Methods and compositions for transducing lymphocytes and regulating the activity thereof

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.

What is claimed is: 
     
         1 . A replication incompetent recombinant retroviral particle, comprising:
 A. 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 Jak pathway, and promoting proliferation and/or survival of T cells; and 
 ii. a second engineered signaling polypeptide comprising an antigen-specific targeting region, a transmembrane domain, and an intracellular activating domain; and 
   B. an activation element on the surface of the replication incompetent recombinant retroviral particle, wherein the activation element comprises a means for binding to CD3.   
     
     
         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 EF1α promoter, the MSCV promoter, or the CD3ζ 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 EF1α 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 signaling domain that is capable of activating a Jak pathway activates a STAT pathway, 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 9 , wherein the signaling domain of the cytokine receptor polypeptide is the intracellular signaling domain of an IL-7 receptor, and wherein the IL-7 receptor is the IL-7Rα, or a functional fragment thereof. 
     
     
         11 . The replication incompetent recombinant retroviral particle of  claim 9 , wherein the signaling domain of the cytokine receptor polypeptide is the intracellular signaling domain of an IL-15 receptor, wherein the IL-15 receptor is the IL-2/IL-15Rβ or the common γchain, or a functional fragment thereof. 
     
     
         12 . The replication incompetent recombinant retroviral particle of  claim 1 , wherein the lymphoproliferative element is constitutively active. 
     
     
         13 . The replication incompetent recombinant retroviral particle of  claim 12 , wherein the constitutively active lymphoproliferative element is an IL-7Rα mutant, or a functional fragment thereof. 
     
     
         14 . The replication incompetent recombinant retroviral particle of  claim 13 , 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. 
     
     
         15 . 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. 
     
     
         16 . The replication incompetent recombinant retroviral particle of  claim 1 , further comprising a means for binding CD28. 
     
     
         17 . The replication incompetent recombinant retroviral particle of  claim 1 , wherein the intracellular activating domain comprises the CD3ζ intracellular activating domain. 
     
     
         18 . The replication incompetent recombinant retroviral particle of  claim 17 , wherein the ASTR comprises a single chain antibody. 
     
     
         19 . 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. 
     
     
         20 . A mammalian packaging cell line comprising a packageable RNA genome for a replication incompetent retroviral particle, wherein said packageable RNA genome comprises:
 A. a 5′ long terminal repeat, or active fragment thereof;   B. a nucleic acid sequence encoding a retroviral cis-acting RNA packaging element;   C. a polynucleotide comprising one or more nucleic acid sequences operatively linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acids encodes two or more inhibitory RNA molecules directed against one or more RNA targets and a second nucleic acid sequence of the one or more nucleic acid sequences encodes comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain; and   D. a 3′ long terminal repeat, or active fragment thereof.