EP3735460A1 - Verfahren und zusammensetzungen zur genetischen modifikation und expansion von lymphozyten und regulierung der aktivität davon - Google Patents

Verfahren und zusammensetzungen zur genetischen modifikation und expansion von lymphozyten und regulierung der aktivität davon

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Publication number
EP3735460A1
EP3735460A1 EP18856963.6A EP18856963A EP3735460A1 EP 3735460 A1 EP3735460 A1 EP 3735460A1 EP 18856963 A EP18856963 A EP 18856963A EP 3735460 A1 EP3735460 A1 EP 3735460A1
Authority
EP
European Patent Office
Prior art keywords
cell
cells
polypeptide
replication incompetent
recombinant retroviral
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18856963.6A
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English (en)
French (fr)
Other versions
EP3735460A4 (de
Inventor
Gregory Ian Frost
James Joseph ONUFFER
Farzad Haerizadeh
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Exuma Biotech Corp
Original Assignee
Exuma Biotech Corp
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Filing date
Publication date
Priority claimed from PCT/US2018/020818 external-priority patent/WO2018161064A1/en
Application filed by Exuma Biotech Corp filed Critical Exuma Biotech Corp
Publication of EP3735460A1 publication Critical patent/EP3735460A1/de
Publication of EP3735460A4 publication Critical patent/EP3735460A4/de
Pending legal-status Critical Current

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/86—Viral vectors
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    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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    • A61K39/4631—Chimeric Antigen Receptors [CAR]
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
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    • C07K2319/00—Fusion polypeptide
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    • C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag
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    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
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    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
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    • C12N2740/00—Reverse transcribing RNA viruses
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    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16051—Methods of production or purification of viral material
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • This disclosure relates to the field of immunology, or more specifically, to the genetic modification of T lymphocytes or other immune cells, and methods of making replication incompetent recombinant retroviral particles and controlling the expression of genes therein.
  • Lymphocytes isolated from a subject can be activated in vitro and genetically modified to express synthetic proteins that enable redirected engagement with other cells and
  • CAR chimeric antigen receptor
  • An example of such a synthetic protein is a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • One CAR that is currently used is a fusion of an extracellular recognition domain (e.g., an antigen-binding domain), a transmembrane domain, and one or more intracellular signaling domains encoded by a replication incompetent recombinant retrovirus.
  • CAR therapies further cannot be controlled for propagation rate in vivo once introduced into the body, nor safely directed towards targets that are also expressed outside the tumor.
  • CAR therapies today are typically infused from cells expanded ex vivo from 12 to 28 days using doses from 1 x 10 5 to 1 x 10 8 cells/kg and are directed towards targets, for example tumor targets, for which off tumor on target toxicity is generally acceptable.
  • kits that help overcome issues related to the effectiveness and safety of methods for transducing and/or genetically modifying lymphocytes such as T cells and/or NK cells. Certain embodiments of such methods are useful for performing adoptive cell therapy with these cells. Accordingly, in some aspects, provided herein are methods, compositions, and kits for genetically modifying and/or transducing lymphocytes, especially T cell and/or NK cells, and/or for regulating the activity of transduced and/or genetically modified T cells and/or NK cells.
  • Transduced and/or genetically modified T cells and/or NK cells that are produced by and/or used in methods provided herein, include functionality and combinations of functionality, in illustrative embodiments delivered from retroviral (e.g. lentiviral) genomes via retroviral (e.g. lentiviral) particles, that provide improved features for such cells and for methods that utilize such cells, such as research methods, commercial production methods, and adoptive cellular therapy.
  • retroviral e.g. lentiviral
  • retroviral particles e.g. lentiviral particles
  • such cells can be produced in less time ex vivo, and that have improved growth properties that can be better regulated.
  • regulatory elements for regulating the expression of CARs, mRNA, inhibitory RNA(s), and/or lymphoproliferative elements, for example chimeric
  • lymphoproliferative elements in lymphocytes such as B cells, T cells and NK cells.
  • lymphocytes such as B cells, T cells and NK cells.
  • recombinant retroviruses that express various functional elements and that carry various functional elements on their surface, and methods and packaging cell lines for producing the recombinant retroviruses. These recombinant retroviruses and methods and cells for producing the same, overcome prior art limitations with respect to the number and size in a genome, of different functional elements that provide benefits when delivered into a T cell and/or NK cells.
  • methods are provided for transducing and/or genetically modifying lymphocytes such as T cells and/or NK cells, and in illustrative embodiments, ex vivo methods for transducing and/or genetically modifying resting T cells and/or NK cells.
  • Some of these aspects can be performed much more quickly than previous methods, which can facilitate more efficient research, more effective commercial production, and improved methods of patient care.
  • methods that in some embodiments utilize recombinant retroviruses provided herein in some aspects along with pharmacologic agents, to provide improved safety mechanisms to help modulate the activity of transduced and/or genetically modified lymphocytes such as T cells and/or NK cells.
  • Such methods, compositions, and kits can be used as research tools, in commercial production, and in adoptive cellular therapy with transduced and/or genetically modified T cells and/or NK cells expressing a CAR.
  • FIG. 1 shows a schematic of illustrative compositions including a packaging cell (100) and a replication incompetent recombinant retroviral particle (200) of one exemplary, non-limiting embodiment of the present disclosure, produced by the packaging cell (100).
  • various vectors referred to as recombinant polynucleotides (110)
  • a recombinant retroviral particle (200) that includes in its genome a first engineered signaling polypeptide that includes one or more lymphoproliferative elements and in some embodiments, a second engineered signaling polypeptide that is a chimeric antigen receptor, or a CAR.
  • the replication incompetent recombinant retroviral particle expresses on its membrane, a pseudotyping element (in a non-limiting embodiment, a Measles Virus hemagglutinin (H) polypeptide and a Measles Virus fusion (F) polypeptide, or cytoplasmic domain deletion variants thereof) (240) that allows the replication incompetent recombinant retroviral particle to bind to and fuse with a target cell; an activation element (in non-limiting embodiments an activation element that has a polypeptide capable of binding to CD28 and a polypeptide capable of binding to CD3) (210 and 220, respectively) that is capable of binding to and activating a resting T cell; and a membrane-bound cytokine (in a non-limiting embodiment, an IL-7 DAF fusion polypeptide) (230).
  • a pseudotyping element in a non-limiting embodiment, a Measles Virus hemagglutinin (H) polypeptide
  • FIG. 2 shows a schematic of non-limiting illustrative compositions including a replication incompetent recombinant retroviral particle (200), produced by a packaging cell (100) and a resting T cell (300) transfected by the replication incompetent recombinant retroviral particle (200).
  • the elements on the surface of the replication incompetent recombinant retroviral particle (200), bind to receptors and or ligands on the surface of a resting T cell.
  • the pseudotyping element can include, in non-limiting embodiments, a binding polypeptide and a fusogenic polypeptide (in non-limiting embodiments, a Measles Virus hemagglutinin (H) polypeptide and a Measles Virus fusion (F) polypeptide, or cytoplasmic domain deletion variants thereof) that facilitate the binding and fusion of the replication incompetent recombinant retroviral particle (200), to the T cell.
  • a binding polypeptide and a fusogenic polypeptide in non-limiting embodiments, a Measles Virus hemagglutinin (H) polypeptide and a Measles Virus fusion (F) polypeptide, or cytoplasmic domain deletion variants thereof
  • the replication incompetent recombinant retroviral particle (200) includes on its surface an activation element (in non- limiting embodiments an activation element that has a polypeptide capable of binding to CD28 and a polypeptide capable of binding to CD3) that is capable of activating the resting T cell by engaging the T- cell receptor complex and optionally a co-receptor (320).
  • an activation element in non-limiting embodiments an activation element that has a polypeptide capable of binding to CD28 and a polypeptide capable of binding to CD3
  • membrane-bound cytokines in non-limiting embodiments, an IL-7 DAF fusion polypeptide
  • present on the surface of the replication incompetent recombinant retroviral particle (200) bind to IL-7R ⁇ (310) on the surface of the resting T cell.
  • the replication incompetent recombinant retroviral particle (200) fuses with the T cell, and polynucleotides that encode the first engineered signaling polypeptide
  • lymphoproliferative element in illustrative embodiments, a constitutively active IL-7R ⁇ (370), are reverse transcribed in the cytosol prior to migrating to the nucleus to be incorporated into the DNA of the activated T cell.
  • Src-FLAG-Vpx (250) packaged with the virus enters the cytosol of the resting T cells and promotes the degradation of
  • the polynucleotides can also encode a second engineered signaling polypeptide that includes a CAR (360).
  • the lymphoproliferative element is expressed when a compound binds to a control element that regulates its expression (in non-limiting examples, the control element is a riboswitch that binds a nucleoside analog).
  • expression of the CAR is also regulated by the control element.
  • Part (330) is SLAM and CD46.
  • Part (340) is CD3.
  • FIGs. 3A-3E show schematics of non-limiting, exemplary vector constructs for transfecting packaging cells to produce replication incompetent recombinant retroviral particles described herein.
  • FIG. 3A shows a construct containing a polynucleotide sequence encoding an FRB domain fused to the NFKB p65 activator domain (p65 AD) and ZFHD1 DNA binding domain fused to three FKBP repeats that is constitutively expressed.
  • the construct in FIG. 3A also includes FflVl REV and Vpx as a SrcFlagVpx fusion under the rapamycin-inducible ZFHDl/p65 AD promoter.
  • FIG. 1 shows a construct containing a polynucleotide sequence encoding an FRB domain fused to the NFKB p65 activator domain (p65 AD) and ZFHD1 DNA binding domain fused to three FKBP repeats that is constitutively expressed.
  • the construct in FIG. 3A also includes
  • FIG. 3B shows a construct containing a polynucleotide encoding an rtTA sequence under the control of the ZFHDl/p65 AD promoter.
