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Publication Number
US-12281147-B2
Publication Date
2025-04-22
Expiration Date
Abstract
The disclosure provides interleukin 21 (IL-21) variants that have (i) decreased or no binding to a human interleukin 21 receptor (IL-21R) as compared to a wild-type human IL-21 polypeptide; or (ii) increased stability as compared to a wild-type human IL-21 polypeptide; or (iii) decreased or no binding to a human interleukin 21 receptor (IL-21R) as compared to a wild-type human IL-21 polypeptide and increased stability as compared to a wild-type human IL-21 polypeptide. The disclosure also provides fusion proteins including an antibody or fragment thereof bound, e.g., covalently linked, to an IL-21 variant.
Core Innovation
The invention relates to human interleukin 21 (IL-21) variants. Human IL-21 variant polypeptides are defined by polypeptide sequences provided by SEQ ID NOs: 462, 463, 1192, 950, 933, and 894, or by introducing one or more amino acid substitutions into a reference IL-21 sequence (SEQ ID NO: 1) selected from the group consisting of R38W and R105W.
The disclosure further provides fusion proteins comprising an antibody or binding fragment thereof and a human IL-21 variant covalently linked to the antibody or binding fragment thereof. The antibody component can include an Fc domain and effectorless Fc mutations, and the linkage is implemented via a polypeptide linker as described in the document.
The disclosed constructs are intended to selectively modulate immune activation by reducing IL-21R activity outside targeted antibody-positive cells and to minimize systemic adverse effects by decreasing or abrogating IL-21R binding. The document further describes validation through binding and biophysical assessments, including affinity measurements, and biological relevance in tumor model outcomes such as measurements associated with immune signaling and STAT3 phosphorylation.
Claims Coverage
The independent claims define three main inventive scopes: a human IL-21 variant, a fusion protein comprising an antibody or binding fragment covalently linked to the IL-21 variant, and a cell line that produces the fusion protein. Across these scopes, the inventive content centers on IL-21 variant definition by specified SEQ ID NOs or R38W/R105W substitutions, and on covalent linkage of the IL-21 variant to an antibody or binding fragment.
Human IL-21 variant defined by specified sequences or R38W/R105W substitutions
A human interleukin 21 (IL-21) variant comprising a polypeptide sequence as set forth in any one of SEQ ID NOs: 462, 463, 1192, 950, 933, and 894, or comprises one or more amino acid substitutions at one or more positions of SEQ ID NO: 1 selected from the group consisting of R38W and R105W.
Covalently linked IL-21 variant fusion protein with antibody or binding fragment
A fusion protein comprising an antibody or binding fragment thereof and a human IL-21 variant comprising a polypeptide sequence as set forth in any one of SEQ ID NOs: 462, 463, 1192, 950, 933, and 894, or comprises one or more amino acid substitutions at one or more positions of SEQ ID NO: 1 selected from the group consisting of R38W and R105W, wherein the human IL-21 variant is covalently linked to the antibody or binding fragment thereof.
Cell line producing covalently linked IL-21 variant antibody fusion protein
A cell line that produces a fusion protein comprising an antibody or binding fragment thereof and a human IL-21 variant comprising a polypeptide sequence as set forth in any one of SEQ ID NOs: 462, 463, 1192, 950, 933, and 894, or comprises one or more amino acid substitutions at one or more positions of SEQ ID NO: 1 selected from the group consisting of R38W and R105W, wherein the IL-21 variant is covalently linked to the antibody or binding fragment thereof.
Overall, the claim coverage is concentrated on IL-21 variants defined by specific SEQ ID NOs and/or R38W and R105W substitutions, and on fusion proteins where those IL-21 variants are covalently linked to an antibody or binding fragment, including a cell line configured to produce such fusion proteins. Dependent claims further narrow antibody features, including an Fc domain and effectorless Fc mutations, and specify covalent linkage via polypeptide linkers.
Stated Advantages
Selectively modulate immune activation by reducing IL-21R activity outside targeted antibody-positive cells.
Minimize systemic adverse effects by decreasing or abrogating IL-21R binding.
Increased stability properties for the IL-21 variant, including manufacturing expression and stability measures described in the document.
Detuned IL-21 fused to anti-CD8 localized similarly tumor growth inhibition to stabilized non-detuned IL-21.
Anti-CD8 targeted non-detuned cytokine caused lethal toxicity after the third dose, whereas detuned IL-21 produced no such toxicity.
Detuned IL-21 had a wider therapeutic index.
Documented Applications
Use of IL-21 variant/fusion constructs to selectively modulate immune activation by limiting IL-21R activity outside targeted antibody-positive cells.
Cancer treatment, including use contexts described for engineered immune cells and CAR T cells.
Biological relevance evaluation in tumor model outcomes, including MC38 tumor model outcomes and measurements associated with immune signaling such as STAT3 phosphorylation.
Binding-affinity measurement of IL-21 variants using AlphaSeq synthetic yeast agglutination protein-protein interaction assay, with possible BLI confirmation.
Example 6 models efficacy/toxicity of a detuned IL-21 (mouse surrogate) fused to anti-CD8 in a syngeneic tumor growth inhibition setting, with evaluation of tumor growth inhibition and lethal toxicity/therapeutic index.
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