Methods and compositions for genetically modifying lymphocytes to express polypeptides comprising the intracellular domain of MPL
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-11325948-B2
Publication Date
2022-05-10
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 relates to a replication incompetent recombinant retroviral particle that carries one or more pseudotyping elements on its surface and includes a polynucleotide with one or more transcriptional units operatively linked to a promoter active in T cells. The transcriptional units encode engineered signaling polypeptides and a chimeric antigen receptor (CAR). The engineered particle framework is positioned to support T-cell signaling through defined intracellular and extracellular components and to combine those components with CAR expression.
A first engineered signaling polypeptide is a chimeric polypeptide that includes an extracellular domain, a transmembrane domain, and a first intracellular signaling domain. The transmembrane domain is covalently attached to the extracellular domain and is between 15 and 100 amino acids, and the first intracellular signaling domain comprises a cytokine receptor polypeptide capable of activating a cellular signaling pathway. The first intracellular signaling domain comprises an intracellular signaling domain of thrombopoietin receptor having at least 90% sequence identity to the sequence of SEQ ID NO:283 and includes a JAK-2 binding domain comprising a Box1 motif according to SEQ ID NO:306.
The extracellular domain of the first engineered signaling polypeptide comprises a leucine zipper dimerizing motif. The disclosed sequence-related embodiments also present additional modular intracellular and co-stimulatory signaling domain options, and multiple signaling polypeptides may be arranged in combinatorial ways. The overall engineered signaling polypeptide concepts are associated with transcriptional, cytokine, and cytotoxic responses, together with expansion and survival properties in the immune receptor frameworks described alongside CARs.
Claims Coverage
The independent claim coverage centers on a replication incompetent recombinant retroviral particle with surface pseudotyping elements and T-cell-active transcriptional units that co-express a first engineered signaling polypeptide and a second engineered signaling polypeptide comprising a CAR. The dependent claims refine the first engineered signaling polypeptide with sequence and structural constraints and add optional membrane-bound activation elements and selectable pseudotyping element sets.
Replication-incompetent recombinant retroviral particle with T-cell-active transcriptional units
A replication incompetent recombinant retroviral particle comprising one or more pseudotyping elements on the surface and a polynucleotide having 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 CAR.
Chimeric first engineered signaling polypeptide with thrombopoietin receptor intracellular signaling
A first engineered signaling polypeptide as a chimeric polypeptide comprising an extracellular domain, a transmembrane domain, and a first intracellular signaling domain comprising a cytokine receptor polypeptide capable of activating a cellular signaling pathway, where the transmembrane domain is between 15 and 100 amino acids and the first intracellular signaling domain comprises an intracellular signaling domain of thrombopoietin receptor having at least 90% sequence identity to SEQ ID NO:283 and includes a JAK-2 binding domain comprising a Box1 motif according to SEQ ID NO:306.
Extracellular leucine zipper dimerizing motif coupled through covalent transmembrane attachment
The first engineered signaling polypeptide where the extracellular domain comprises a leucine zipper dimerizing motif and the transmembrane domain is covalently attached to the extracellular domain.
CAR-encoding second engineered signaling polypeptide in the same retroviral transcriptional units
A second engineered signaling polypeptide comprising a chimeric antigen receptor (CAR), encoded by the transcriptional units of the polynucleotide carried by the replication incompetent recombinant retroviral particle.
The claims define a replication incompetent recombinant retroviral particle that uses surface pseudotyping elements and T-cell-active transcriptional units to co-express a chimeric first engineered signaling polypeptide with a thrombopoietin receptor intracellular signaling domain including a Box1 motif and a CAR. The dependent claims further narrow sequence identity thresholds and motif placement, and add optional membrane-bound activation elements and selectable pseudotyping element sets.
Stated Advantages
Rapidly genetically modifying resting lymphocytes (T cells/NK cells).
Contacting lymphocytes in whole blood within a closed system.
Reducing ex vivo processing time (potentially same day).
Removing free viral particles after contact by PBMC enrichment and/or leukodepletion filtration using a leukodepletion filter assembly.
Associated with transcriptional, cytokine, and cytotoxic responses, together with expansion and survival properties in the immune receptor frameworks described alongside CARs.
Documented Applications
Genetic modification of resting lymphocytes, including T cells and NK cells, using replication-incompetent recombinant retroviral particles for adoptive cell therapy.
Using a closed system to contact lymphocytes in whole blood with a surface-associated T-cell activation element (e.g., anti-CD3) prior to introduction into an adoptive cell therapy workflow.
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