  • FIG. 3C shows a construct containing a polynucleotide encoding a puromycin resistance gene flanked by loxP sites and the extracellular MYC tag flanked by lox2272 sites. Both selectable markers are under the control of a BiTRE promoter, which is flanked by FRT sites.
  • FIG. 3D shows a construct that contains a polynucleotide encoding RFP flanked by loxP sites that is under the control of a TRE promoter and a single FRT site between the TRE promoter and the 5' loxP site of RFP.
  • FIG. 3E shows a construct containing a polynucleotide encoding GFP flanked by loxP sites that is under the control of the TRE promoter and a single FRT site between the TRE promoter and the 5' loxP site of GFP.
  • the constructs in FIGs. 3C-3E function as landing pads for other polynucleotide sequences to insert into the genome of the packaging cell line.
  • FIGs. 4A-4C show schematics of non-limiting, exemplary vector constructs for transfecting packaging cells to produce replication incompetent recombinant retroviral particles described herein.
  • FIG. 4A shows a construct containing a tricistronic polynucleotide encoding anti-CD3 (clone UCHT1) scFvFc with a CD 14 GPI anchor attachment site, CD80 extra cellular domain (ECD) capable of binding CD28 with a CD16B GPI anchor attachment site, and IL-7 fused to decay-accelerating factor (DAF) with transposon sequences flanking the polynucleotide region for integration into the HEK293S genome.
  • FIG. 4A shows a construct containing a tricistronic polynucleotide encoding anti-CD3 (clone UCHT1) scFvFc with a CD 14 GPI anchor attachment site, CD80 extra cellular domain (ECD) capable of binding CD28 with a CD
  • FIG. 4B shows a construct containing a polynucleotide with a BiTRE promoter and a polynucleotide region encoding the gag and pol polypeptides in one direction and a polynucleotide region encoding the measles virus FAx and HAy proteins in the other direction.
  • FIG. 4B shows a construct containing a polynucleotide with a BiTRE promoter and a polynucleotide region encoding the gag and pol polypeptides in one direction and a polynucleotide region encoding the measles virus FAx and HAy proteins in the other direction.
  • FIG. 4C shows a construct containing a polynucleotide sequence encoding a CAR and the lymphoproliferative element IL7R ⁇ -insPPCL under the control of a CD3Z promoter which is not active in HEK293S cells, wherein the CAR and IL7R ⁇ -insPPCL are separated by a polynucleotide sequence encoding a T2A ribosomal skip sequence and the IL7R ⁇ -insPPCL has an acyclovir riboswitch controlled ribozyme.
  • the CAR-containing construct further includes cPPT/CTS, an RRE sequence, and a polynucleotide sequence encoding FflV-l Psi ( ⁇ ). The entire polynucleotide sequence on the CAR-containing construct to be integrated into the genome is flanked by FRT sites.
  • FIG. 5 shows a schematic of the lentiviral expression vector encoding GFP, an anti-CD 19 chimeric antigen receptor, and an eTAG referred to herein as F 1-0-03.
  • FIGs. 6A and 6B show a histogram of the percentage (%) CD3+GFP+ cells in the total CD3+ population (FIG. 6A) and a histogram of the absolute cell count per well of the CD3+GFP+ population (FIG. 6B) at 3, 6, 9, 13 and 17 days after transduction of freshly isolated and unstimulated PBMCs from Donor 12M, for 14h with VSV-G pseudotyped lentiviral particles encoding Fl-0-03 and displaying a GPI-anchored antiCD3 scFvFc on their surface as indicated. Each bar represents the mean +/- SD of duplicates.
  • FIGs. 7A and 7B show a histogram of the percentage (%) CD3+GFP+ cells in the total CD3+ population (FIG. 7A) and a histogram of the absolute cell count per well of the CD3+GFP+ population (FIG. 7B) at 3 and 6 days after transduction of freshly isolated and unstimulated PBMCs from Donor 13F, for 14h, with the indicated lentiviral particles encoding Fl-0-03.
  • A shows results using VSV-G pseudotyped lentiviral particles (triplicate experiments);
  • B shows results using VSV-G pseudotyped lentiviral particles with OKT3 Ab (lug/mL) added to the transduction medium (duplicate experiments);
  • C shows results using VSV-G pseudotyped lentiviral particles expressing GPI-anchored UCHTlscFvFc on their surface (triplicate experiments);
  • D shows results using VSV-G pseudotyped lentiviral particles displaying GPI anchored UCHTlscFvFc and GPI-anchored CD80 on their surface (duplicate experiments).
  • Each bar represents the mean +/- SD of duplicates or triplicates, as indicated in FIG. 7A.
  • FIGs. 8A and 8B show a histogram of the percentage (%) CD3+GFP+ cells in the total CD3+ population (FIG. 8A) and a histogram of the absolute cell count per well of the CD3+GFP+ population (FIG. 8B) at 3, 6 and 9 days after transduction of freshly isolated and unstimulated PBMCs from Donor 12M for the indicated time of exposure (2-20h), with VSV-G pseudotyped lentiviral particles encoding Fl-0-03 and displaying GPI anchored UCHTlscFvFc and GPI-anchored CD80 on their surface as indicated. Transduction was performed in a plate or a shaker flask as indicated. Each bar represents the mean +/- SD of duplicates for lentiviral particles pseudotyped with VSV-G ("[VSV-G]"); the other experiments did not have replicates.
  • FIGs. 9A and 9B show a histogram of the percentage (%) CD3+GFP+ cells in the Live CD3+ population (FIG. 9A) and a histogram of the absolute cell count per uL of the total live population (FIG. 9B) at day 3 post-transduction of freshly isolated and unstimulated PBMCs from Donor 18, with the indicated lentiviral particles encoding Fl-0-03. Each bar represents the mean +/- SD of duplicates.
  • FIGs. 10A and 10B show histograms of the percentage (%) GFP+ cells in the total live cell population (FIG. 10A) and the GFP+ cell number per ⁇ l of culture (FIG. 10B) at Day 6 after transduction of unstimulated PBMCS for 4 hours by the different recombinant lentiviral particles at an MOI of 1.
  • FIGs. 11A and 1 IB show histograms of the percentage (%) FLAG+ cells in the total live cell population (FIG. 11A) and the FLAG+ cell number per ⁇ l of culture (FIG. 1 IB) at Day 6 after transduction of unstimulated PBMCs for 4 hours by the different recombinant lentiviral particles.
  • the lentiviral particles encoded F 1-3 -219 and were used at an MOI of 1.
  • the lentiviral particles were pseudotyped with VSV-G [VSV-G], BaEV [BaEV], or BaEV in which the fusion inhibitory R peptide was deleted Untransduced PBMCs ("Cells”) were included as a control.
  • FIG. 12A and 12B show histograms of the percentage of CD3+ FLAG+ cells in the total live cell population (FIG. 12A) and the CD3+ FLAG+ cell number per ⁇ of culture (FIG. 12B) at Day 6 after transduction of unstimulated PBMCs for 4 hours by the different recombinant lentiviral particles.
  • the lentiviral particles were pseudotyped with VSV-G [VSV-G], MuLV [MuLV], or a fusion of UCHTlscFv to MuLV [U-MuLV].
  • the lentiviral particles [VSV-G + U] and [MuLV + U] were pseudotyped with VSV-G and MuLV, respectively, and further displayed UCHTlscFvFc-GPI. Untransduced PBMCs ("Cells") were included as a control.
  • FIG. 13 is a histogram showing the CD3+FLAG+ cell number per ⁇ of culture at Day 6 after transduction of unstimulated PBMCs by the different recombinant lentiviral particles at an MOI of lfor the indicated period of time.
  • F 1-3 -253 encoded an antiCD19 CAR
  • F 1-3 -451 encoded a CLE in addition to the same CAR.
  • the lentiviral particles were pseudotyped with VSV-G [VSV-G] and optionally displayed UCHTlScFvFc-GPI [VSV-G + U] as indicated.
  • Samples were treated with dapivirine, an inhibitor of reverse transcription (RT inb) or dolutegravir, an inhibitor to integration (INT Inb), as indicated.
  • FIG. 14A provides a schematic of IL7R ⁇ variants tested for lymphoproliferative/survival activity when expressed in PBMCs.
  • FIG. 14B provides a bar graph showing percent viability of PBMCs in the presence and absence of IL-2.
  • FIG. 15 is a schematic of a non-limiting, exemplary transgene expression cassette containing a polynucleotide sequence encoding a CAR and a candidate CLE of Libraries 3A, 3B, 3.1A, and 3. IB.
  • FIG. 16 is a schematic of a non-limiting, exemplary transgene expression cassette containing a polynucleotide sequence encoding a CAR and a candidate chimeric lymphoproliferative element (CLE) of Libraries 1A, 1.1 A, and 1.1B.
  • CLE lymphoproliferative element
  • FIG. 17 is a schematic of a non-limiting, exemplary transgene expression cassette containing a polynucleotide sequence encoding a candidate CLE of Libraries 2B and 2. IB.
  • FIG. 18 is a schematic of a non-limiting, exemplary transgene expression cassette containing a polynucleotide sequence encoding a candidate CLE of Libraries 4B and 4.1 B.
  • FIG. 19 is a graph showing the fold expansion of PBMCs transduced with lentiviral particles encoding individual CLEs and cultured for 35 days in the absence of exogenous cytokines.
  • FIG. 20 is a graph showing the fold expansion of PBMCs transduced with lentiviral particles encoding an anti-CD19 CAR construct and individual CLEs and cultured for 35 days in the presence of donor matched PBMCs but in the absence of exogenous cytokines.
  • FIG. 21 is a graph showing the efficiency by which the indicated lentiviral particle transduced resting PBMCs in 4 hours. Transduction efficiency was measured as the % CAR+ PBMCs after 6 days in culture in the absence of exogenous cytokines as determined by FACS. Each lentiviral particle encoded a CAR and a CLE. Lentiviral particles F1-1-228U and F1-3-219U displayed UCHTlscFvFc-GPI on their surface.
