Truncated guinea pig L-asparaginase variants and methods of use
Inventors
Lavie, Arnon • NGUYEN, Hien-Anh • Schalk, Amanda
Assignees
US Department of Veterans Affairs • University of Illinois System
Publication Number
US-11578315-B2
Publication Date
2023-02-14
Expiration Date
2038-08-10
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Abstract
Variant guinea pig L-asparaginases which are truncated and humanized are described as are fusion proteins containing the L-asparaginase and use of the L-asparaginases in the treatment of cancers such as acute lymphoblastic leukemia and acute myeloid leukemia.
Core Innovation
Certain cancers, such as acute lymphoblastic leukemia (ALL), are dependent upon scavenging L-asparagine (Asn) from blood, commonly due to their low expression of asparagine synthetase. L-asparaginases are critical in treating these cancers by hydrolyzing Asn into L-aspartic acid and ammonia. Existing bacterial L-asparaginase therapeutics exhibit dual activity including L-glutaminase activity, which has been associated with clinical toxicity and immunogenicity that limits treatment effectiveness.
The invention addresses these disadvantages by providing C-terminally truncated and humanized guinea pig L-asparaginase (GpA) variants that retain or improve catalytic activity compared to wild-type GpA, which lacks L-glutaminase activity and has a low Km for asparagine. The truncated variants comprise the N-terminal catalytic domain of approximately 359 to 396 residues, improving stability and therapeutic potential. Humanization is achieved via amino acid substitutions, directed evolution, or structure-based mutation to reduce immunogenicity and increase sequence identity with human L-asparaginase while maintaining low Km and activity.
Further modifications include adding tags such as histidine tags, SUMO tags, albumin-binding domains, or fusion to three tandem soluble domains of TRAIL to increase in vivo circulation time, stability, and enhance anti-tumor efficacy through combined intrinsic and extrinsic apoptotic pathways. The invention encompasses nucleic acid constructs, expression vectors, host cells, and pharmaceutical compositions containing these truncated and humanized GpA variants or fusion proteins for use in treating cancers dependent on asparagine depletion.
Claims Coverage
The patent includes multiple independent claims covering the truncated and humanized GpA variants, their nucleic acid sequences, expression constructs, host cells, pharmaceutical compositions, and methods of cancer treatment. There are several inventive features corresponding to structural variants, functional enhancements, and therapeutic applications.
GpA variant with specific truncation and amino acid substitutions
A GpA variant comprising an amino acid sequence with at least 85% identity to residues 1 to 359 of SEQ ID NO:1, including one or more specific amino acid substitutions from a defined group (e.g., H10R, Q23R, K25E, etc.) optionally further having substitutions such as C198A, C198S, C198V, or cysteine substitutions at E49C, R52C, K225C, Q257C, Q281C, E340C, and truncated between residues 359 and 396, preferably at residue 369.
GpA variant with catalytic activity equal or greater than wild-type
The GpA variant exhibits catalytic activity equal to or greater than that of wild-type GpA as defined by SEQ ID NO:1.
GpA variant comprising one or more affinity and solubility tags
The GpA variant further comprises affinity or stability enhancing tags such as a histidine tag, a SUMO tag, an albumin-binding domain, or combinations thereof to improve in vivo stability and circulation.
Nucleic acid and expression constructs encoding the GpA variants
Nucleic acid molecules encode the defined GpA variants, incorporated into expression vectors suitable for host cell transformation, enabling recombinant protein production.
Pharmaceutical compositions and methods of cancer treatment using GpA variants
Pharmaceutical compositions contain the GpA variants with pharmaceutically acceptable excipients, and methods comprise administering effective amounts of these variants to treat cancers such as non-Hodgkin's lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, and related malignancies.
Specific GpA variant amino acid sequences and fusion proteins
GpA variants comprising sequences as set forth in SEQ ID NOs: 45-50, 51, 54-57, 58-65, and 86, optionally further comprising histidine tags, SUMO tags, albumin-binding domains, or fused to three tandem soluble domains of TRAIL, wherein TRAIL comprises residues 115-281 of human TRAIL.
The claims cover truncated and humanized guinea pig L-asparaginase variants with defined amino acid modifications, fusion tags for improved stability and pharmacokinetics, nucleic acid constructs for recombinant expression, pharmaceutical formulations, and therapeutic methods for treating various cancers reliant on asparagine depletion, including specific protein variants and fusions with TRAIL.
Stated Advantages
Reduced immunogenicity compared to bacterial L-asparaginases.
Lack of L-glutaminase activity, reducing clinical toxicity.
Catalytic efficiency with low Km for asparagine similar or superior to wild-type GpA.
Increased in vivo circulation time and stability via truncation, humanization, PEGylation, or fusion tags.
Enhanced anti-tumor efficacy through fusion with TRAIL domains inducing intrinsic and extrinsic apoptosis.
Improved therapeutic potential for extended patient populations including pediatric and adult leukemia patients.
Documented Applications
Use in the treatment of cancers such as acute lymphoblastic leukemia, acute myeloid leukemia, non-Hodgkin's lymphoma, B cell lymphoma, Burkitt's lymphoma, chronic myelocytic leukemia, chronic lymphocytic leukemia, and hairy cell leukemia.
Treatment of malignancies dependent upon exogenous asparagine, including certain hematologic cancers and potential solid tumors such as pancreatic and ovarian cancers.
Combination chemotherapy regimens for leukemia treatment.
Fusion protein use for enhanced killing of cancer cells including in vitro human leukemia cell lines and in vivo mouse models.
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