Compositions and methods for enhancing adoptive T cell therapeutics
Inventors
Roybal, Kole • GARCIA, Julie • Zhu, Iowis • Choi, Jaehyuk • Daniels, Jay
Assignees
Northwestern University • University of California San Diego UCSD
Publication Number
US-12295971-B2
Publication Date
2025-05-13
Expiration Date
2043-10-02
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Abstract
The present disclosure relates generally to compositions and methods for improving T cell therapy. In particular, the disclosure provides polypeptides and recombinant nucleic acid constructs and/or recombinant nucleic acids encoding polypeptides having mutations capable of altering T cell signaling, cytokine production, and/or in vivo persistence in tumors of therapeutic T cells comprising the mutation. The T cell signaling can be by NFAT, NF-κB and/or AP-1 pathways. The disclosure also provides vectors and cells including the polypeptides and/or recombinant nucleic acid constructs and/or recombinant nucleic acids of the disclosure as well as methods of preparing a T cell for use in cell therapy, and methods of identifying a mutation useful for improving T cell therapy.
Core Innovation
The invention discloses compositions and methods for improving adoptive T cell therapy by engineering T cells to express specific mutated polypeptides or fusion proteins, notably those comprising a caspase-associated recruitment domain (CARD) containing protein (such as CARD11) fused to a Src Homology region 2 (SH2) domain. These polypeptides, and nucleic acid constructs encoding them, are designed to enhance therapeutic efficacy by modulating key T cell signaling pathways including NFAT, NF-κB, and AP-1, and by improving cytokine production, persistence, and fitness of engineered T cells in tumors.
The invention addresses the need for more effective adoptive T cell therapies, specifically targeting the major limitation of poor efficacy against solid tumors and the challenge of T cell exhaustion, lack of persistence, and insufficient anti-tumor activity. Current rational design or genome-wide screens have not adequately overcome these bottlenecks. The patent approaches this by introducing or exploiting genetic mutations—identified from T cell lymphoma or clonal T cell expansion data—that naturally alter T cell signaling or enhance the fitness and persistence of T cells.
In the disclosed methods, T cells are engineered ex vivo to express identified gain-of-function mutations or gene fusions—such as the CARD11-PIK3R3 fusion—which can tune specific T cell transcriptional signaling outputs. These engineered T cells can include additional receptors such as chimeric antigen receptors (CARs) or TCRs for specificity and are suitable for use in cell therapy, particularly for the treatment of cancer and autoimmune diseases. The patent further describes how such mutations may be identified, introduced, and the resulting T cells formulated for therapeutic administration.
Claims Coverage
The claims define several inventive features centered around nucleic acid constructs, recombinant proteins, engineered cells, and methods associated with polypeptides containing a CARD domain and an SH2 domain, especially constructs highly similar to SEQ ID NO: 206.
Nucleic acid constructs encoding CARD-SH2 fusion polypeptides with sequence similarity to SEQ ID NO: 206
A nucleic acid construct comprising a sequence encoding a polypeptide with: - A caspase-associated recruitment domain (CARD) containing protein - A Src Homology region 2 (SH2) domain Where the encoded polypeptide has at least 85% identity to SEQ ID NO: 206.
Polypeptides with CARD and SH2 domains and high similarity to SEQ ID NO: 206
A polypeptide comprising a CARD containing protein and an SH2 domain, encoded by a nucleic acid construct, with at least 85% (and in certain claims, at least 95%) identity to SEQ ID NO: 206.
Vectors encoding the CARD-SH2 fusion
A vector selected from plasmid, retrovirus vector, adenovirus vector, or adeno-associated virus vector, containing the nucleic acid construct encoding the CARD-SH2 fusion polypeptide having at least 85% or 95% identity to SEQ ID NO: 206.
Engineered cells comprising CARD-SH2 fusion polypeptides
Isolated, in vitro, or ex vivo engineered cells containing the polypeptide or nucleic acid encoding the CARD-SH2 fusion, where the polypeptide has at least 85% identity to SEQ ID NO: 206. The engineered cell can be any immune cell type as specified in the claims.
Engineered cells further comprising a CAR, TCR, cytokine, chemokine, or growth factor
Engineered cells as above, where the cell also comprises a CAR, a TCR, a cytokine, chemokine, or a growth factor, with CAR or TCR specificity for a broad range of specified tumor or immune antigens.
Promoter-controlled expression of the CARD-SH2 fusion
Engineered cells where expression of the CARD-SH2 fusion is driven by a specified promoter, with a wide selection of T cell-specific and other promoters enumerated in the claim text.
Methods of making engineered cells introducing CARD-SH2 fusion nucleic acid constructs
A method of making an isolated, in vitro, or ex vivo engineered cell comprising introducing a nucleic acid construct encoding a polypeptide having a CARD domain and SH2 domain (at least 85% identity to SEQ ID NO: 206) into the cell.
Engineered cells with specific genomic modifications
Engineered cells as above where the nucleic acid is inserted via RNA, plasmid, retrovirus, adenovirus, adeno-associated virus vector, or by CRISPR/Cas system, and/or in the TCR alpha locus. Also includes cells with reduced or eliminated endogenous TCR expression, or specified as autologous or allogeneic cells.
The claims collectively cover constructs, polypeptides, engineered cells, and methods relating to CARD-SH2 domain fusion proteins, their nucleic acid sequences, vectors, and the therapeutic use and manufacture of immune cells expressing such constructs.
Stated Advantages
Enables engineered T cells with enhanced in vivo persistence and accumulation in tumors compared to prior approaches that overexpress wild-type genes.
Reduces T cell exhaustion, increases proliferation, improves effector function, and increases fitness and anti-tumor activity of therapeutic T cells.
Allows tunable and specific modification of key signaling pathways (such as NFAT, NF-κB, AP-1) unachievable by wild-type gene overexpression.
Improves efficacy of adoptive T cell therapies in solid tumors, which are typically resistant to current CAR-T therapies.
Provides a platform for engineering a broad range of immune cell types beyond T cells, including macrophages and NK cells.
Engineered cells show durable tumor control and long-term memory characteristics, enabling protection from tumor relapse.
CARD11-PIK3R3 expressing cells demonstrated a favorable safety profile with no evidence of malignant transformation in long-term animal studies.
Documented Applications
Therapeutic use of engineered T cells expressing CARD-SH2 fusion proteins in adoptive T cell therapy for cancer, including solid and hematological tumors.
Preparation and administration of immune cells (including CAR-T and TCR-T cells) genetically modified to express CARD-SH2 fusion polypeptides for enhanced persistence and anti-tumor activity.
Use in treatment of autoimmune diseases in subjects, where enhanced engineered T cell function is beneficial.
Engineering of a wide variety of immune cell types—including T cells, macrophages, monocytes, NK cells, gamma delta T cells, invariant NKT cells, and MAIT cells—using the disclosed polypeptides or constructs.
Engineered cells for cell-based therapies against tumors expressing a wide range of target antigens, as extensively listed in the document.
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