Methods and compositions for transducing lymphocytes and regulating the activity thereof
Inventors
Frost, Gregory Ian • Onuffer, James Joseph • Guibinga, Ghiabe H. • Haerizadeh, Farzad • Kundu, Anirban
Assignees
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Publication Number
US-11111505-B2
Publication Date
2021-09-07
Expiration Date
Abstract
The present disclosure provides methods for genetically modifying lymphocytes and methods for performing adoptive cellular therapy that include transducing T cells and/or NK cells. The methods can include inhibitory RNA molecule(s) and/or engineered signaling polypeptides that can include a lymphoproliferative element, and/or a chimeric antigen receptor (CAR), for example a microenvironment restricted biologic CAR (MRB-CAR). Additional elements of such engineered signaling polypeptides are provided herein, such as those that drive proliferation and regulatory elements therefor, as well as replication incompetent recombinant retroviral particles and packaging cell lines and methods of making the same. Numerous elements and methods for regulating transduced and/or genetically modified T cells and/or NK cells are provided, such as, for example, those including riboswitches, MRB-CARs, recognition domains, and/or pH-modulating agents.
Core Innovation
The invention provides a replication incompetent recombinant retroviral particle that is pseudotyped with one or more pseudotyping elements on the surface, including a vesicular stomatitis virus (VSV-G) envelope protein, a feline endogenous virus (RD114) envelope protein, or a Paramyxoviridae envelope protein. The particle is configured to deliver engineered T-cell signaling polypeptides into T cells and to activate resting T cells through a surface activation element.
The particle includes one or more transcriptional units operatively linked to a promoter active in T cells and encoding a first engineered signaling polypeptide comprising a lymphoproliferative element with a cytokine receptor polypeptide having a signaling domain capable of activating a Stat pathway and promoting proliferation and/or survival of T cells. The particle also encodes a second engineered signaling polypeptide comprising a chimeric antigen receptor with an antigen-specific targeting region, a transmembrane domain, and an intracellular activating domain.
The particle further includes an activation element on the surface that is fused to a heterologous membrane attachment sequence and is capable of binding to CD3 on the surface of a resting T cell to activate the resting T cell. The activation element comprises an anti-CD3 antibody and is not encoded by a polynucleotide in the recombinant retroviral particle.
Claims Coverage
The independent claim identified in the provided material covers a replication-incompetent recombinant retroviral particle with three inventive features: specific pseudotyping elements, T-cell-active transcriptional units encoding a Stat-activating lymphoproliferative cytokine receptor element and a CAR, and a non-genome-encoded surface activation element that binds CD3 on resting T cells.
Replication incompetent retroviral particle with defined pseudotyping elements
A replication incompetent recombinant retroviral particle comprising one or more pseudotyping elements on the surface, wherein the one or more pseudotyping elements comprises a vesicular stomatitis virus (VSV-G) envelope protein, a feline endogenous virus (RD114) envelope protein, or a Paramyxoviridae envelope protein.
T-cell-active transcriptional units encoding lymphoproliferative and CAR signaling polypeptides
A polynucleotide comprising one or more transcriptional units, wherein each transcriptional unit is operatively linked to a promoter active in T cells and encodes a first engineered signaling polypeptide comprising a lymphoproliferative element with a cytokine receptor polypeptide comprising a signaling domain capable of activating a Stat pathway and promoting proliferation and/or survival of T cells, and a second engineered signaling polypeptide comprising a chimeric antigen receptor comprising an antigen-specific targeting region, a transmembrane domain, and an intracellular activating domain.
Surface CD3 activation element fused to a heterologous membrane attachment sequence
An activation element on the surface of the replication incompetent recombinant retroviral particle, wherein the activation element is fused to a heterologous membrane attachment sequence, is capable of binding to CD3 on the surface of a resting T cell and activating the resting T cell, is not encoded by a polynucleotide in the recombinant retroviral particle, and comprises an anti-CD3 antibody.
The inventive scope centers on a pseudotyped replication-incompetent recombinant retroviral particle carrying T-cell-active transcriptional units for a Stat-activating lymphoproliferative cytokine receptor element and an antigen-specific CAR element, together with a separately provided surface anti-CD3 activation element fused to a heterologous membrane attachment sequence and not encoded by the particle.
Stated Advantages
Promotes proliferation and/or survival of T cells via a cytokine receptor signaling domain capable of activating a Stat pathway.
Activates resting T cells through a surface activation element capable of binding CD3.
Potential elimination of prior ex vivo stimulation and lymphodepletion is emphasized.
Safety via compound-dependent activation/termination of lymphoproliferation is emphasized.
Reduced ex vivo time (as short as hours) is emphasized.
Regulates constitutively active T-cell survival and lymphoproliferative IL-7R pathways in vivo without ex vivo stimulation.
Reduces on-target/off-tumor toxicity using microenvironment-restricted CAR binding with pH modulation concepts.
Documented Applications
Adoptive cellular therapy using rapid ex vivo transduction of resting T cells and/or NK cells with replication-incompetent retroviral particles.
Adoptive cell therapy using engineered T cells and NK cells with CAR expression and additional inhibitory/lymphoproliferative and safety/activation elements.
Microenvironment-restricted CAR binding in the tumor microenvironment using pH modulation concepts to reduce on-target/off-tumor toxicity.
In vivo use without ex vivo stimulation to regulate a constitutively active T-cell survival/lymphoproliferative IL-7R pathway for expansion/persistence.
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