  • FIGs. 22A and 22B are graphs showing a time course of the total number of viable cells after resting PBMCs were transduced with the indicated lentiviral particle for 4 hours and cultured in vitro in the absence of exogenous cytokines for 6 days.
  • Each lentiviral particle encoded a CAR and a CLE.
  • Lentiviral particles F1-1-228U and F1-3-219U displayed UCHTlscFvFc-GPI on their surface.
  • FIGs. 23 A, 23B, and 23C are graphs showing a time course of the copies of lentiviral genome per ⁇ g of genomic DNA from the blood of tumor-bearing NSG mice dosed with human PBMCs transduced with the indicated lentiviral particle for 4 hours and injected intravenously without the PBMCs having been expanded ex vivo.
  • Fl-1-228, F1-1-228U, Fl-3-219, and Fl- 3-219U also encoded a CLE.
  • Lentiviral particles F1-1-228U and F1-3-219U also displayed
  • FIG. 24 is a graph showing the number of CAR+ cells per 200 ⁇ l of blood of tumor-bearing NSG mice dosed with human PBMCs transduced with the indicated lentiviral particle for 4 hours and injected intravenously without the PBMCs having been expanded ex vivo. Blood was sampled at the time the mice were euthanized.
  • Fl-1-228, F1-1-228U, Fl-3-219, and Fl-3- 219U also encoded a CLE.
  • Lentiviral particles F1-1-228U and F1-3-219U also displayed UCHTlscFvFc- GPI on their surface.
  • FIG. 25 A is a graph showing the mean tumor volume of CHO-ROR2 tumors in NSG mice dosed intravenously with PBS or human PBMCs transduced with the indicated lentiviral particle encoding an anti-ROR2 MRB CAR and a CLE for 4 hours without the PBMCs having been expanded ex vivo.
  • FIG. 25B is a graph showing the mean tumor volume of Raji tumors in NSG mice dosed intravenously with PBS or human PBMCs transduced with the indicated lentiviral particle encoding an anti-CD 19 CAR and a CLE for 4 hours without the PBMCs having been expanded ex vivo.
  • Lentiviral particles F1-1-228U and F1-3-219U displayed UCHTlscFvFc-GPI on their surface.
  • FIG. 26A is a schematic of the lentiviral vector backbone F 1-0-02 including a transgene expression cassette driving expression of GFP and eTag and a synthetic EF-lalpha promoter and intron A upstream of the GFP.
  • FIG. 26B shows insertion of the miRNAs into EFlalpha intron A of the F 1-0-02 backbone.
  • FIG. 27 is a graph showing that the miR As targeting CD3zeta that are in the EF-lalpha promoter intron are able to knockdown expression of the CD3 complex.
  • FIG. 28 is a histogram showing the AACt of samples transduced with miR-TCRa containing replication incompetent lentiviral particles.
  • the AACt values are representative of the amount of processed miR-TCRa miRNA in each transduced sample relative to the non-transduced control.
  • chimeric antigen receptor or “CAR” or “CARs” refers to engineered receptors, which graft an antigen specificity onto cells, for example T cells, NK cells, macrophages, and stem cells.
  • the CARs of the invention include at least one antigen-specific targeting region (ASTR), a transmembrane domain (TM), and an intracellular activating domain (IAD) and can include a stalk, and one or more co-stimulatory domains (CSDs).
  • ASTR antigen-specific targeting region
  • TM transmembrane domain
  • IAD intracellular activating domain
  • the CAR is a bispecific CAR, which is specific to two different antigens or epitopes. After the ASTR binds specifically to a target antigen, the IAD activates intracellular signaling.
  • the IAD can redirect T cell specificity and reactivity toward a selected target in a non-MHC -restricted manner, exploiting the antigen-binding properties of antibodies.
  • the non-MHC-restricted antigen recognition gives T cells expressing the CAR the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape.
  • CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.
  • the term "microenvironment” means any portion or region of a tissue or body that has constant or temporal, physical, or chemical differences from other regions of the tissue or regions of the body.
  • a tumor microenvironment refers to the environment in which a tumor exists, which is the non-cellular area within the tumor and the area directly outside the tumorous tissue but does not pertain to the intracellular compartment of the cancer cell itself.
  • the tumor microenvironment can refer to any and all conditions of the tumor milieu including conditions that create a structural and or functional environment for the malignant process to survive and/or expand and/or spread.
  • the tumor microenvironment can include alterations in conditions such as, but not limited to, pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, concentration of electrolytes, concentration of glucose, concentration of hyaluronan, concentration of lactic acid or lactate, concentration of albumin, levels of adenosine, levels of R-2-hydroxyglutarate, concentration of pyruvate, concentration of oxygen, and/or presence of oxidants, reductants, or co-factors, as well as other conditions a skilled artisan will understand.
  • conditions such as, but not limited to, pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, concentration of electrolytes, concentration of glucose, concentration of hyaluronan, concentration of lactic acid or lactate, concentration of albumin, levels of adenosine, levels of R-2-hydroxyglutar
  • polynucleotide and “nucleic acid” refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • antibody includes polyclonal and monoclonal antibodies, including intact antibodies and fragments of antibodies which retain specific binding to antigen.
  • the antibody fragments can be, but are not limited to, fragment antigen binding (Fab) fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fab'-SH fragments, (Fab')2 Fv fragments, Fd fragments, recombinant IgG (rlgG) fragments, single-chain antibody fragments, including single-chain variable fragments (scFv), divalent scFv's, trivalent scFv's, and single domain antibody fragments (e.g., sdAb, sdFv, nanobody).
  • the term includes genetically engineered and or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, single-chain antibodies, fully human antibodies, humanized antibodies, fusion proteins including an antigen-specific targeting region of an antibody and a non- antibody protein, heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv's, and tandem tri-scFv's.
  • antibody should be understood to include functional antibody fragments thereof.
  • the term also includes intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and subclasses thereof, IgM, IgE, IgA, and IgD.
  • antibody fragment includes a portion of an intact antibody, for example, the antigen binding or variable region of an intact antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab” fragments, each with a single antigen-binding site, and a residual "Fe” fragment, a designation reflecting the ability to crystallize readily.
  • Pepsin treatment yields an F(ab')2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.
  • the terms "single-chain Fv,” “scFv,” or “sFv” antibody fragments include the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further includes a polypeptide linker or spacer between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding.
  • VH and VL domains refer to VH and VL domains that have been isolated from a host without further molecular evolution to change their affinities when generated in an scFv format under specific conditions such as those disclosed in US patent 8709755 B2 and application WO/2017/033331A1.
  • affinity refers to the equilibrium constant for the reversible binding of two agents and is expressed as a dissociation constant (Kd).
  • Kd dissociation constant
  • Affinity can be at least 1-fold greater, at least 2-fold greater, at least 3 -fold greater, at least 4-fold greater, at least 5 -fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20- fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater, or more, than the affinity of an antibody for unrelated amino acid sequences.
  • Affinity of an antibody to a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more.
  • nM nanomolar
  • pM picomolar
  • fM femtomolar
  • the term “avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution.
  • the terms “immunoreactive” and “preferentially binds” are used interchangeably herein with respect to antibodies and/or antigen-binding fragments.
  • binding refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
  • Non-specific binding would refer to binding with an affinity of less than about 10 -7 M, e.g., binding with an affinity of 10 -6 M, 10 -5 M, 10 -4 M, etc.
  • cell surface expression system or “cell surface display system” refers to the display or expression of a protein or portion thereof on the surface of a cell.
  • a cell is generated that expresses proteins of interest fused to a cell-surface protein.
  • a protein is expressed as a fusion protein with a transmembrane domain.
  • the term “element” includes polypeptides, including fusions of polypeptides, regions of polypeptides, and functional mutants or fragments thereof and polynucleotides, including microRNAs and shRNAs, and functional mutants or fragments thereof.
  • region is any segment of a polypeptide or polynucleotide.
  • a "domain” is a region of a polypeptide or polynucleotide with a functional and/or structural property.
  • the terms "stalk” or “stalk domain” refer to a flexible polypeptide connector region providing structural flexibility and spacing to flanking polypeptide regions and can consist of natural or synthetic polypeptides.
  • a stalk can be derived from a hinge or hinge region of an
  • immunoglobulin e.g., IgGl
  • IgGl immunoglobulin
  • Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain disulfide (S-S) bonds in the same positions.
  • the stalk may be of natural occurrence or non-natural occurrence, including but not limited to an altered hinge region, as disclosed in U.S. Pat. No. 5,677,425.
  • the stalk can include a complete hinge region derived from an antibody of any class or subclass.
  • the stalk can also include regions derived from CD8, CD28, or other receptors that provide a similar function in providing flexibility and spacing to flanking regions.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • polypeptide is a single chain of amino acid residues linked by peptide bonds. A polypeptide does not fold into a fixed structure nor does it have any posttranslational modification.
  • a “protein” is a polypeptide that folds into a fixed structure. “Polypeptides” and “proteins” are used interchangeably herein.
  • a polypeptide may be "purified" to remove contaminant components of a polypeptide's natural environment, e.g. materials that would interfere with diagnostic or therapeutic uses for the polypeptide such as, for example, enzymes, hormones, and other proteinaceous or
  • a polypeptide can be purified (1) to greater than 90%, greater than 95%, or greater than 98%, by weight of antibody as determined by the Lowry method, for example, more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or nonreducing conditions using Coomassie blue or silver stain.
  • SDS-PAGE sodium dodecyl sulfate- polyacrylamide gel electrophoresis
  • immune cells generally includes white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow.
  • HSC hematopoietic stem cells
  • Immune cells includes, e.g., lymphocytes (T cells, B cells, natural killer (NK) cells) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells).
