Truncated epiderimal growth factor receptor (EGFRt) for transduced t cell selection
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Publication Number
US-12421294-B2
Publication Date
2025-09-23
Expiration Date
Abstract
A non-immunogenic selection epitope may be generated by removing certain amino acid sequences of the protein. For example, a gene encoding a truncated human epidermal growth factor receptor polypeptide (EGFRt) that lacks the membrane distal EGF-binding domain and the cytoplasmic signaling tail, but retains an extracellular epitope recognized by an anti-EGFR antibody is provided. Cells may be genetically modified to express EGFRt and then purified without the immunoactivity that would accompany the use of full-length EGFR immunoactivity. Through flow cytometric analysis, EGFRt was successfully utilized as an in vivo tracking marker for genetically modified human T cell engraftment in mice. Furthermore, EGFRt was demonstrated to have cellular depletion potential through cetuximab mediated antibody dependent cellular cytotoxicity (ADCC) pathways. Thus, EGFRt may be used as a non-immunogenic selection tool, tracking marker, a depletion tool or a suicide gene for genetically modified cells having therapeutic potential.
Core Innovation
The invention relates to a population of human T-cells that harbor a nucleic acid molecule encoding an EGFR truncated protein (EGFRt) comprising amino acids 23-357 of SEQ ID NO:3 while not encoding an EGFR Domain I, an EGFR Domain II, an EGFR Juxtamembrane Domain, or an EGFR Tyrosine Kinase Domain. The truncated EGFR retains an anti-EGFR extracellular epitope while lacking EGF-binding distal domains and the cytoplasmic signaling tail.
The truncated EGFR marker (EGFRt) is associated with anti-EGFR antibody-mediated extracellular binding that enables selection, purification, and detection. The document describes genetic modification of human T-cells to express EGFRt for immunomagnetic/FACS selection and purification using biotinylated cetuximab (Erbitux) and anti-biotin reagents, and for in vivo tracking of engraftment using cetuximab-mediated detection.
The document further describes cetuximab-mediated cytotoxic pathways in connection with EGFRt-expressing T-cells, including ADCC and CDC. In this context, EGFRt-expressing CAR T-cells are associated with potential depletion/suicide activity mediated through cetuximab, while construct examples link EGFRt with CAR components including tumor-associated antigen targeting such as CD19/CD20/CD22.
Claims Coverage
The independent claims cover two related human T-cell populations defined by the specific encoded EGFR truncated sequence and the presence of or exclusions from EGFR functional domains. Across the two independent claims, there are inventive features that specify an EGFRt encoding region that excludes EGFR Domain I, EGFR Domain II, EGFR Juxtamembrane Domain, and EGFR Tyrosine Kinase Domain, and a second encoded sequence defined as SEQ ID NO:6.
EGFR truncated encoding without EGFR functional domains
A population of human T-cells harboring a nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence comprising amino acids 23-357 of SEQ ID NO:3 and not encoding an EGFR Domain I, an EGFR Domain II, an EGFR Juxtamembrane Domain, and an EGFR Tyrosine Kinase Domain.
Human T-cells encoding SEQ ID NO:6
A population of human T-cells harboring a nucleic acid molecule encoding the amino acid sequence comprising SEQ ID NO:6.
Overall, the claim coverage defines human T-cell populations by the precise EGFR truncated encoding sequence(s), including exclusion of EGFR Domain I, EGFR Domain II, EGFR Juxtamembrane Domain, and EGFR Tyrosine Kinase Domain for one independent claim, and by the encoded amino acid sequence comprising SEQ ID NO:6 for the other.
Stated Advantages
EGFRt inactivity, including no EGF binding and no EGFR phosphorylation.
Enrichment of the EGFRt-expressing T-cells from low purity to >90% purity.
Preservation of CAR effector phenotype after expansion.
Cetuximab-mediated cytotoxicity (including ADCC/CDC pathways) of EGFRt-associated T-cells.
In vivo detection of EGFRt-associated cells for tracking engraftment.
Documented Applications
Immunomagnetic/FACS selection and purification of EGFRt-expressing human T-cells using biotinylated cetuximab with anti-biotin reagents.
In vivo tracking of T cell engraftment in mice using cetuximab-mediated detection of EGFRt.
Cetuximab-mediated depletion/suicide activity associated with EGFRt-expressing T-cells via ADCC/CDC pathways.
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