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 • US Department of Health and Human Services
Publication Number
US-11339198-B2
Publication Date
2022-05-24
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 or in combination with whole IL-15 Receptor alpha (IL15Ra) or its soluble form (IL15sRa), leading to increased IL-15 stability and potency in vitro and in vivo. It further provides methods for expressing these optimized sequences in mammalian cells, enhancing IL-15 production and biological effects such as lymphocyte expansion and activation.
The problem addressed concerns the inefficient natural expression of IL-15 due to inhibitory signals within native RNA sequences such as potential splice sites, A/T-rich content reducing stability, and low codon optimization, resulting in low protein yield from native mammalian sequences. Additionally, the natural soluble form of IL-15 Receptor alpha has been suggested to antagonize IL-15, whereas its sushi domain fused to IL-15 can be agonistic. There is a need to develop expression vectors and nucleic acid sequences that improve IL-15 production, stability, and potency, as well as co-expression systems with IL15Ra to enhance the cytokine's bioavailability and biological activity for therapeutic applications.
Claims Coverage
The patent includes one independent claim focusing on a method of improving IL-15 stability and potency by co-administering IL-15 and IL-15 receptor alpha polynucleotides with specific optimized sequence features.
Optimized IL-15 nucleic acid sequence encoding mature IL-15
A method administration involving an IL-15 polynucleotide with at least 85% sequence identity to nucleotides 145-489 of SEQ ID NO:3, encoding a mature IL-15 polypeptide, with a high proportion of non-native nucleotides at specified positions increasing GC content and expression.
IL-15 receptor alpha polynucleotide
Co-administration of an IL-15 receptor alpha (IL-15Rα) polynucleotide encoding an IL-15Rα polypeptide, where the IL-15Rα nucleic acid can be a codon-optimized human IL-15Rα with at least 95% sequence identity to SEQ ID NO:47 or SEQ ID NO:49.
Operable linkage to promoters and vector designs
Both IL-15 and IL-15Rα nucleic acid sequences are operably linked to promoters and can be present either on the same vector with different promoters or on separate vectors, enabling coordinated expression.
Use of heterologous signal peptides or signal peptide-propeptides
The IL-15 and/or IL-15Rα polynucleotides may include heterologous signal peptide-propeptide (SIG-PRO) or signal peptide (SIG) sequences from proteins such as tissue plasminogen activator (tPA), granulocyte-macrophage colony stimulating factor (GM-CSF), growth hormone, or immunoglobulin to enhance secretion and expression.
Therapeutic and administration contexts
The method applies to individuals with cancer or immunodeficiency, with co-administration routes including subcutaneous, intramuscular, or parenteral delivery, and use as adjuvants to augment immune responses.
The claim covers a method utilizing optimized IL-15 and IL-15Rα nucleic acid sequences for co-administered expression to improve IL-15 stability, potency, and biological activity with flexibility in vector configuration, sequence optimization, signal peptide usage, and therapeutic context.
Stated Advantages
Increased expression and secretion levels of IL-15 by approximately 8- to 100-fold compared to wild-type sequences due to codon optimization and RNA sequence modification.
Enhanced stability of IL-15 in circulation when co-expressed or co-administered with IL-15 receptor alpha, resulting in prolonged bioavailability and increased biological effects.
Greater potency in vitro and in vivo, demonstrated by robust expansion and activation of lymphocyte subsets including NK cells, T cells, B cells, and NK T cells.
Co-expression with full-length or soluble IL-15 receptor alpha dramatically increases IL-15 secretion, stability, and steady-state protein levels by factors exceeding 10- to 1000-fold.
Improved versions of IL-15 with heterologous signal peptides from proteins like tissue plasminogen activator further enhance extracellular accumulation and protein production.
Combination delivery of IL-15 with IL-15 receptor alpha nucleic acids results in enhanced immune response augmentation in animal models, including increased antigen-specific multifunctional T cells after vaccination.
Documented Applications
Treatment of immunodeficiency conditions by promoting in vivo or in vitro expansion of lymphocytes such as B cells, T cells, NK cells, and NK T cells using improved IL-15 and/or IL15Ra nucleic acid sequences.
Use as vaccine adjuvants co-delivered with one or more vaccine antigens to enhance antigen-specific immune responses, including therapeutic vaccination against infectious diseases such as HIV/SIV.
Cancer immunotherapy by administering improved IL-15 and IL-15Rα sequences or proteins to increase immune cell function and numbers.
Administration of DNA vaccines encoding IL-15 and IL-15Rα, optionally by injection and/or electroporation, to produce enhanced systemic IL-15 levels and improved lymphocyte responses in mammalian hosts including humans and non-human primates.
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