  • T cell includes all types of immune cells expressing CD3 including T-helper cells (CD4 + cells), cytotoxic T cells (CD8 + cells), T-regulatory cells (Treg) and gamma-delta T cells.
  • a "cytotoxic cell” includes CD8 + T cells, natural-killer (NK) cells, NK-T cells, ⁇ T cells, a subpopulation of CD4 + cells, and neutrophils, which are cells capable of mediating cytotoxicity responses.
  • stem cell generally includes pluripotent or multipotent stem cells.
  • stem cells includes, e.g., embryonic stem cells (ES); mesenchymal stem cells (MSC); induced- pluripotent stem cells (iPS); and committed progenitor cells (hematopoietic stem cells (HSC); bone marrow derived cells, etc.).
  • ES embryonic stem cells
  • MSC mesenchymal stem cells
  • iPS induced- pluripotent stem cells
  • HSC hematopoietic stem cells
  • bone marrow derived cells etc.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • the terms “individual”, “subject”, “host”, and “patient” refer to a mammal, including, but not limited to, humans, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non- human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.
  • murines e.g., rats, mice
  • lagomorphs e.g., rabbits
  • non- human primates humans
  • canines felines
  • ungulates e.g., equines, bovines, ovines, porcines, caprines
  • the terms “therapeutically effective amount” or “efficacious amount” refers to the amount of an agent, or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to affect such treatment for the disease.
  • the “therapeutically effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.
  • the term “evolution” or “evolving” refers to using one or more methods of mutagenesis to generate a different polynucleotide encoding a different polypeptide, which is itself an improved biological molecule and/or contributes to the generation of another improved biological molecule.
  • Physiological or "normal” or “normal physiological” conditions are conditions such as, but not limited to, pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, concentration of electrolytes, concentration of glucose, concentration of hyaluronan, concentration of lactic acid or lactate, concentration of albumin, levels of adenosine, levels of R-2-hydroxyglutarate, concentration of pyruvate, concentration of oxygen, and/or presence of oxidants, reductants, or co-factors, as well as other conditions, that would be considered within a normal range at the site of administration, or at the tissue or organ at the site of action, to a subject.
  • a "genetically modified cell” includes cells that contain exogenous nucleic acids whether or not the exogenous nucleic acids are integrated into the genome of the cell.
  • a "polypeptide” as used herein can include part of or an entire protein molecule as well as any posttranslational or other modifications.
  • a pseudotyping element as used herein can include a "binding polypeptide” that includes one or more polypeptides, typically glycoproteins, that identify and bind the target host cell, and one or more "fusogenic polypeptides” that mediate fusion of the retroviral and target host cell membranes, thereby allowing a retroviral genome to enter the target host cell.
  • the "binding polypeptide” as used herein can also be referred to as a "T cell and or NK cell binding polypeptide” or a "target engagement element,” and the "fusogenic polypeptide” can also be referred to as a "fusogenic element”.
  • a "resting" lymphocyte such as for example, a resting T cell, is a lymphocyte in the GO stage of the cell cycle that does not express activation markers such as Ki-67. Resting lymphocytes can include naive T cells that have never encountered specific antigen and memory T cells that have been altered by a previous encounter with an antigen. A “resting" lymphocyte can also be referred to as a "quiescent" lymphocyte.
  • lymphodepletion involves methods that reduce the number of lymphocytes in a subject, for example by administration of a lymphodepletion agent. Lymphodepletion can also be attained by partial body or whole body fractioned radiation therapy.
  • a lymphodepletion agent can be a chemical compound or composition capable of decreasing the number of functional lymphocytes in a mammal when administered to the mammal.
  • One example of such an agent is one or more chemotherapeutic agents.
  • Such agents and dosages are known, and can be selected by a treating physician depending on the subject to be treated.
  • lymphodepletion agents include, but are not limited to, fludarabine, cyclophosphamide, cladribine, denileukin diftitox, or combinations thereof
  • RNA interference is a biological process in which RNA molecules inhibit gene expression or translation by neutralizing targeted RNA molecules.
  • the RNA target may be mRNA, or it may be any other RNA susceptible to functional inhibition by RNAi.
  • an "inhibitory RNA molecule” refers to an RNA molecule whose presence within a cell results in RNAi and leads to reduced expression of a transcript to which the inhibitory RNA molecule is targeted.
  • An inhibitory RNA molecule as used herein has a 5' stem and a 3' stem that is capable of forming an RNA duplex.
  • the inhibitory RNA molecule can be, for example, a miRNA (either endogenous or artificial) or a shRNA, a precursor of a miRNA (i.e. a Pri-miRNA or Pre-miRNA) or shRNA, or a dsRNA that is either transcribed or introduced directly as an isolated nucleic acid, to a cell or subject.
  • a miRNA either endogenous or artificial
  • shRNA a precursor of a miRNA (i.e. a Pri-miRNA or Pre-miRNA) or shRNA
  • a dsRNA that is either transcribed or introduced directly as an isolated nucleic acid, to a cell or subject.
  • double stranded RNA or “dsR A” or "RNA duplex” refers to RNA molecules that are comprised of two strands. Double-stranded molecules include those comprised of two RNA strands that hybridize to form the duplex RNA structure or a single RNA strand that doubles back on itself
  • the duplex region comprises a sequence complementary to a target RNA.
  • the sequence complementary to a target RNA is an antisense sequence, and is frequently from 18 to 29, from 19 to 29, from 19 to 21, or from 25 to 28 nucleotides long, or in some embodiments between 18, 19, 20, 21, 22, 23, 24, 25 on the low end and 21, 22, 23, 24, 25, 26, 27, 28 29, or 30 on the high end, where a given range always has a low end lower than a high end.
  • Such structures typically include a 5' stem, a loop, and a 3' stem connected by a loop which is contiguous with each stem and which is not part of the duplex.
  • the loop comprises, in certain embodiments, at least 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • the loop comprises from 2 to 40, from 3 to 40, from 3 to 21, or from 19 to 21 nucleotides, or in some embodiments between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 on the low end and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 on the high end, where a given range always has a low end lower than a high end.
  • microRNA flanking sequence refers to nucleotide sequences including microRNA processing elements.
  • MicroRNA processing elements are the minimal nucleic acid sequences which contribute to the production of mature microRNA from precursor microRNA. Often these elements are located within a 40 nucleotide sequence that flanks a microRNA stem-loop structure. In some instances the microRNA processing elements are found within a stretch of nucleotide sequences of between 5 and 4,000 nucleotides in length that flank a microRNA stem-loop structure.
  • linker when used in reference to a multiplex inhibitory RNA molecule refers to a connecting means that joins two inhibitory RNA molecules.
  • a "recombinant retrovirus” refers to a non-replicable, or "replication
  • retrovirus incompetent
  • retroviral particle recombinant retrovirus
  • retrovirus/retroviral particle can be any type of retroviral particle including, for example, gamma retrovirus, and in illustrative embodiments, lentivirus.
  • retroviral particles for example lentiviral particles
  • such retroviral particles typically are formed in packaging cells by transfecting the packing cells with plasmids that include packaging components such as Gag, Pol and Rev, an envelope or pseudotyping plasmid that encodes a pseudotyping element, and a transfer, genomic, or retroviral (e.g. lentiviral) expression vector, which is typically a plasmid on which a gene(s) or other coding sequence of interest is encoded.
  • a retroviral (e.g. lentiviral) expression vector includes sequences (e.g. a 5' LTR and a 3' LTR flanking e.g. a psi packaging element and a target heterologous coding sequence) that promote expression and packaging after transfection into a cell.
  • sequences e.g. a 5' LTR and a 3' LTR flanking e.g. a psi packaging element and a target heterologous coding sequence
  • a "framework" of a miRNA consists of "5' microRNA flanking sequence” and/or "3' microRNA flanking sequence” surrounding a miRNA and, in some cases, a loop sequence that separates the stems of a stem-loop structure in a miRNA.
  • the "framework” is derived from naturally occurring miRNAs, such as, for example, miR-155.
  • the terms “5' microRNA flanking sequence” and “5' arm” are used interchangeably herein.
  • the terms “3' microRNA flanking sequence” and “3' arm” are used interchangeably herein.
  • miRNA precursor refers to an RNA molecule of any length which can be enzymatically processed into an miRNA, such as a primary RNA transcript, a pri-miRNA, or a pre- miRNA.
  • construct refers to an isolated polypeptide or an isolated
  • polynucleotide encoding a polypeptide.
  • a polynucleotide construct can encode a polypeptide, for example, a lymphoproliferative element.
  • a skilled artisan will understand whether a construct refers to an isolated polynucleotide or an isolated polypeptide depending on the context.
  • the present disclosure overcomes prior art challenges by providing improved methods and compositions for genetically modifying lymphocytes, for example NK cells and in illustrative embodiments, T cells.
  • some of these methods do not include prior-activation of the lymphocyte, and some of these methods are performed in less time than prior methods.
  • compositions that have many uses, including their use in these improved methods are provided.
  • Some of these compositions are genetically modified lymphocytes that have improved proliferative and survival qualities, including in in vitro culturing, for example in the absence of growth factors.
  • Such genetically modified lymphocytes will have utility for example, as research tools to better understand factors that influence T cell proliferation and survival, and for commercial production, for example for the production of certain factors, such as growth factors and immunomodulatory agents, that can be harvested and tested or used in commercial products.
  • Some embodiments provided herein are methods for performing adoptive cellular therapy that include transducing T cells and/or NK cells, that require far less time ex vivo, for example, 24, 12, or 8 hours or less, and in some embodiments without prior ex vivo stimulation. These methods are well-suited for closed system ex vivo processing of blood from a subject, and can be performed with the subject present in the same room as and/or in some embodiments, within their line of sight of their blood or isolated blood cells thereof at all times during performance of the method.
