefore, variants with reduced L-glutaminase activity but retained L-asparaginase effectiveness and improved stability are needed.
The invention includes not only the engineered ErA variants with substitutions at key positions that reduce L-glutaminase activity but maintain or improve stability and efficacy, but also fusion proteins linking these variants to three tandem soluble domains of TRAIL which induce apoptosis. These fusion proteins exhibit enhanced cytotoxic activity against leukemia and lymphoma cells. Additionally, modifications such as PEGylation, histidine tags, SUMO tags, and albumin-binding domains further enhance in vivo circulation time, decreasing the frequency or dose needed for therapeutic efficacy.
Claims Coverage
The claims cover three main inventive features: ErA variants with specific amino acid substitutions and improved enzymatic properties; PEGylated ErA variants with cysteine residues enabling site-specific PEG attachment; and fusion proteins combining ErA variants with three tandem soluble TRAIL domains.
ErA variants with substitutions at positions 31, 63, and/or 254
Erwinia chrysanthemi L-asparaginase variants comprising an amino acid substitution at position 31 selected from isoleucine, valine, leucine, or threonine, and at position 63 or 254 selected from glutamine, asparagine, or aspartate/asparagine/glutamine. The variants exhibit an asparaginase reaction rate (kcat) at least 75% of wild-type, a Km for L-asparagine less than 250 μM, a glutaminase reaction rate (kcat) less than 60% of wild-type, and a Km for L-glutamine greater than 3 mM. These variants may further include histidine, SUMO, albumin-binding tags, or combinations thereof.
PEGylated ErA variants with introduced cysteine residues for site-specific PEGylation
ErA variants having amino acid substitutions at one or more of positions 31, 63, and 254 and further modified by PEGylation via engineered cysteine residues at specific positions (such as 72, 76, 79, 84, 85, 206, 210, 215, 216, 235, 239, 240, 261, 264, 265, 268, 269, 318, or 322) enabling site-specific maleimide-based PEG conjugation. These variants may also include histidine, SUMO, or albumin-binding domains.
Fusion proteins comprising L-asparaginase linked to three tandem soluble TRAIL domains
Fusion proteins of an L-asparaginase (from Erwinia chrysanthemi or Escherichia coli, optionally variants with substitutions at positions 31, 63, and 254) linked to three tandem soluble domains of TRAIL (residues 115-281 of human TRAIL). The three TRAIL domains are linked by peptide linkers of 1 to 8 amino acids, and the TRAIL trimer is linked to L-asparaginase via a peptide linker of 1 to 20 amino acids, including linkers rich in glycine and serine. These fusion proteins may further include histidine, SUMO, or albumin-binding tags, and can be PEGylated with cysteine residues introduced at specified positions.
The claims comprehensively cover engineered ErA variants with specific amino acid substitutions that reduce glutaminase activity while maintaining asparaginase function and stability, their PEGylated forms via engineered cysteine sites, and fusion proteins with three soluble TRAIL domains, all designed to improve efficacy and reduce toxicity in cancer treatment.
Stated Advantages
Reduced L-glutaminase activity diminishes toxic side effects commonly associated with L-asparaginase treatment.
Enhanced in vivo circulation time of L-asparaginase due to addition of tags (histidine, SUMO, albumin-binding domain) and/or PEGylation increases therapeutic half-life and efficacy.
Fusion of L-asparaginase with three tandem soluble TRAIL domains significantly enhances cytotoxic activity against cancer cells.
Variants maintain high L-asparaginase reaction rates (kcat) and favorable Km values for asparagine, ensuring efficacy in depleting asparagine to treat cancers.
PEGylation via site-specific cysteine mutations allows for conjugation that minimally impacts enzyme activity, providing controlled modification for improved pharmacokinetics.
Documented Applications
Treatment of cancers dependent on external asparagine supply including acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), non-Hodgkin's lymphoma, B cell lymphoma, Burkitt's lymphoma, chronic myelocytic leukemia, chronic lymphocytic leukemia, and hairy cell leukemia.
Use as a component of multi-agent chemotherapy regimens for leukemias, including combination with glucocorticoids, corticosteroids, methotrexate, vincristine, cyclophosphamide, and anthracyclines.
Pharmaceutical compositions comprising the ErA variants or fusion proteins for intravenous or other routes of administration for cancer therapy.
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