Methods and compositions for genetically modifying lymphocytes to express polypeptides comprising the intracellular domain of MPL
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.
1. A replication incompetent recombinant retroviral particle comprising:
A. one or more pseudotyping elements on the surface of the replication incompetent recombinant retroviral particle; and
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, wherein said first engineered signaling polypeptide is a chimeric polypeptide comprising an extracellular domain, a transmembrane domain, and a first intracellular signaling domain comprising a cytokine receptor polypeptide that is capable of activating a cellular signaling pathway, wherein said transmembrane domain is covalently attached to said extracellular domain and said first intracellular signaling domain,
wherein said transmembrane domain is between 15 and 100 amino acids,
wherein said 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 comprises a JAK-2 binding domain comprising a Box1 motif according to SEQ ID NO:306, and
wherein said extracellular domain comprises a leucine zipper dimerizing motif, and
ii. a second engineered signaling polypeptide comprising a chimeric antigen receptor (CAR).
2. The replication incompetent recombinant retroviral particle of claim 1 , wherein the leucine zipper dimerizing motif is from a jun polypeptide.
3. The replication incompetent recombinant retroviral particle of claim 1 , wherein a linker of between 1 and 4 alanine residues is present between the extracellular domain and the transmembrane domain.
4. 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 of the replication incompetent recombinant retroviral particle, in the genome of the replication incompetent recombinant retroviral particle.
5. The replication incompetent recombinant retroviral particle of claim 1 , wherein the first intracellular signaling domain comprises the Box1 motif having amino acids 17-20 of SEQ ID NO:283.
6. The replication incompetent recombinant retroviral particle of claim 1 , wherein the intracellular signaling domain does not comprise a ubiquitin targeting motif having a lysine residue corresponding to K40 of SEQ ID NO:283 or a lysine residue corresponding to K60 of SEQ ID NO:283.
7. The replication incompetent recombinant retroviral particle of claim 1 , wherein the intracellular signaling domain comprises a tyrosine residue corresponding to Y8 of SEQ ID NO:283, Y29 of SEQ ID NO:283, Y78 of SEQ ID NO:283, Y113 of SEQ ID NO:283, and Y118 of SEQ ID NO:283, and wherein the intracellular signaling domain contains the Shc phosphotyrosine-binding motif NXXY (SEQ ID NO:307), where each X can be any amino acid.
8. The replication incompetent recombinant retroviral particle of claim 1 , wherein the intracellular signaling domain comprises a STAT3 consensus binding sequence YXXQ (SEQ ID NO:308) where each X can be any amino acid, corresponding to amino acids 118-121 of SEQ ID NO:283.
9. The replication incompetent recombinant retroviral particle of claim 5 , wherein the intracellular signaling domain comprises the sequence YLXL (SEQ ID NO:309) corresponding to amino acid 113-116 of SEQ ID NO:283, where X can be any amino acid.
10. The replication incompetent recombinant retroviral particle of claim 1 , wherein the intracellular signaling domain of said first intracellular signaling domain is at least 95% identical to SEQ ID NO:283.
11. The replication incompetent recombinant retroviral particle of claim 1 , wherein the transmembrane domain does not comprise the transmembrane domain of the thrombopoietin receptor.
12. The replication incompetent recombinant retroviral particle of claim 1 , wherein the replication incompetent recombinant retroviral particle is a lentivirus, and wherein the first engineered signaling polypeptide comprises a second intracellular signaling domain, wherein the second intracellular signaling domain is a signaling domain from a gene other than the thrombopoietin receptor, that promotes proliferation, survival, and/or provides a co-stimulatory signal that enhances a differentiation state, proliferative potential, or resistance to cell death.
13. The replication incompetent recombinant retroviral particle of claim 12 , wherein the second intracellular signaling domain is from a gene selected from CD27, CD40, CRLF2, CSF2RA, CSF3R, EPOR, FCER1G, FCGR2A, FCGR2C, GHR, IFNAR1, IFNAR2, IFNGR2, IL1R1, IL1RL1, IL2RA, IL2RG, IL3RA, IL5RA, IL6R, IL7R, IL9R, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL18R1, IL18RAP, IL20RB, IL22RA1, IL27RA, IL31RA, LEPR, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18.
