Nucleic acids comprising polynucleotides encoding chimeric antigen receptors
Inventors
Tanaka, Shinya • Hirano, Naoto • Kagoya, Yuki
Assignees
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Publication Number
US-10822392-B2
Publication Date
2020-11-03
Expiration Date
Abstract
Disclosed herein are chimeric antigen receptors (CARs) comprising an intracellular segment comprising an interleukin receptor chain, a JAK-binding motif, a Signal Transducer and Activator of Transcription (STAT) 5 association motif and/or a CD3ζ intracellular signaling domain comprising an exogenous STAT3 association motif, as well as cells and 5 compositions comprising said CARs and uses thereof.
Core Innovation
The invention provides CARs encoded by a nucleic acid, where the CAR includes an extracellular domain capable of binding to a predetermined antigen, a transmembrane domain, and an intracellular segment. The intracellular segment includes one or more intracellular signaling domains selected from a cytoplasmic domain of an interleukin receptor chain and/or a cytoplasmic co-stimulatory domain, together with a CD3zeta intracellular signaling domain engineered to comprise an exogenous STAT3-association motif.
The intracellular segment is further characterized by inclusion of an endogenous or exogenous Janus Kinase (JAK)-binding motif. The described components include defined STAT3/STAT5 motif concepts and definitions related to receptor and co-stimulatory domains, with an optional signal peptide at the N terminus of the CAR.
The disclosed architecture is directed toward enhanced intracellular JAK/STAT signaling using interleukin receptor chain cytoplasmic domains combined with a CD3zeta domain containing an exogenous STAT3-association motif. The described content highlights functional outcomes including enhanced STAT3/STAT5 phosphorylation, increased proliferation/viability, maintenance of a stem cell-like memory phenotype, and improved in vivo anti-leukemic efficacy versus earlier-generation CARs.
Claims Coverage
The independent claim covers a nucleic acid encoding a CAR with an intracellular signaling architecture combining interleukin receptor cytoplasmic domains, a CD3zeta domain with an exogenous STAT3-association motif, and an endogenous or exogenous JAK-binding motif, optionally including an N-terminal signal peptide. The dependent claims refine this architecture by selecting particular interleukin receptor chains, particular co-stimulatory cytoplasmic domains, and additional motif and positional constraints.
Car intracellular signaling architecture with STAT3-association motif
A nucleic acid encoding a CAR in which the intracellular segment comprises a CD3zeta intracellular signaling domain comprising an exogenous STAT3-association motif, together with one or more intracellular signaling domains selected from a cytoplasmic domain of an interleukin receptor chain and/or a cytoplasmic co-stimulatory domain.
JAK-binding motif in the intracellular segment
The nucleic acid wherein the intracellular segment comprises an endogenous or exogenous Janus Kinase (JAK)-binding motif.
Optional N-terminal signal peptide
The nucleic acid optionally encodes a signal peptide at the N terminus of the CAR.
STAT3 motif within a defined distance of the CAR C terminus
The nucleic acid where an exogenous STAT3-association motif is positioned less than 100 amino acid residues from the C terminus of the CAR.
Specific STAT3-association motif sequences
The nucleic acid wherein the exogenous STAT3-association motif is YXXQ (SEQ ID NO: 13), optionally YRHQ (SEQ ID NO: 22).
Interleukin receptor chain selection for the CAR intracellular segment
The nucleic acid where the interleukin receptor chain is either the IL-2Rbeta chain or the IL-21Ralpha chain.
Co-stimulatory cytoplasmic domain options
The nucleic acid where the cytoplasmic co-stimulatory domain is a cytoplasmic domain of CD28, CD2, CD4, CD5, CD8alpha, CD8beta, CD134 or CD137.
Across the independent claim and refinements shown, the claims center on an intracellular CAR design that combines interleukin receptor cytoplasmic domain signaling and/or co-stimulatory cytoplasmic domains with a CD3zeta intracellular signaling domain containing an exogenous STAT3-association motif, together with an endogenous or exogenous JAK-binding motif. Additional refinements specify the interleukin receptor chain choice, selectable co-stimulatory cytoplasmic domains, and STAT3 motif sequence and position constraints.
Stated Advantages
Enhanced STAT3/STAT5 phosphorylation.
Increased proliferation/viability.
Maintained stem cell-like memory phenotype.
Improved in vivo anti-leukemic efficacy versus earlier-generation CARs.
Documented Applications
In vivo anti-leukemic efficacy testing in NSG mice using an anti-CD19 CAR context.
Anti-CD19 CAR functional evaluation in a leukemia setting with NALM-6 leukemia.
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