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-9790261-B2
Publication Date
2017-10-17
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
Interleukin-15 (IL-15) is a pleiotropic cytokine important for both innate and adaptive immune systems, promoting activation and development of immune cells such as neutrophils, macrophages, dendritic cells, natural killer (NK) cells, and CD8+ T cells. IL-15 administration has therapeutic potential for immunodeficiencies, vaccine adjuvants, and expansion of immune cells, especially in diseases such as HIV/AIDS.
The natural soluble form of IL-15 Receptor alpha (IL15Ra) has been suggested to antagonize IL-15 activity, but its sushi domain fused to IL-15 acts as an agonist. Optimizing IL-15 expression vectors and nucleic acid sequences is crucial to enhance IL-15 bioavailability, stability, and potency for therapeutic use. The invention addresses the need for efficient expression of IL-15 alone or combined with IL15Ra or its soluble form (IL15sRa) in mammalian cells.
The invention provides nucleic acid sequences, expression vectors, and mammalian cells optimized for high-level expression of IL-15, alone or combined with IL15Ra or IL15sRa. The nucleic acid sequences are codon optimized to differ significantly from native sequences, notably increasing GC content and minimizing adverse RNA signals to improve mRNA stability and protein production. Further improvements include replacement of native IL-15 or IL15Ra signal peptides with heterologous signal peptides such as those from tissue plasminogen activator (tPA), growth hormone, or immunoglobulins, leading to synergistic enhancement of IL-15 expression.
Co-expression of IL-15 with whole IL15Ra or IL15sRa stabilizes IL-15, increasing its secretion, bioactivity, and half-life in vitro and in vivo. Expression vectors allow adjustable expression ratios to maximize IL-15 stability and lymphocyte expansion effects. The methods and compositions are useful for immunotherapy, vaccine adjuvants, treatment of immunodeficiencies, and expansion of lymphocyte populations expressing IL-2/IL-15 beta gamma receptors, including CD4+, CD8+, and multifunctional T cells.
Claims Coverage
The patent includes multiple claims with independent claims focusing on expression vectors containing codon optimized IL-15 nucleic acid sequences combined with IL-15 receptor alpha sequences, and host cells expressing these sequences. The inventive features revolve around nucleic acid sequence optimization, co-expression strategies, heterologous signal peptides, promoter linkage, and methods for producing biologically stable IL-15 and IL-15Rα proteins.
Expression vector comprising codon-optimized IL-15 and IL-15 receptor alpha nucleic acid sequences
An expression vector contains an IL-15 nucleic acid sequence having at least 85% identity to nucleotides 145-489 of SEQ ID NO:3 encoding IL-15, and an IL-15 receptor alpha (IL-15Rα) nucleic acid sequence encoding IL-15Rα, with each sequence operably linked to a promoter.
Nucleic acid sequence optimization at specific nucleotide positions
The IL-15 nucleic acid sequence has non-native nucleotides at 80% to 95% or more of 80 specified nucleotide positions within SEQ ID NO:3 to increase expression, particularly incorporating guanine or cytosine at these locations, raising GC content.
Use of heterologous signal peptides fused to IL-15 and/or IL-15Rα sequences
The expression vector includes nucleic acid sequences encoding signal peptide-propeptides or signal peptides from heterologous proteins such as GM-CSF, tissue plasminogen activator (tPA), growth hormone, or immunoglobulins fused to IL-15 and/or IL-15Rα coding sequences to improve secretion and expression.
Separate promoters for IL-15 and IL-15Rα expression
The IL-15 and IL-15Rα nucleic acid sequences are each operably linked to different promoters, allowing independent regulation of expression levels.
Codon-optimized IL-15Rα nucleic acid sequences
The IL-15Rα nucleic acid sequence is codon optimized with at least 95% sequence identity to SEQ ID NO:47 or SEQ ID NO:49, or comprises these exact sequences.
Host cells comprising the expression vector and methods of heterodimer expression
Host cells, particularly mammalian cells such as HEK293, contain the expression vector and produce IL-15 and IL-15Rα as biologically active heterodimers. Methods for culturing these cells under conditions producing the heterodimer are claimed.
The claims primarily cover expression vectors with codon optimized IL-15 and IL-15Rα sequences operably linked to promoters, with specific nucleotide optimizations, use of heterologous signal peptides, independent promoter systems, and methods and host cells producing stable IL-15/IL-15Rα heterodimers, enabling high-level expression, increased stability, and bioactivity of IL-15.
Stated Advantages
Dramatically increased expression levels of IL-15 protein in mammalian cells compared to wild-type sequences, with up to 100-fold or more increase.
Increased stability and bioavailability of IL-15 in vitro and in vivo due to co-expression with IL-15 Receptor alpha or its soluble form.
Enhanced biological function of IL-15, including activation and expansion of lymphocyte subsets such as B cells, T cells, NK cells, and multifunctional T cells.
Ability to produce IL-15 with prolonged circulation half-life or sharper peak levels depending on use of whole or soluble IL-15 receptor alpha.
Improved secretion and expression efficiency conferred by replacing native signal peptides with heterologous signal peptides from proteins like tissue plasminogen activator.
Documented Applications
Treatment of immunodeficiency conditions by promoting the expansion of various lymphocyte populations, including B cells, T cells, NK cells, and NK T cells.
Use as vaccine adjuvants to enhance immune responses against one or more antigens via co-administration with antigens.
Cancer immunotherapy involving enhanced delivery and stability of biologically active IL-15 cytokines.
In vitro and in vivo expansion of lymphocyte populations expressing IL-2/IL-15 beta gamma receptors and multifunctional T cell populations.
Therapeutic vaccination enhancement demonstrated in macaques, leading to increased antigen-specific cytokine-producing and effector memory T cells.
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