14. The replication incompetent recombinant retroviral particle of claim 13 , wherein the second intracellular signaling domain is a CD40 intracellular signaling domain.
15. The replication incompetent recombinant retroviral particle of claim 14 , wherein the second intracellular signaling domain is between 50 and 66 amino acids in length, comprises the TRAF binding sites from the CD40 intracellular signaling domain, and is at least 90% identical to the CD40 intracellular signaling domain.
16. The replication incompetent recombinant retroviral particle of claim 1 , wherein the replication incompetent recombinant retroviral particle further comprises a membrane-bound T cell activation element, and wherein the membrane-bound T cell activation element is an anti-CD3 antibody.
17. The replication incompetent recombinant retroviral particle of claim 16 , wherein the anti-CD3 antibody is an anti-CD3 scFvFc antibody, wherein the membrane-bound T cell activation element is fused to a heterologous membrane attachment sequence, and wherein the heterologous membrane attachment sequence is a GPI anchor attachment sequence.
18. The replication incompetent recombinant retroviral particle of claim 1 , wherein one of the one or more pseudotyping elements is selected from a MuLV envelope protein, a BaEV envelope protein, a VSV-G envelope protein, an influenza HA, an influenza NA, a paramyxovirus Measles envelope protein H, and a paramyxovirus Measles envelope protein F.
19. The replication incompetent recombinant retroviral particle of claim 1 , wherein one of the one or more pseudotyping elements is MuLV fused to an anti-CD3 antibody.
20. The replication incompetent recombinant retroviral particle of claim 1 , wherein the first engineered signaling polypeptide is a fusion polypeptide comprising a recognition domain that is recognized by a monoclonal antibody approved biologic.
21. The replication incompetent recombinant retroviral particle of claim 1 , wherein the first engineered signaling polypeptide possesses the property of:
a) improving expansion of pre-activated PBMCs transduced with a retroviral particle comprising a nucleic acid construct encoding the first engineered signaling polypeptide when transduced along with a nucleic acid encoding an anti-CD19 CAR comprising a CD3 zeta intracellular activating domain but no co-stimulatory domain, between day 7 and day 21 of in vitro culturing in the absence of exogenously added cytokines, compared to a control construct identical to the nucleic acid construct comprising the first engineered signaling polypeptide but without the first engineered signaling polypeptide, under identical conditions; and/or
b) expanding pre-activated PBMCs transduced with a nucleic acid construct encoding the first engineered signaling polypeptide at least 2-fold between day 7 and day 21 of in vitro culturing in the absence of exogenously added cytokines, when transduced along with a nucleic acid encoding an anti-CD19 CAR comprising a CD3 zeta intracellular activating domain but no costimulatory domain.
22. The replication incompetent recombinant retroviral particle of claim 1 , wherein the replication incompetent recombinant retroviral particle further comprises a membrane-bound cytokine on the surface of the replication incompetent 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:462.
23. A method for genetically modifying a T cell of a subject, wherein the method comprises:
contacting a population of T cells comprising the T cell ex vivo, with a population of replication incompetent recombinant retroviral particles according to claim 16 , wherein said contacting is performed for less than 12 hours to facilitate membrane fusion of the T cell to the replication incompetent recombinant retroviral particle, thereby genetically modifying the T cell.
24. The method of claim 23 , wherein the method further comprises:
collecting blood comprising the population of T cells from a subject before contacting the population of T cells ex vivo with the population of replication incompetent recombinant retroviral particles; and
reintroducing the genetically modified T cell into the subject.
25. The method of claim 24 , wherein the genetically modified T cell is not expanded ex vivo after said contacting and before being reintroduced into the subject.
26. The method of claim 23 , wherein the population of T cells comprises between 90% and 100% resting T cells.
27. The method of claim 24 , wherein the population of T cells undergoes 4 or fewer cell divisions ex vivo prior to being reintroduced into the subject.