Codon optimized IL-15 and IL-15R-alpha genes for expression in mammalian cells
Inventors
Felber, Barbara K. • Pavlakis, George N.
Assignees
National Institutes of Health NIH
Publication Number
US-9303080-B2
Publication Date
2016-04-05
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 comprising a codon optimized 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 for the high-level expression of interleukin-15 (IL-15), alone and combined with whole IL-15 Receptor alpha (IL15Ra) or the soluble form of IL15Ra (IL15sRa). It discloses methods for expressing IL-15 in mammalian cells by transfecting with codon optimized IL-15 sequences and combinations of IL-15 with its receptor alpha that increase IL-15 stability and potency in vitro and in vivo. The invention further includes nucleic acid modifications that improve mRNA stability and expression through the alteration of codons and removal of inhibitory signals in RNA such as splice sites and low stability AT-rich sequences, resulting in substantially increased IL-15 protein expression compared to native sequences.
The problem addressed is the inefficient native expression of IL-15 due to embedded negative regulatory signals in the native nucleic acid sequences. Native soluble forms of IL-15 Receptor alpha were previously suggested to antagonize IL-15; however, the invention shows that co-expression of IL-15 with whole or soluble IL-15 receptor alpha stabilizes IL-15, enhancing its bioavailability and biological effects such as lymphocyte expansion and activation. Thus, the invention solves issues of low IL-15 expression and stability, enabling improved therapeutic utility by optimizing coding sequences and co-expressing IL-15 with receptor alpha to produce biologically active, stable cytokine complexes.
Claims Coverage
The patent includes one independent claim focused on nucleic acid sequences encoding IL-15 comprising specific nucleotide regions. The claims cover improved polynucleotides, expression vectors, mammalian cells, and nucleic acid configurations incorporating signal peptides and RNA export elements.
Isolated polynucleotide encoding IL-15 with defined nucleotide sequence
A polynucleotide comprising a nucleic acid sequence encoding an IL-15 protein, wherein the sequence comprises nucleotides 145-489 of SEQ ID NO:3.
Polynucleotide including heterologous signal peptide or propeptide sequence
The polynucleotide comprises a nucleic acid sequence encoding a signal peptide propeptide or a signal peptide from a heterologous protein linked to nucleotides 145-489 of SEQ ID NO:3.
Use of specific heterologous proteins for signal peptide source
The heterologous protein for the signal peptide is selected from granulocyte-macrophage colony stimulating factor (GM-CSF), tissue plasminogen activator (tPA), growth hormone, and an immunoglobulin.
Operable linkage to RNA export elements to enhance expression
The polynucleotide is operably linked to a nucleic acid encoding an RNA export element.
Expression vector comprising the improved IL-15 polynucleotide
An expression vector comprising the isolated polynucleotide encoding the IL-15 protein.
Mammalian cell comprising improved IL-15 polynucleotide
An isolated mammalian cell comprising the polynucleotide encoding the IL-15 protein.
The claims cover isolated IL-15 encoding polynucleotides with specific sequence regions, inclusion of heterologous signal peptides from defined proteins, linkage to RNA export elements to enhance expression, and their incorporation into expression vectors and mammalian cells, focusing on improved IL-15 expression via codon optimization and sequence modification.
Stated Advantages
Increased IL-15 protein expression levels in mammalian cells by at least 4- to over 100-fold compared to native sequences.
Enhanced stability and potency of IL-15 in vitro and in vivo when co-expressed with whole or soluble IL-15 Receptor alpha.
Improved bioavailability and biological effects of IL-15, including expansion and activation of lymphocyte populations such as NK cells and T cells.
Increased steady-state levels and prolonged plasma presence of IL-15 leading to stronger immune responses.
Capability to coordinate expression ratios of IL-15 and receptor alpha to optimize cytokine potency and stability.
Applications in prophylactic and therapeutic vaccinations, cancer immunotherapy, and treatment of immunodeficiency conditions.
Documented Applications
Therapeutic use of IL-15 for immunodeficiency, promoting lymphocyte expansion and activation (B cells, T cells, NK cells, NK T cells).
Use as adjuvant in vaccines, including anti-HIV vaccines, to enhance immune responses.
Cancer immunotherapy via increased delivery of biologically active cytokines to tissues.
In vitro expansion of T cells and NK cells for immunotherapy.
Administration as DNA, RNA, or protein to increase IL-15 bioavailability and biological effects in mammalian subjects.
Use in therapeutic vaccination of macaques to increase antigen-specific multifunctional T cell responses.
Enhancement of immune response against one or more antigens by administering improved IL-15 nucleic acids alone or in combination with IL15Ra.
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