Method of refolding an interleukin-2 (IL-2) protein
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Publication Number
US-11091525-B2
Publication Date
2021-08-17
Expiration Date
Abstract
Conjugates of an interleukin-2 (“IL-2”) moiety and one or more nonpeptidic, water-soluble polymers are provided. Typically, the non-peptidic, water-soluble polymer is poly(ethylene glycol) or a derivative thereof. Also provided, among other things, are compositions comprising conjugates, methods of making conjugates, methods of administering compositions to an individual, nucleic acid sequences, expression systems, host cells, and methods for preparing IL-moieties.
Core Innovation
The disclosure relates to interleukin-2 (IL-2) conjugates in which an IL-2 moiety residue is covalently attached to a nonpeptidic, water-soluble polymer, including poly(ethylene glycol) (PEG) and PEG derivatives. The IL-2 moiety is linked to the polymer by releasable or stable linkages, and the document describes activated PEG polymeric reagents and coupling to IL-2 lysine or N-terminal sites. The disclosure also describes attachment through activated esters, including succinimidyl derivatives, reductive amination concepts involving a Schiff base, and thiol-targeted attachment to IL-2 cysteine residues, including a cysteine residue at position 125.
The disclosed subject matter further describes PEG reagent chemistries such as mPEG acetal, mPEG piperidone, mPEG methylketone, mPEG tresylate, mPEG maleimide, and mPEG epoxide, including forked and branched forms. The linkage concepts presented include ester, carbonate, phosphate, thio-ester-type, amide, urethane, amine, and urea-type linkages, and the document includes recombinant IL-2 moiety obtained from host cells such as expression in E. coli. A set of thiol-selective polymeric reagents is also described for attaching IL-2 through thiol-reactive polymer linkages, including maleimide-like reagents, vinyl sulfone reagents, thiol and disulfide reagents, and thioether linkages.
The disclosure further indicates a preference to avoid disrupting IL-2 disulfide bonds in forming the thiol-directed conjugates. It also describes a possibility of adding a cysteine via synthetic or genetic engineering to enable the thiol-directed conjugation while working with the selected IL-2 sequences.
Claims Coverage
The partial content identifies one independent claim centered on a dialysis-based refolding workflow for SEQ ID NO:3 interleukin-2 using a guanidine/Tris/Cu2+ system with controlled equilibration and slow guanidine reduction at 4°C. No independent claims are provided for the polymer conjugate subject matter summarized in the core innovation.
Dialysis-based refolding of denatured interleukin-2 in a dialysis bag
Placing a solution comprising a denatured interleukin-2 protein dissolved in 6 M guanidine and 100 mM Tris buffer at pH 8, having a Cu2+ concentration of 0.1 mM, within a dialysis bag having a molecular weight pore size of 3.5 kilodaltons, wherein the interleukin-2 protein has an amino acid sequence of SEQ ID NO:3.
Equilibration in a guanidine/Tris reservoir followed by slow guanidine reduction by adding water
Placing the interleukin-2 protein-containing dialysis bag into a reservoir containing an aqueous solution comprising 4.8 M guanidine and 0.1 M Tris pH 8 buffer, allowing the dialysis bag to equilibrate in the reservoir, and slowly further reducing the concentration of guanidine in the reservoir by adding water to provide the interleukin-2 protein as a refolded interleukin-2 protein.
Temperature-controlled refolding at 4°C
Carrying out steps (i)-(iv) at 4°C to refold the denatured interleukin-2 protein.
The claim coverage centers on a dialysis-bag refolding workflow for a SEQ ID NO:3 interleukin-2 protein using guanidine/Tris solutions, Cu2+, controlled equilibration, slow guanidine reduction by adding water, and performing the steps at 4°C.
Stated Advantages
Improved IL-2 conjugates are motivated by limitations of aldesleukin toxicity, as described in the document.
Extended half-life and improved efficacy at lower dosing frequency/amount are reported in the described in vivo and pharmacokinetic model results.
Enables formation of IL-2 conjugates with water-soluble polymeric reagents using specific linkage chemistries and defined spacer moieties (X).
Provides carboxyl-specific conjugation concepts for attaching the IL-2 residue to the polymeric reagent using hydrazide/hydrazone linkage and optional reduction.
Documented Applications
PEGylated IL-2 conjugates are described as being characterized and preliminarily evaluated for functional activity using a CTLL-2 proliferation assay and for pharmacokinetic and in vivo efficacy in lung metastatic melanoma and subcutaneous melanoma model descriptions.
In vivo model descriptions include a lung metastatic melanoma model and a subcutaneous melanoma model.
IL-2 moiety conjugate forms are evaluated using a CTLL-2 proliferation assay, as described in the document.
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