Codon optimized IL-15 and IL-15R-alpha genes for expression in mammalian cells
Inventors
Felber, Barbara K. • Pavlakis, George N.
Assignees
US Department of Health and Human Services
Publication Number
US-10428133-B2
Publication Date
2019-10-01
Expiration Date
2027-01-12
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Abstract
The present invention provides for nucleic acids improved for the expression of interleukin-15 (IL-15) in mammalian cells. The invention further provides for methods of expressing IL-15 in mammalian cells by transfecting the cell with a nucleic acid sequence encoding an improved IL-15 sequence.The present invention further provides expression vectors, and IL-15 and IL-15 receptor alpha combinations (nucleic acid and protein) that increase IL-15 stability and potency in vitro and in vivo. The present methods are useful for the increased bioavailability and biological effects of IL-15 after DNA, RNA or protein administration in a subject (e.g. a mammal, a human).
Core Innovation
The invention provides nucleic acid sequences, expression vectors, and mammalian cells optimized for high-level expression of interleukin-15 (IL-15), alone and in combination with whole IL-15 Receptor alpha (IL15Ra) or its soluble form (IL15sRa). It further details methods to express IL-15 in mammalian cells through recombinant modification to increase protein production, stability, and biological activity. Importantly, the invention includes improved IL-15 and IL15Ra nucleic acid sequences with significant nucleotide changes that enhance GC content and minimize negative RNA elements, as well as strategies of replacing IL-15 and IL15Ra signal peptides with heterologous sequences to improve secretion and expression efficiency.
The invention addresses the problem that native IL-15 sequences express poorly in mammalian cells due to RNA instability, potential splice sites, and low stability elements embedded in the coding regions. This low expression limits the therapeutic and immunological applications of IL-15. Additionally, while IL-15 receptor alpha can act as an antagonist in soluble form, co-expression with IL-15 enhances IL-15 stability and potency. Therefore, there is a need for improved IL-15 and IL15Ra nucleic acid sequences and vectors that increase expression, stability, bioactivity, and bioavailability both in vitro and in vivo.
Claims Coverage
The patent includes one independent claim focused on methods of expanding lymphocytes via co-administration of improved IL-15 and IL-15 receptor alpha nucleic acids. The main inventive features relate to the codon optimization of IL-15 sequences, co-expression with IL15Ra sequences, and specific configurations of the nucleic acids for expression and administration.
Method for lymphocyte expansion by co-administration of codon-optimized IL-15 and IL-15Rα nucleic acids
A method of expanding lymphocytes by co-administering (i) an IL-15 polynucleotide with an IL-15 nucleic acid sequence having at least 85% identity to nucleotides 145-489 of SEQ ID NO:3 encoding mature IL-15, and (ii) an IL-15 receptor alpha polynucleotide encoding an IL-15Rα polypeptide, where both sequences are operably linked to promoters.
High GC content and codon optimization at defined nucleotide positions
The IL-15 nucleic acid sequence comprises non-native nucleotides at 80-95% or more of the 80 specified nucleotide positions (156, 159, 162,...486 of SEQ ID NO:3), many being G or C nucleotides, increasing the GC content to at least 50%, to improve expression.
Use of codon-optimized IL-15Rα sequences
IL-15Rα nucleic acid sequence comprises codon-optimized sequences with at least 95% identity to SEQ ID NO:47 or SEQ ID NO:49 for whole or soluble forms of IL-15 receptor alpha.
Provision for expression vectors and promoters
The IL-15 and IL-15Rα nucleic acid sequences are operably linked to promoters, with the polynucleotides present in the same or separate vectors, optionally linked to different promoters to adjust expression levels.
Inclusion of heterologous signal peptide-propeptide sequences
The IL-15 and/or IL-15Rα sequences optionally comprise signal peptide-propeptide (SIG-PRO) or signal peptide (SIG) sequences from heterologous proteins such as tPA, GM-CSF, growth hormone, or immunoglobulins for improved secretion.
Administration and therapeutic use
The method is applicable for individuals with cancer or immunodeficiency and involves subcutaneous, intramuscular, or parenteral administration of IL-15 and IL-15Rα polynucleotides, including use as vaccine adjuvants or immunotherapy agents.
The claim covers methods and compositions that improve expression and bioactivity of IL-15 by codon-optimizing IL-15 nucleic acids, co-expressing IL-15 with optimized IL-15 receptor alpha sequences, using heterologous secretion signals, and administering these molecules in vectors or protein form to expand lymphocytes for therapeutic purposes.
Stated Advantages
Improved expression vectors and nucleic acid sequences increase IL-15 protein production by 8- to 100-fold compared to wild-type sequences.
Co-expression of IL-15 with IL-15 receptor alpha or its soluble form dramatically enhances IL-15 stability, bioavailability, and biological effects, including expansion of natural killer and T cells.
Use of heterologous signal peptide and propeptide sequences further increases extracellular accumulation and secretion of IL-15 protein.
The combination of IL-15 and IL-15 receptor alpha sequences allows generation of biologically active complexes that maintain prolonged activity in vivo, offering tunable pharmacokinetics.
The optimized nucleic acid sequences are designed to reduce negative RNA elements, thus increasing mRNA stability and expression efficiency.
Documented Applications
Treatment of immunodeficiency by expansion and activation of lymphocytes including B cells, T cells, NK cells, and NK T cells in vivo or in vitro.
Use as a vaccine adjuvant to enhance immune responses against one or more antigens when co-administered with antigen-encoding nucleic acids or proteins.
Cancer immunotherapy by increasing lymphocyte numbers and function.
Therapeutic vaccination to generate antigen-specific multifunctional T cells producing IL-2 and IFN-gamma, improving viral control in macaques.
Co-expression of IL-15 and IL-15 receptor alpha in mammalian cells for increased cytokine delivery and therapeutic outcomes.
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