Methods and compositions for transducing lymphocytes and regulating the activity thereof
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.
1. A replication incompetent recombinant retroviral particle, comprising:
A. one or more pseudotyping elements on the surface of the replication incompetent recombinant retroviral particle, 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;
B. a polynucleotide comprising one or more transcriptional units, wherein each of the one or more transcriptional units is operatively linked to a promoter active in T cells, and wherein the one or more transcriptional units encode
i. a first engineered signaling polypeptide comprising a lymphoproliferative element wherein the lymphoproliferative element comprises a cytokine receptor polypeptide comprising a signaling domain that is capable of activating a Stat pathway, and promoting proliferation and/or survival of T cells; and
ii. a second engineered signaling polypeptide comprising a chimeric antigen receptor comprising an antigen-specific targeting region, a transmembrane domain, and an intracellular activating domain; and
C. 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, wherein the activation element is a polypeptide capable of binding to CD3 on the surface of a resting T cell and activating the resting T cell and is not encoded by a polynucleotide in the recombinant retroviral particle and wherein the activation element comprises an anti-CD3 antibody.
2. The replication incompetent recombinant retroviral particle of claim 1 , wherein the first engineered signaling polypeptide is encoded in a reverse orientation with respect to a cis-acting RNA packaging element in the genome of the recombinant retroviral particle.
3. The replication incompetent recombinant retroviral particle of claim 1 , wherein the promoter of at least one of the one or more transcriptional units is a T cell specific promoter.
4. The replication incompetent recombinant retroviral particle of claim 1 , wherein the promoter of at least one of the one or more transcriptional units is the EF1a promoter, the MSCV promoter, or the CD3z promoter.
5. The replication incompetent recombinant retroviral particle of claim 1 , wherein the polynucleotide further comprises an intron, and wherein the intron encodes an shRNA or one or more miRNAs.
6. The replication incompetent recombinant retroviral particle of claim 5 , wherein the intron is an EF1a intron.
7. The replication incompetent recombinant retroviral particle of claim 1 , wherein the first engineered signaling polypeptide coding sequence is operably linked to an intron in a first transcription unit of the one or more transcriptional units, and wherein the first transcriptional unit is in reverse orientation with respect to a cis-acting RNA packaging element in the genome of the recombinant retroviral particle.
8. The replication incompetent recombinant retroviral particle of claim 1 , wherein the Stat pathway is a Stat1 pathway, a Stat3 pathway, a Stat4 pathway, or a Stat5 pathway.
9. The replication incompetent recombinant retroviral particle of claim 1 , wherein the signaling domain is an intracellular signaling domain of an IL-7 receptor, an intracellular signaling domain of an IL-12 receptor, an intracellular signaling domain of an IL-15 receptor, or an intracellular signaling domain of an IL-21 receptor.
10. The replication incompetent recombinant retroviral particle of claim 1 , wherein the lymphoproliferative element is constitutively active.
11. The replication incompetent recombinant retroviral particle of claim 10 , wherein the constitutively active lymphoproliferative element is an IL-7Rα mutant, or a functional fragment thereof.
12. The replication incompetent recombinant retroviral particle of claim 11 , wherein the IL-7Rα mutant comprises amino acids 229 to 292 of SEQ ID NO:229 with an insertion of PPCL at amino acid 243 of SEQ ID NO:229.
13. The replication incompetent recombinant retroviral particle of claim 1 , wherein the lymphoproliferative element is a fusion polypeptide comprising a recognition domain that is recognized by a monoclonal antibody approved biologic.
14. The replication incompetent recombinant retroviral particle of claim 1 , wherein the transcriptional unit encoding the first engineered signaling polypeptide further comprises a riboswitch.
15. The replication incompetent recombinant retroviral particle of claim 1 , wherein the activation element further comprises a second polypeptide, wherein the second polypeptide is capable of binding CD28.
16. The replication incompetent recombinant retroviral particle of claim 1 , wherein the polypeptide capable of binding to CD3 is fused to a heterologous GPI anchor attachment sequence.
17. The replication incompetent recombinant retroviral particle of claim 1 , wherein the anti-CD3 antibody is a GPI-anchored anti-CD3scFvFc.
18. The replication incompetent recombinant retroviral particle of claim 1 , wherein the Paramyxoviridae envelope protein is a Measles Virus F polypeptide, or a Measles Virus H polypeptide.
19. The replication incompetent recombinant retroviral particle of claim 1 , wherein the replication incompetent recombinant retroviral particle is a lentivirus or a gammaretroviral particle.
20. The replication incompetent recombinant retroviral particle of claim 1 , wherein the genome of the recombinant retroviral particle is less than 10,000 kb, wherein the replication incompetent recombinant retroviral particle is a lentiviral particle, wherein the lymphoproliferative element is a constitutively active cytokine receptor that is not tethered to a cytokine, and wherein the replication incompetent recombinant retroviral particle further encodes a polypeptide that is recognized by a monoclonal antibody approved biologic.
21. The replication incompetent recombinant retroviral particle of claim 1 , wherein the recombinant retroviral particle further comprises a membrane-bound cytokine on the surface of the recombinant retroviral particle, wherein the membrane-bound cytokine comprises a fusion polypeptide of IL-7 and DAF, and wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:286.