  • the aspects and embodiments of the disclosure herein overcome problems associated with current adoptive cellular therapies by providing methods for transducing resting T cells and/or resting NK cells, that typically utilize a pseudotyping element that facilitates binding and fusion of a replication incompetent recombinant retroviral particle to a resting T cell and/or a resting NK cell, to facilitate genetic modification of the resting T cells and/or NK cells by the replication incompetent recombinant retroviral particles.
  • a chimeric antigen receptor and one or more lymphoproliferative elements whose expression is under the control of a control element, such that exposure of the subject to a compound that binds the control element, or termination of such exposure, promotes expansion of the genetically modified T cells and/or NK cells in vivo.
  • a method for genetically modifying resting T cells and/or resting NK cells of a subject such as a patient having a disease or disorder, wherein blood from the subject is collected; resting T cells and/or NK cells are genetically modified by contacting them with a replication incompetent recombinant retroviral particle; and the genetically modified cells are reintroduced into the subject typically within a shorter period of time than prior methods, for example within 24 hours and in some non-limiting embodiments, within 12 hours and/or without further expanding the population of genetically modified T cells and/or NK cells ex vivo, for example such that the genetically modified resting T cells and/or NK cells do not undergo more than 4 cell divisions ex vivo.
  • methods provided herein can be performed in much less time than current CAR therapies, thereby providing processes by which a subject can remain in a clinic for the entire time of the ex vivo steps. This facilitates performance of the ex vivo steps in a closed system, which reduces the chances for contamination and mixing of patient samples and can be performed more readily by clinical labs.
  • FIGs. 1 and 2 provide schematic diagrams of illustrative compositions used in methods provided herein.
  • FIG. 1 provides a diagram of a packaging cell (100) and a replication incompetent recombinant retroviral particle, produced by such a packaging cell (200).
  • the packaging cell (100) includes recombinant polynucleotides (110) incorporated into its genome that include recombinant transcriptional elements that express retroviral proteins and various different membrane -bound polypeptides under the control of inducible promoters that are regulated by transactivators, which bind and are activated by ligands.
  • transactivators inducible promoters, and ligands are used to induce the sequential expression and accumulation of cell membrane -bound polypeptides that will be incorporated into the membrane of the replication incompetent recombinant retroviral particle as well as retroviral components necessary for packaging and assembly of the replication incompetent recombinant retroviral particles.
  • the illustrative packaging cell (100) illustrated in FIG. 1 is produced, and can be used in illustrative methods to produce replication incompetent recombinant retroviral particles used in methods of transfecting resting T cells and/or NK cells ((300) in FIG. 2) provided herein.
  • the packaging cell (100) in non-limiting illustrative embodiments, includes in its genome nucleic acids encoding a packageable retroviral RNA genome that includes at least some of the elements of a retroviral genome necessary for packaging and assembly of the replication incompetent recombinant retroviral particle (as non-limiting illustrative examples, a retroviral psi element, a retroviral gag polypeptide and a retroviral pol polypeptide).
  • the packaging cell and replication incompetent recombinant retroviral particles formed therefrom can include a retroviral Vpx polypeptide (250), which in non-limiting illustrative examples can be expressed as a membrane associated fusion protein, for example a Src-Flag-Vpx polypeptide; a pseudotyping element that can include a binding polypeptide and a fusogenic polypeptide (240), which in a non-limiting embodiment includes a Measles Virus hemagglutinin (H) polypeptide and a Measles Virus fusion (F) polypeptide, or cytoplasmic domain deletion variants thereof; optionally, one or more activation elements (210, 220), which in a non- limiting embodiment includes a membrane-
  • RNA retroviral genome inside of and typically integrated into the genome of the packaging cell that becomes the genome of the replication incompetent recombinant retroviral particle includes retroviral components (as non-limiting illustrative examples, retroviral Gag and Pol polynucleotides) that are necessary for retroviral production, infection and integration into the genome of a host cell, which is typically a resting T cell and/or NK cell.
  • retroviral components as non-limiting illustrative examples, retroviral Gag and Pol polynucleotides
  • the retroviral genome furthermore includes polynucleotides encoding one or typically two engineered signaling polypeptides provided herein.
  • One of the engineered signaling polypeptides typically encodes at least one lymphoproliferative element (in non-limiting examples a constitutive interleukin 7 receptor mutant) and the other engineered signaling polypeptide typically encodes a chimeric antigen receptor.
  • the replication incompetent recombinant retroviral particle, (200) is then used to transduce a resting T cell and/or resting NK cell (300) in methods provided herein. As shown in FIG. 2, after the resting T cell and/or NK cell (300) is contacted with the replication incompetent recombinant retroviral particle (200), membrane polypeptides discussed above on the surface of the replication incompetent recombinant retroviral particle bind to receptors and/or ligands on the surface of the resting T cell and/or NK cell (300).
  • the pseudotyping element which as indicated above can include a binding polypeptide that binds to molecules on the surface of resting T cells and/or resting NK cells and a fusogenic polypeptide, facilitates the binding and fusion of replication incompetent recombinant retroviral particle (200) to the T cell and/or NK cell membrane.
  • the activation element(s) (210, 220) activate the resting T cell and/or NK cell (300) by engaging the T-cell receptor complex, a process which occurs over the time course of the contacting or an incubation thereafter.
  • the membrane-bound cytokines (230) can be present on the surface of replication incompetent recombinant retroviral particle and bind cytokine receptors (310) on the surface of the resting T cell and/or NK cell (300), thus further promoting binding and activation.
  • cytokine receptors 310) on the surface of the resting T cell and/or NK cell (300)
  • ex vivo stimulation or activation by an element that is not already in or on the replication incompetent recombinant retroviral particle (200) is not required. This in turn, helps to cut down the ex vivo time that is required for completion of the methods in these illustrative methods provided herein.
  • the replication incompetent recombinant retroviral particle Upon binding to the T cell and/or NK cell (200), the replication incompetent recombinant retroviral particle then fuses with the T cell and/or NK cell (300), and polypeptides and nucleic acids in the replication incompetent recombinant retroviral particle enter the T cell and/or NK cell (300).
  • one of these polypeptides in the replication incompetent recombinant retroviral particle is the Vpx polypeptide (250).
  • the Vpx polypeptide (250) binds to and induces the degradation of the SAMHDl restriction factor (350), which degrades free dNTPs in the cytoplasm.
  • the concentration of free dNTPs in the cytoplasm increases as Vpx degrades SAMHDl, and reverse transcription activity is increased, thus facilitating reverse transcription of the retroviral genome and integration into the T cell and/or NK cell genome.
  • the T cell and/or NK cell genome After integration of the retroviral genome into the T cell and/or NK cell (200), the T cell and/or NK cell genome includes nucleic acids encoding the signaling polypeptide encoding the
  • T cells and/or NK cells that are transduced with replication incompetent recombinant retroviral particles herein have one or more signals that drive proliferation and/or inhibit cell death, which in turn in illustrative embodiments, avoids the requirements of prior methods to lymphodeplete a host before returning transduced T cells and/or NK cells back into the subject. This in turn, in illustrative embodiments, further reduces the requirement for days of processing before transduced T cells and/or NK cells are reintroduced into a subject.
  • no more than 36 hours, 24 hours, 12 hours, or in some instances even 8 hours, of time is required from collection of blood from the subject to reintroduction of the blood to the subject, which fundamentally changes the CAR-T process from prior methods.
  • control element provides one of the safety mechanisms provided herein as well. For example, ceasing administration of the compound can down-regulate or even terminate expression of the lymphoproliferative element and optionally the CAR, thus ending a proliferation and/or survival signal to the transduced T cell and/or NK cell and its progeny.
  • PBMC peripheral blood mononuclear cell
  • a lymphocyte typically a T cell and/or an NK cell
  • a resting T cell and/or resting NK cell that includes contacting the lymphocyte with a replication incompetent recombinant retroviral particle, wherein the replication incompetent recombinant retroviral particle typically comprises a pseudotyping element on its surface, wherein said contacting (and incubation under contacting conditions) facilitates transduction of the resting T cell and/or NK cell by the replication incompetent recombinant retroviral particle, thereby producing the genetically modified T cell and/or NK cell.
  • PBMC peripheral blood mononuclear cell
  • a lymphocyte typically a T cell and/or an NK cell
  • a resting T cell and/or resting NK cell that includes contacting the lymphocyte with a replication incompetent recombinant retroviral particle, wherein the replication incompetent recombinant retroviral particle typically comprises
  • the pseudotyping element is typically capable of binding the resting T cell and/or NK cell and can facilitate membrane fusion on its own or in conjunction with other protein(s) of the replication incompetent recombinant retroviral particles.
  • Various elements or steps of such method aspects for transducing and/or genetically modifying a PBMC, lymphocyte, T cell and/or NK cell are provided herein, for example in this section and the Exemplary Embodiments section, and such methods include embodiments that are provided throughout this specification, as further discussed herein,
  • embodiments of any of the aspects for transducing and/or genetically modifying a PBMC or a lymphocyte, for example an NK cell or in illustrative embodiments, a T cell provided for example in this section and in the Exemplary Embodiments section, can include any of the embodiments of replication incompetent recombinant retroviral particles provided herein, including those that include one or more lymphoproliferative element, CAR, pseudotyping element, ribo
  • the retroviral particle is a lentiviral particle.
  • a method for genetically modifying and/or transducing a PBMC or a lymphocyte, such as a T cell and/or NK cell can be performed in vitro or ex vivo.
  • a skilled artisan will recognize that details provided herein for transducing and/or genetically modifying PBMCs or lymphocytes, such as T cell(s) and/or NK cell(s) can apply to any aspect that includes such step(s).
  • the cell is genetically modified and/or transduced without requiring prior activation or stimulation, whether in vivo, in vitro, or ex-vivo.
  • the cell is activated during the contacting and is not activated at all or for more than 15 minutes, 30 minutes, 1, 2, 4, or 8 hours before the contacting.
