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-9725492-B2
Publication Date
2017-08-08
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 the high-level expression of interleukin-15 (IL-15), alone or combined with whole IL-15 Receptor alpha (IL15Ra) or the soluble form of IL15Ra (IL15sRa). These improvements involve codon optimization of IL-15 sequences that differ significantly from native mammalian IL-15 sequences to increase mRNA stability and expression efficiency in mammalian cells.
The invention further provides methods of co-expressing IL-15 with IL15Ra or IL15sRa to enhance IL-15 stability and potency both in vitro and in vivo. Co-expression results in a coordinated production of IL-15 and its receptor that increases IL-15 secretion, stability, and biological activity. Expression vectors are provided that allow controlled expression ratios of IL-15 and IL15Ra to optimize immune activation effects in mammalian cells.
The problem being addressed is the suboptimal expression of native IL-15 due to RNA instability and inhibitory signals embedded in the native sequences, including potential splice sites and low stability elements. Additionally, the natural soluble form of IL-15 Receptor alpha may act as an antagonist, and efficient therapeutic use requires development of nucleic acid and protein forms that improve expression, stability, and activity of IL-15 and its receptor to treat immunodeficiencies and enhance immune responses.
Claims Coverage
The patent includes 20 claims with independent claims focusing on polynucleotides encoding optimized IL-15 sequences, expression vectors containing these sequences, and host cells including these vectors. There are two main inventive features related to the codon-optimized IL-15 sequences and their improvements for expression and function.
Optimized IL-15 coding sequences with specific nucleotide changes and increased GC content
The invention claims polynucleotides encoding IL-15 polypeptides wherein the nucleic acid sequences have at least 85% sequence identity to a defined region of SEQ ID NO:3 and include non-native nucleotides at a majority of specified nucleotide positions. These sequences show increased GC content and codon optimization to improve expression. The sequences can encode mature IL-15 and may include a fused signal peptide-propeptide from a heterologous protein such as tissue plasminogen activator (tPA).
Expression vectors and host cells comprising the optimized IL-15 sequences
The invention claims expression vectors comprising the optimized IL-15 polynucleotides and isolated host mammalian cells, including specific cell lines such as HEK293, COS, CHO, HeLa, NIH3T3, RD, or PC12, that contain these vectors. These systems enable high-level expression of biologically active IL-15 protein with improved production compared to wild-type sequences.
In summary, the independent claims cover nucleic acid compositions encoding highly optimized IL-15 with specific nucleotide and codon changes that improve GC content and expression, associated expression vectors, and mammalian host cells incorporating these nucleic acids for production of IL-15.
Stated Advantages
Dramatically increased IL-15 expression levels in mammalian cells compared to wild-type sequences, achieving 5-fold to over 100-fold improvements.
Increased stability and bioavailability of IL-15 through co-expression with IL-15 Receptor alpha or its soluble form, significantly enhancing IL-15 potency in vitro and in vivo.
Enhanced expansion and activation of lymphocyte subsets, including B cells, T cells, natural killer (NK) cells, and NK T cells, leading to improved immune responses.
Improved vaccine adjuvant effect and immunotherapy potential for treating immunodeficiency and enhancing antigen-specific immune responses.
Documented Applications
Use of optimized IL-15 and IL-15 Receptor alpha nucleic acid sequences and proteins as vaccine adjuvants to enhance immune responses against antigens.
Therapeutic applications for immunodeficiency by expanding lymphocyte populations in vivo or in vitro, including B cells, T cells, NK cells, and NK T cells.
Cancer immunotherapy through increased IL-15 biological effects by co-delivery and expression of IL-15 and IL-15 receptor alpha.
Expansion of multifunctional T cells expressing IL-2 and IFN-gamma following antigenic stimulation.
Delivery of IL-15 and IL15Ra encoding DNA constructs by injection and electroporation in mammals for immunotherapy.
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