Methods and compositions for genetically modifying lymphocytes to express polypeptides comprising the intracellular domain of CD79A and CD79B
Inventors
Frost, Gregory Ian • Onuffer, Jr., James Joseph • Guibinga, Ghiabe H. • Haerizadeh, Farzad • Vigant, Frederic • Kundu, Anirban
Assignees
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Publication Number
US-12325728-B2
Publication Date
2025-06-10
Expiration Date
Abstract
The present disclosure provides methods and compositions for genetically modifying lymphocytes, for example T cells and/or NK cells, in shorter times than previously and/or in whole blood or a component thereof. In some embodiments a lymphodepletion filter assembly is used before or after forming a reaction mixture where lymphocytes are contacted with recombinant retroviral particles in a closed system, to genetically modify the lymphocytes.
Core Innovation
The invention provides a vector having one or more transcriptional units operatively linked to a promoter active in T cells. Each transcriptional unit encodes a first engineered signaling polypeptide and a second engineered signaling polypeptide comprising a chimeric antigen receptor (CAR) or a recombinant TCR.
The first engineered signaling polypeptide is a chimeric polypeptide having an extracellular domain, a transmembrane domain, and a first intracellular signaling domain capable of activating a cellular signaling pathway. The transmembrane domain is covalently attached to the extracellular domain and the first intracellular signaling domain, is between 15 and 100 amino acids, and the first intracellular signaling domain includes a first ITAM motif with the sequence YX1X2L/I, where X1 and X2 are any amino acids.
The first intracellular signaling domain is selected from CD79A and CD79B, where the selected intracellular domain has at least 90% sequence identity to specified sequences. The extracellular domain comprises a leucine zipper dimerizing motif.
The document also describes replication-incompetent recombinant retroviral particle formats, including lentiviral particles, and constructs further comprising T cell activation elements that bind to CD3 and membrane-bound cytokines such as IL-7 and/or IL-2 and IL-15 with anchoring concepts.
Claims Coverage
The provided content contains one independent claim. It covers a vector architecture that combines a T-cell-active transcriptional unit encoding two engineered signaling polypeptides, with 5 inventive features centered on the first engineered signaling polypeptide and the second engineered signaling polypeptide, and additional dependent features relating to delivery particle formats.
T-cell-active transcriptional units encoding two engineered signaling polypeptides
A vector comprising one or more transcriptional units operatively linked to a promoter active in T cells, wherein the transcriptional units encode a first engineered signaling polypeptide and a second engineered signaling polypeptide comprising a chimeric antigen receptor (CAR) or a recombinant TCR.
Chimeric signaling polypeptide with covalently attached extracellular, transmembrane, and ITAM intracellular signaling
A first engineered signaling polypeptide that is a chimeric polypeptide comprising an extracellular domain, a transmembrane domain, and a first intracellular signaling domain capable of activating a cellular signaling pathway, wherein the transmembrane domain is covalently attached to the extracellular domain and the first intracellular signaling domain.
ITAM motif defined by YX1X2L/I with CD79A/CD79B selection
The first intracellular signaling domain comprises a first ITAM motif with the sequence YX1X2L/I, where X1 is any amino acid and X2 is any amino acid, and is selected from CD79A having at least 90% sequence identity to SEQ ID NO:210 and CD79B having at least 90% sequence identity to SEQ ID NO:211.
Extracellular leucine zipper dimerizing motif with defined transmembrane length
The extracellular domain comprises a leucine zipper dimerizing motif, and the transmembrane domain is between 15 and 100 amino acids.
Second engineered signaling polypeptide comprising CAR or recombinant TCR
A second engineered signaling polypeptide comprising a chimeric antigen receptor (CAR) or a recombinant TCR.
Replication-incompetent recombinant retroviral particle format
The vector is a replication-incompetent recombinant retroviral particle, including lentiviral particles.
CD3-binding T-cell activation element on the particle
The replication-incompetent recombinant retroviral particle includes a T cell activation element with a binding means that binds to CD3.
Membrane-bound cytokine on the particle surface
A replication-incompetent recombinant retroviral particle further includes a membrane-bound cytokine on its surface, including IL-7 and/or IL-2 and IL-15 with anchoring concepts.
Overall, the claim coverage centers on a vector design that specifies a T-cell-active transcriptional unit encoding two engineered signaling polypeptides, a first chimeric signaling polypeptide with covalently attached extracellular, transmembrane, and intracellular regions, an ITAM motif defined by YX1X2L/I with selection from CD79A or CD79B at defined identity thresholds, and an extracellular leucine zipper dimerizing motif together with a bounded transmembrane length.
Stated Advantages
Rapid genetic modification of resting T cells and/or NK cells using closed systems.
Reduced ex vivo time and reduced ex vivo steps via combined leukodepletion filtration assembly and retroviral particle concepts for point-of-care workflows.
Documented Applications
Adoptive cell therapy involving genetically modified T cells and/or NK cells.
Whole-blood transduction of T cells without prior PBMC enrichment in a closed blood processing system.
Point-of-care workflows using a leukodepletion filtration assembly in combination with retroviral particles.
Ex vivo genetic modification of a subject’s T cell population by contacting with replication-incompetent recombinant retroviral particles to promote membrane fusion and thereby effect genetic modification of the T cell.
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