  • activation by elements that are not present on the retroviral particle surface is not required for genetically modifying and/or transducing the cell. Accordingly, such activation or stimulation elements are not required other than on the retroviral particle, before, during, or after the contacting.
  • these illustrative embodiments that do not require pre-activation or stimulation provide the ability to rapidly perform in vitro experiments aimed at better understanding T cells and the biologicals mechanisms, therein. Furthermore, such methods provide for much more efficient commercial production of biological products produced using PBMCs, lymphocytes, T cells, or NK cells, and development of such commercial production methods. Finally, such methods provide for more rapid ex vivo processing of PBMCs for adoptive cell therapy, fundamentally simplifying the delivery of such therapies, for example by providing point of care methods.
  • the contacting step of a method for transducing and/or a method for genetically mnodifying provided herein typically includes an initial step in which the retroviral particle, typically a population of retroviral particles, are brought into contact with the cell, typically a population of cells while in suspension in a liquid buffer and/or media to form a transduction reaction mixture, followed by an optional incubating period in this reaction mixture that includes the retroviral particles and cells in suspension.
  • the contacting can be performed for example in a chamber of a closed system adapted for processing of PMBCs, as discussed in more detail herein.
  • the transduction reaction mixture can include one or more buffers and ions, and in illustrative embodiments includes a culture media, such as those known in the art for ex vivo processes involving lymphocytes as provided in further detail herein.
  • the transduction reaction mixture can be incubated at between 23 and 39 °C, and in some illustrative embodiments at 37 °C. In certain embodiments, the transduction reaction can be carried out at 37-39 °C for faster fusion/transduction.
  • the cells and retroviral particles when brought into contact in the transduction reaction mixture can be immediately processed to remove the retroviral particles that remain free in suspension and not associated with cells, from the cells.
  • the cells in suspension and retroviral particles whether free in suspension or associated with the cells in suspension can be incubated for various lengths of timep, as provided herein for a contacting step in a method provided herein.
  • a wash can be performed, such as a wash in the media used in the transduction reaction, regardless of whether such cells will be studied in vitro, ex vivo or introduced into a subject.
  • the replication incompetent recombinant retroviral particle can further include an activation element, which can be any activation element provided herein.
  • the activation element can be anti-CD3, such as anti-CD3 scFv, or anti-CD3 scFvFc.
  • the contacting step in a method provided herein of transducing and/or genetically modifying a PBMC or a lymphocyte, typically a T cell and/or an NK cell can be performed (or can occur) for between 1 and 24 hours, for example, between 1 and 12 hours, or between 1 and 6 hours.
  • the contacting can be performed for less than 24 hours, for example, less than 12 hours, less than 8 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes or less than 15 minutes, but in each case there is at least an in initial contacting step in which retroviral particles and cells are brought into contact in suspension in a transduction reaction mixture.
  • Such suspension can include allowing cells and retroviral particles to settle or causing such settling through application of a force, such as a centrifugal force, to the bottom of a vessel or chamber.
  • a force such as a centrifugal force
  • the contacting can be performed (or can occur) for between 30 seconds or 1, 2, 5, 10, 15, 30 or 45 minutes, or 1, 2, 3, 4, 5, 6, 7, or 8 hours on the low end of the range, and between 10 minutes, 15 minutes, 30 minutes, or 1, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, and 72 hours on the high end of the range.
  • the contacting step can be performed for between 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, or 30 minutes on the low end of the range and 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, or 12 hours on the high end of the range.
  • the contacting step is performed for between 30 seconds, 1 minute, and 5 minutes on the low end of the range, and 10 minutes on the high end of the range. In another illustrative embodiment, the contacting is performed for between an initial contacting step only (without any further incubating in the reaction mixture including the retroviral particles free in suspension and cells in suspension) without any further incubation in the reaction mixture, or a 5 minute, 10 minute, 15 minute, 30 minute, or 1 hour incubation in the reaction mixture.
  • Methods of genetic modification and/or transduction of a cell typically include insertion into the cell, of a polynucleotide comprising one or more transcriptional units encoding a CAR or a lymphoproliferative element, or in illustrative embodiments encoding both a CAR and a
  • lymphoproliferative element according to any of the CAR and lymphoproliferative element embodiments provided herein. As illustrated in the Examples herein, the lymphoproliferative element, promotes survival and/or proliferation, which results in survival and/or expansion of the genetically modified and/or transduced cells under appropriate conditions including, but not limited to those provided in the Examples provided herein.
  • PBMC(s), lymphocyte(s), NK cell(s), or T cell(s) that are genetically modified herein with one or more nucleic acids that encode a CAR or both a CAR and a lymphoproliferative element are capable of, adapted for, possesses the property of, and/or are modified for improved survival or expansion in ex vivo or in vitro culture in culture media in the absence of one or more added cytokines such as IL-2, IL-15, or IL-7, or an added lymphocyte mitogenic agent, compared to a control cell(s) identical to the genetically modified and/or transduced cell(s) before it was genetically modified and/or transduced, or in some embodiments compared to a control cell that is modified and/or transduced with a control recombinant retroviral particle identical to the recombinant retroviral particle used to produce the genetically modified and/or transduced cell capable of improved survival or expansion, except that the control retroviral particle does not comprise a
  • lymphoproliferative element or the intracellular domain(s) thereof, or both the CAR and the
  • control cells and conditions for culturing are chosen such that under these conditions, survival and/or proliferation of said control cell(s) would be promoted by adding said one or more cytokines, such as IL-2, IL-15, or IL-7, or said lymphocyte mitogenic agent to the culture media, although such cytokines or mitogenic agent is not actually added for the comparison.
  • cytokines such as IL-2, IL-15, or IL-7
  • methods for genetically modifying and/or transducing herein provide a rapid means to provide new functionality to the transduced cells by inserting into the cell, nucleic acids that when expressed provide the functions of a CAR and/or a lymphoproliferative element as discussed herein.
  • nucleic acids that when expressed provide the functions of a CAR and/or a lymphoproliferative element as discussed herein.
  • such cells are able to survive and/or expand better in vitro and in vivo than control cells that are identical to the genetically modified cells but do not comprise a nucleic acid that encodes the lymphoproliferative element and optionally the CAR.
  • the Examples herein demonstrate that PBMCs that are genetically modified to express a CAR or to express both a CAR and a lymphoprol ferative element survive and/or proliferate more during the first 7 days in culture after contacting, wherein the culturing is in the absence of any added cytokine such as, but not limited to, IL-2, IL-15, or IL-7, or an added lymphocyte mitogenic agent, than identical control cells that were not genetically modified.
  • this Example shows that T cells that are genetically modified to express a CAR and a lymphoproiiferative element have increased survival between day 7 and day 14 or day 21 post contacting under these conditions, than identical control cells that were not genetically modified.
  • T cells that were genetically modified to express both a CAR (e.g. an anti-CD 19 CAR) and a driver (as exemplified for a number of drivers identified herein) had increased survival properties between day 7 and day 21 post-contacting under these conditions, over identical cells that were genetically modified to express the CAR but not a CAR (e.g. an anti-CD 19 CAR) and a driver (as exemplified for a number of drivers identified herein) had increased survival properties between day 7 and day 21 post-contacting under these conditions, over identical cells that were genetically modified to express the CAR but not a
  • a CAR e.g. an anti-CD 19 CAR
  • a driver as exemplified for a number of drivers identified herein
  • lymphoproiiferative element
  • the genetically modified and/or transduced cell exhibits, is capable of, is adapted for, possesses the property of, or is modified for improved expansion in a culture media in the absence of any exogenously added T cell stimulation agents (e.g. IL-2, IL-7, IL-15, anti-CD3, or anti- CD28,) or other exogenously added lymphocyte mitogenic agents compared to a control cell identical to the genetically modified and/or transduced cell(s) before it was genetically modified and/or transduced.
  • T cell stimulation agents e.g. IL-2, IL-7, IL-15, anti-CD3, or anti- CD28,
  • the genetically modified cell exhibits, is capable of, is adapted for, possesses the property of, or is modified to expand better than a control cell between day 3 and day 6 of ex vivo culture after the contacting in the absence of exogenously added cytokines, in the absence of antigen bound by an optional CAR encoded by a genome of the retroviral particle, and/or in the absence of a lymphocyte mitogenic agent.
  • the genetically modified cell is capable of, adapted for, possesses the property of, or modified for expanding at least two-fold between day 3 and day 6 of ex vivo culture after the contacting, in the absence of exogenously added cytokines and in the absence of antigen bound by an optional CAR encoded by a genome of the retroviral particle.
  • Media that can be included in a contacting step for example when the cells and retroviral particles are initially brought into contact or during optional incubation periods with the reaction mixture thereafter that include retroviral particles and cells in suspension in the media, or media that can be used during cell culturing and/or during various wash steps, can include base media such as commercially available media for ex vivo T cell and/or NK cell culture.
  • Non-limiting examples of such media include, X-VIVOTM 15 Chemically Defined, Serum -free Hematopoietic Cell Medium (Lonza) (2018 catalog numbers BE02-060F, BE02-00Q, BE-02-061Q, 04-744Q, or 04-418Q), ImmunoCultTM-XF T Cell Expansion Medium (STEMCELL Technologies) (2018 catalog number 10981), PRIME-XV ® T Cell Expansion XSFM (Irvine Scientific) (2018 catalog number 91141), AIM V ® Medium CTSTM
  • Thermo Fisher Scientific (Referred to herein as "Thermo Fisher"), or CTSTM OptimizerTM media (Thermo Fisher) (2018 catalog numbers A 10221-01 (basal media (bottle)), and A10484-02 (supplement), A10221-03 (basal media (bag)), A1048501 (basal media and supplement kit (bottle)) and, A1048503 (basal media and supplement kit (bag)).
  • Such media can be a chemically defined, serum-free formulation manufactured in compliance with cGMP.
