Chimeric antigen receptors targeting glypican-3 or mesothelin
Inventors
Ho, Mitchell • Li, Nan • Li, Dan
Assignees
US Department of Health and Human Services
Publication Number
US-11802163-B2
Publication Date
2023-10-31
Expiration Date
2038-11-07
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Abstract
Nucleic acid constructs encoding a chimeric antigen receptor (CAR) and a truncated human epidermal growth factor receptor (huEGFRt) are described. The encoded CARs include a tumor antigen-specific monoclonal antibody, such as a glypican-3 (GPC3)-specific, a GPC2-specific or a mesothelin-specific monoclonal antibody, fused to a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory domain and a CD3ζ signaling domain. Isolated host cells, such as isolated T cells that co-express the disclosed CARs and huEGFRt are also described. T cells transduced with the disclosed CAR constructs can be used for cancer immunotherapy.
Core Innovation
Disclosed herein are nucleic acid constructs encoding chimeric antigen receptors (CARs) and a truncated human epidermal growth factor receptor (huEGFRt). The encoded CARs include tumor antigen-specific monoclonal antibodies, such as glypican-3 (GPC3)-specific, GPC2-specific or mesothelin-specific antibodies, fused to a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory domain and a CD3ζ signaling domain. Host cells, such as isolated T cells, co-expressing these CARs and huEGFRt are also described, wherein T cells transduced with the CAR constructs can be used for cancer immunotherapy.
The invention addresses the problem that, while CARs have shown promise in treating hematological malignancies, their application to solid tumors like hepatocellular carcinoma is limited. There is a need for effective CARs targeting solid tumor antigens with controllable off-target effects and enhanced T cell tracking and elimination capabilities.
The disclosed CAR constructs encoding both tumor antigen-specific binding fragments and a truncated EGFR (huEGFRt) allow for selection, tracking and elimination of CAR-expressing T cells in vivo via commonly used anti-EGFR antibodies, addressing safety and monitoring challenges. Furthermore, the design of these CARs with specific extracellular hinge, transmembrane, co-stimulatory and signaling domains provides targeted cytotoxicity against tumors expressing GPC3, GPC2 or mesothelin, thereby enhancing cancer immunotherapy efficacy.
Claims Coverage
The patent includes multiple inventive features related to nucleic acid constructs encoding CARs targeting glypican-3 or mesothelin, methods of treatment, and associated vectors and host cells.
Nucleic acid molecule encoding a CAR with specified components
The nucleic acid molecule encodes a chimeric antigen receptor comprising a first granulocyte-macrophage colony stimulating factor receptor signal sequence (GMCSFRss), a tumor antigen-specific antibody or antigen-binding fragment specific for GPC3 or mesothelin with defined VH and VL domain complementarity determining regions, an extracellular hinge region, a transmembrane domain, an intracellular co-stimulatory domain, an intracellular signaling domain, a self-cleaving 2A peptide, a second GMCSFRss, and a truncated human epidermal growth factor receptor (huEGFRt).
Extracellular hinge region composition
The extracellular hinge region comprises a CD8a hinge region or a CD28 hinge region.
Specific domain compositions in CAR
The CAR includes a CD8a hinge region as extracellular hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain intracellularly, and a CD3ζ signaling domain as intracellular signaling domain.
Defined nucleic acid sequences for hinge and signal sequences
The nucleic acid encoding the CD8a hinge comprises the sequence of SEQ ID NO: 3. The nucleic acids encoding the first and second GMCSFRss comprise the sequence of SEQ ID NO: 1.
Promoter inclusion for expression
The nucleic acid molecule further comprises a human elongation factor 1α (EF1α) promoter sequence 5' of the nucleic acid encoding the first GMCSFRss.
Antigen-binding fragment format
The antigen-binding fragment is a single-chain variable fragment (scFv).
Nucleic acid sequences for GPC3-specific antibodies
Where the tumor antigen is GPC3, the nucleic acid encoding the antibody or fragment comprises the VH domain CDR1, CDR2 and CDR3 from SEQ ID NO: 25 and the VL domain CDR1, CDR2 and CDR3 from SEQ ID NO: 27, including specific nucleotide ranges detailed in claims.
Vectors and host cells
Vectors, including viral vectors such as lentiviral vectors, comprising the nucleic acid molecule and isolated host cells comprising these vectors.
Methods of treating GPC3-positive and mesothelin-positive cancers
Methods comprising administering a therapeutically effective amount of isolated host cells expressing the nucleic acid molecule to treat GPC3-positive cancers (e.g., hepatocellular carcinoma, melanoma, ovarian clear-cell carcinoma) or mesothelin-positive cancers (e.g., mesothelioma, prostate, lung, stomach cancer).
Types of host cells used in treatment methods
Isolated host cells used for treatment are T lymphocytes or natural killer cells, which may be autologous or allogeneic.
The claims cover nucleic acid molecules encoding CAR constructs targeting GPC3 or mesothelin with defined signal sequences, hinge, transmembrane, co-stimulatory and signaling domains, vectors and host cells comprising these, and methods of treating cancers positive for these tumor antigens by administering such CAR-expressing cells.
Stated Advantages
CAR constructs allow selection and tracking of CAR-expressing T cells via binding to the truncated human EGFR using an FDA-approved antibody, facilitating in vivo monitoring and ablation of CAR T cells.
CAR T cells targeting GPC3, GPC2, or mesothelin exhibit high specificity and cytotoxicity against tumor cells expressing these antigens, sparing antigen-negative cells.
GPC3-targeted CAR T cells show robust activation, proliferation, polyfunctionality, and in vivo persistence leading to durable tumor control and regression in hepatocellular carcinoma models.
The huEGFRt component enhances safety by enabling elimination of CAR T cells through antibody-dependent cellular cytotoxicity when needed.
Documented Applications
Cancer immunotherapy by administering isolated host cells expressing CARs targeting glypican-3 (GPC3), glypican-2 (GPC2), or mesothelin.
Treatment of GPC3-positive cancers including hepatocellular carcinoma, melanoma, ovarian clear-cell carcinoma, yolk sac tumor, neuroblastoma, hepatoblastoma, and Wilms' tumor.
Treatment of GPC2-positive cancers including neuroblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, and osteosarcoma.
Treatment of mesothelin-positive cancers including mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, triple negative breast cancer, and ovarian cancer.
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