  • the media can be xeno-free and complete.
  • the base media has been cleared by regulatory agencies for use in ex vivo cell processing, such as an FDA 510(k) cleared device.
  • the media is the basal media with or without the supplied T cell expansion supplement of 2018 catalog number A1048501 (CTSTM OpTmizerTM T Cell Expansion SFM, bottle format) or A1048503 (CTSTM OpTmizerTM T Cell Expansion SFM, bag format) both available from Thermo Fisher (Waltham, MA).
  • Additives such as human serum albumin, human AB+ serum, and/or serum derived from the subject can be added to the transduction reaction mixture.
  • Supportive cytokines can be added to the transduction reaction mixture, such as IL2, IL7, or IL 15, or those found in human sera.
  • dGTP can be added to the transduction reaction in certain embodiments.
  • the genetically modified T cell or NK cell is capable of engraftment in vivo in mice and/or enrichment in vivo in mice for at least 7, 14, or 28 days.
  • mice may be treated or otherwise genetically modified so that any immunological differences between the genetically modified T cell and/or NK cell do not result in an immune response being elicited in the mice against any component of the lymphocyte transduced by the replication incompetent recombinant retroviral particle.
  • a packaging cell and in illustrative embodiments a packaging cell line, and in particularly illustrative embodiments a packaging cell provided in certain aspects herein, is used to produce the replication incompetent recombinant retroviral particle.
  • the packaging cell line can be a suspension cell line.
  • the packaging cell line can be grown in serum- free media.
  • the lymphocyte can be from a subject. In illustrative embodiments, the lymphocyte can be from blood of a subject.
  • a method for transducing (and/or genetically modifying) lymphocytes comprising:
  • PBMCs peripheral blood mononuclear cells
  • the cells can be contacted with a retroviral particle without prior activation.
  • the T cells and/or NK cells have not been incubated on a substrate that adheres to monocytes for more than 4 hours in one embodiment, or for more than 6, hours in another embodiment, or for more than 8 hours in another embodiment before the transduction.
  • the T cells and/or NK cells have been incubated overnight on an adherent substrate to remove monocytes before the transduction.
  • the method can include incubating the T cells and/or NK cells on an adherent substrate that binds monocytes for no more than 30 minutes, 1 hour, or 2 hours before the transduction.
  • the T cells and/or NK cells are exposed to no step of removing monocytes by an incubation on an adherent substrate before said transduction step.
  • the T cells and/or NK cells are not incubated with or exposed to a bovine serum, such as a cell culturing bovine serum, for example fetal bovine serum before or during the transduction.
  • a method for genetically modifying or transducing a lymphocyte of a subject in illustrative embodiments, a T cell and/or and NK cell or a population of T cells or NK cells, that includes contacting the T cell(s) and/or NK cell(s) of, typically of a subject ex vivo, with a replication incompetent recombinant retroviral particle comprising in its genome 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 acid sequences 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 a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain,
  • CAR chimeric antigen receptor
  • lymphocyte e.g. a T cell or an NK cell
  • a method for genetically modifying or transducing a lymphocyte comprising contacting the lymphocyte (e.g. the T cell or NK cell) or a population thereof, of the subject ex vivo, with a replication incompetent recombinant retroviral particle comprising in its genome a polynucleotide comprising one or more nucleic acid sequences operatively linked to a promoter active in lymphocytes (e.g. T cells and/or NK cells), wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g.
  • RNA molecules directed against one or more RNA targets and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain, wherein said contacting facilitates genetic modification and/or transduction of the lymphocyte (e.g. T cell or NK cell), or at least some of the lymphocytes (e.g. T cells and/or NK cells) by the replication incompetent recombinant retroviral particle, thereby producing a genetically modified and/or transduced lymphocyte (e.g. T cell and/or NK cell).
  • CAR chimeric antigen receptor
  • the genetically modified and/or transduced lymphocyte e.g. T cell and/or NK cell
  • the genetically modified and/or transduced lymphocyte e.g. T cell and/or NK cell
  • the lymphocyte(s) are resting T cells and/or resting NK cells that are in contact with the replication incompetent recombinant retroviral particles for between 1 hour and 12 hours.
  • any of the method aspects provided immediately above that include 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 acid sequences encodes one or more (e.g.
  • the polynucleotide may further include a third nucleic acid sequence that encodes at least one lymphoproliferative element that is not an inhibitory RNA molecule.
  • the lymphoproliferative element can be a cytokine or cytokine receptor polypeptide, or a fragment thereof comprising a signaling domain.
  • the lymphoproliferative element is constitutive ly active.
  • the lymphoproliferative element can be an IL-7 receptor or a fragment thereof.
  • the lymphoproliferative element can be a
  • constitutively active IL-7 receptor or a constitutively active fragment thereof.
  • an inhibitory RNA molecule can in some embodiments include a 5 ' strand and a 3 ' strand that are partially or fully complementary to one another, wherein said 5' strand and said 3' strand are capable of forming an 18-25 nucleotide RNA duplex.
  • the 5' strand can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length
  • the 3' strand can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
  • the 5' strand and the 3' strand can be the same or different lengths.
  • the RNA duplex can include one or more mismatches. In alternate embodiments, the RNA duplex has no mismatches.
  • an inhibitory RNA molecule can be a miRNA or an shRNA.
  • the inhibitory molecule can be a precursor of a miRNA, such as for example, a Pri-miRNA or a Pre-miRNA, or a precursor of an shRNA.
  • the inhibitory molecule can be an artificially derived miRNA or shRNA.
  • the inhibitory RNA molecule can be a dsRNA (either transcribed or artificially introduced) that is processed into an siRNA or the siRNA itself.
  • the inhibitory RNA molecule can be a miRNA or shRNA that has a sequence that is not found in nature, or has at least one functional segment that is not found in nature, or has a combination of functional segments that are not found in nature.
  • at least one or all of the inhibitory RNA molecules are miR-155.
  • an inhibitory RNA molecule in some embodiments, can comprises from 5 ' to 3 ' orientation: a 5 ' arm, a 5 ' stem, a loop, a 3' stem that is partially or fully complementary to said 5' stem, and a 3' arm. In some embodiments, at least one of two or more inhibitory RNA molecules has this arrangement.
  • the 5' stem can be 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
  • the 3' stem can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
  • the loop can be 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,2 5, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.
  • the 5' arm, 3' arm, or both are derived from a naturally occurring miRNA.
  • the 5' arm, 3' arm, or both are derived from a naturally occurring miRNA is selected from the group consisting of: miR-155, miR-30, miR- 17-92, miR-122, and miR-21.
  • the 5' arm, 3' arm, or both are derived from miR-155.
  • the 5' arm, 3' arm, or both are derived from Mus musculus miR- 155 or Homo sapiens miR-155.
  • the 5' arm has the sequence set forth in SEQ ID NO:256 or is a functional variant thereof, such as, for example, a sequence that is the same length as SEQ ID NO:256, or 95%, 90%, 85%, 80%,75%, or 50% as long as SEQ ID NO: 256 or is 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less; and is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%
  • the 3' arm has the sequence set forth in SEQ ID NO:260 or is a functional variant thereof, such as, for example, the same length as SEQ ID NO:260, or 95%, 90%, 85%, 80%,75%, or 50% as long as SEQ ID NO: 260 or is a sequence that is 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less; and is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%
  • the inhibitory RNA molecules can be adjoined to one another either directly or indirectly by non-functional linker sequence(s).
  • the linker sequences can be between 5 and 120 nucleotides in length, or between 10 and 40 nucleotides in length.
  • the one or more (e.g. two or more) inhibitory RNA molecules can be in an intron.
  • the intron is in a promoter.
  • the intron is EF-lalpha intron A.
  • the intron is adjacent to and downstream of a promoter, which in illustrative embodiments, is inactive in a packaging cell used to produce the replication incompetent recombinant retroviral particle.
  • any of the method aspects provided immediately above that include 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 acid sequences encodes two or more inhibitory RNA molecules directed against one or more RNA targets
  • the two or more inhibitory RNA molecules in some embodiments, can be directed against different targets. In an alternate embodiment, the two or more inhibitory RNA molecules are directed against the same target.
  • the RNA targets are mRNAs transcribed from genes that are expressed by T cells such as but not limited to PD-1 (prevent inactivation); CTLA4 (prevent inactivation); TCRa (safety - prevent autoimmunity); TCRb (safety - prevent autoimmunity); CD3Z (safety - prevent autoimmunity); SOCS1 (prevent inactivation); SMAD2 (prevent inactivation); a miR-155 target (promote activation); IFN gamma (reduce CRS); cCBL (prolong signaling); TRAIL2 (prevent death); PP2A (prolong signaling); ABCG1 (increase cholesterol microdomain content by limiting clearance of cholesterol).
  • T cells such as but not limited to PD-1 (prevent inactivation); CTLA4 (prevent inactivation); TCRa (safety - prevent autoimmunity); TCRb (safety - prevent autoimmunity); CD3Z (safe
  • the RNA targets are mRNAs transcribed from genes that encode components of the T cell receptor (TCR) complex.
  • TCR T cell receptor
  • at least one of the two or more of inhibitory RNA molecules can decrease expression of T cell receptors, in illustrative embodiments, one or more endogenous T cell receptor(s) of a T cell.
  • the RNA target can be mRNA transcribed from the endogenous TCRa or TCR gene of the T cell whose genome comprises the first nucleic acid sequence encoding the one or more miRNAs.
  • the RNA target is mRNA transcribed from the TCRa gene.
  • any of the method aspects provided immediately above that include 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 acid sequences encodes one or more (e.g. 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 a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain, in some embodiments, the CAR is a microenvironment restricted biologic (MRB)-CAR.
  • MRB microenvironment restricted biologic
  • the ASTR of the CAR binds to a tumor associated antigen.
  • the ASTR of the CAR is a microenvironment-restricted biologic (MRB)-ASTR.
  • MRB-ASTR preferentially or only binds its cognate antigen under certain aberrant conditions, such as those that exist in the tumor microenvironment.
  • MRB-ASTRs that bind preferentially or exclusively under aberrant conditions of a tumor microenvironment can provide a reduction in on-target off-tumor effects as binding to the antigen in normal physiological conditions is reduced, in some situations to levels below detection by immunoassays.
  • Normal physiological conditions can include those of temperature, pH, osmotic pressure, osmolality, oxidative stress, and electrolyte concentration that would be considered within a normal range at the site of administration, or at the tissue or organ at the site of action, to a subject.
  • An aberrant condition is that which deviates from the normally acceptable range for that condition.
  • the MRB-ASTR is reversibly or irreversibly inactivated at the normal conditions.
  • An MRB-ASTR as used herein can be an antibody, an antigen, a ligand, a receptor binding domain of a ligand, a receptor, a ligand binding domain of a receptor, or an affibody.
  • the MRB-ASTR is an antibody
  • it can be a full-length antibody, a single-chain antibody, an Fab fragment, an Fab' fragment, an (Fab')2 fragment, an Fv fragment, a divalent single-chain antibody, or a diabody, wherein the ASTR comprises a heavy chain and a light chain from an antibody.
  • the MRB-ASTR is a single-chain variable fragment.
  • any of the method aspects provided immediately above that include 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 acid sequences encodes one or more (e.g.
  • any or all of the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence is operably linked to a riboswitch.
  • the riboswitch is capable of binding a nucleoside analog.
  • the nucleoside analog is an antiviral drug.
  • methods are provided for activating and/or genetically modifying, and typically transducing resting T cells or NK cells, in illustrative embodiments resting T cells, by contacting the cells with a retroviral particle disclosed herein and soluble anti-CD3 antibodies at 25-200, 50-150, 75- 125, or 100 ng/ml.
  • such methods are performed without prior activation, and can be carried out, for example, for 8 hours or less, 4 hours or less, or between 2 and 8 hours, 2 and 4 hours, or between 2 and 3 hours.
  • provided herein are methods for performing adoptive cell therapy on a subject. As an illustrative example, the method can include the following:
  • PBMCs peripheral blood mononuclear cells
  • methods with similar steps are referred to as methods for genetically modifying and expanding lymphocytes of a subject.
  • methods for genetically modifying and expanding lymphocytes of a subject are referred to as methods for genetically modifying and expanding lymphocytes of a subject.
  • a skilled artisan will understand that the discussion herein as it applies to methods and compositions for performing adoptive cell therapy apply to methods for genetically modifying and expanding lymphocytes of a subject as well.
  • the adoptive cell therapy methods of the present disclosure are carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject.
  • the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.
  • a subject having a disease or disorder enters a medical facility where the subject's blood is drawn using known methods, such as venipuncture.
  • the volume of blood drawn from a subject is between 10, 15, 20, 25, 30, 35, 40, 50, 75, or 100 ml on the low end of the range and 200, 250, 300, 350, 400, 500, 750, 1000, 2000, or 2500 ml on the high end of the range. In some embodiments, between 10 and 400 ml are drawn from the subject. In some embodiments, between 20 and 250 ml of blood are drawn from the subject. In some embodiments, the blood is fresh when it is processed. In any of the embodiments disclosed herein, fresh blood can be blood that was withdrawn from a subject less than 15, 30, 45, 60, 90, 120, 150, or 180 minutes prior. In some embodiments, the blood is processed in the methods provided herein without storage.
  • a transduced T cell and/or NK cell includes progeny of ex vivo transduced cells that retain at least some of the nucleic acids or polynucleotides that are incorporated into the cell during the ex vivo transduction.
  • methods herein that recite "reintroducing" a transduced cell it will be understood that such cell is typically not in a transduced state when it is collected from the blood of a subject.
  • a subject in any of the aspects disclosed herein can be for example, an animal, a mammal, and in illustrative embodiments a human.
  • the delivery of a polynucleotide encoding a lymphoproliferative element, such as an IL7 constitutively active mutant, to a resting T cell and/or NK cell ex vivo, which can integrate into the genome of the T cell or NK cell, provides that cell with a driver for in vivo expansion without the need for lymphodepleting the host.
  • a lymphoproliferative element such as an IL7 constitutively active mutant
  • the subject is not exposed to a lymphodepleting agent within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days, or within 1 month, 2 months, 3 months or 6 months of performing the contacting, during the contacting, and/or within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days, or within 1 month, 2 months, 3 months or 6 months after the modified T cells and/or NK cells are reintroduced back into the subject.
  • methods provided herein can be performed without exposing the subject to a lymphodepleting agent during a step wherein a replication incompetent recombinant retroviral particle is in contact with resting T cells and/or resting NK cells of the subject and/or during the entire ex vivo method.
  • methods of expanding genetically modified T cells and/or NK cells in a subject in vivo is a feature of some embodiments of the present disclosure.
  • such methods are ex vivo propagation-free or substantially propagation-free.
  • This entire method/process from blood draw from a subject to reintroduction of blood back into the subject after ex vivo transduction of T cells and/or NK cells can occur over a time period less than 48 hours, less than 36 hours, less than 24 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, 2 hours, or less than 2 hours.
  • the entire method/process from blood draw/collection from a subject to reintroduction of blood back into the subject after ex vivo transduction of T cells and/or NK cells occurs over a time period between 1 hour and 12 hours, or between 2 hours and 8 hours, or between 1 hour and 3 hours, or between 2 hours and 4 hours, or between 2 hours and 6 hours, or between 4 hours and 12 hours, or between 4 hours and 24 hours, or between 8 hours and 24 hours, or between 8 hours and 36 hours, or between 8 hours and 48 hours, or between 12 hours and 24 hours, or between 12 hours and 36 hours, or between 12 hours and 48 hours, or over a time period between 15, 30, 60, 90, 120, 180, and 240 minutes on the low end of the range, and 120, 180, and 240, 300, 360, 420, and 480 minutes on the high end of the range.
  • the entire method/process from blood draw/collection from a subject to reintroduction of blood back into the subject after ex vivo transduction of T cells and/or NK cells occurs over a time period between 1, 2, 3, 4, 6, 8, 10, and 12 hours on the low end of the range, and 8, 9, 10, 11, 12, 18, 24, 36, or 48 hours on the high end of the range.
  • the genetically modified T cells and/or NK cells are separated from the replication incompetent recombinant retroviral particles after the time period in which contact occurs.
  • the method from blood collection through transduction of T cells and/or NK cells does not include a step of removing monocytes by an incubation on an adherent substrate of more than 4 hours in one embodiment, or for more than 6, hours in another embodiment, or for more than 8 hours in another embodiment.
  • the method from blood collection through transduction of T cells and/or NK cells does not include an overnight incubation on an adherent substrate to remove monocytes.
  • the method from blood collection through transduction of T cells and/or NK cells includes a step of removing monocytes by an incubation on an adherent substrate for no more than 30 minutes, 1 hour, or 2 hours.
  • the method from blood collection from a subject through transduction of lymphocytes in illustrative embodiments T cells and/or NK cells, including resting T cells and/or NK cells, include no step of removing monocytes by an incubation on an adherent substrate.
  • the method from blood collection from a subject through transduction of lymphocytes includes, the T cells and/or NK cells are not incubated with or exposed to a bovine serum such as a cell culturing bovine serum, for example fetal bovine serum during the method.
  • a bovine serum such as a cell culturing bovine serum, for example fetal bovine serum during the method.
  • the method from blood collection from a subject through reintroduction of T cells and/or NK cells into the subject does not include a step of removing monocytes by an incubation on an adherent substrate of more than 4 hours in one embodiment, or for more than 6, hours in another embodiment, or for more than 8 hours in another embodiment.
  • the method from blood collection from a subject through reintroduction of T cells and/or NK cells into the subject does not include an overnight incubation on an adherent substrate to remove monocytes.
  • the method from blood collection from a subject through reintroduction of T cells and/or NK cells into the subject includes a step of removing monocytes by an incubation on an adherent substrate for no more than 30 minutes, 1 hour, or 2 hours.
  • the method from blood collection from a subject through reintroduction of T cells and/or NK cells into the subject includes no step of removing monocytes by an incubation on an adherent substrate.
  • the method from blood collection from a subject through reintroduction of T cells and/or NK cells into the subject the T cells and/or NK cells are not incubated with or exposed to a bovine serum, such as a cell culturing bovine serum, for example fetal bovine serum during the method.
  • a bovine serum such as a cell culturing bovine serum, for example fetal bovine serum during the method.
  • a subject remains within line of site and/or within 100, 50, 25, or 12 feet or arm's distance of their blood or cells that are being processed, for the entire method/process from blood draw/collection from the subject to reintroduction of blood to the subject after ex vivo transduction of T cells and/or NK cells.
  • a subject remains awake and/or at least one person can continue to monitor the blood or cells of the subject that are being processed, throughout and/or continuously for the entire method/process from blood draw/collection from the subject to reintroduction of blood to the subject after ex vivo transduction of T cells and/or NK cells.
  • the entire method/process for adoptive cell therapy and/or for transducing resting T cells and/or NK cells from blood draw/collection from the subject to reintroduction of blood to the subject after ex vivo transduction of T cells and/or NK cells can be performed with continuous monitoring by a human.
  • the entire method/process from blood draw/collection from the subject to reintroduction of blood to the subject after ex vivo transduction of T cells and/or NK cells are blood cells incubated in a room that does not have a person present.
  • the entire method/process from blood draw/collection from the subject to reintroduction of blood to the subject after ex vivo transduction of T cells and/or NK cells is performed next to the subject and/or in the same room as the subject and/or next to the bed or chair of the subject.
  • sample identity mix-ups can be avoided, as well as long and expensive incubations over periods of days or weeks.
  • methods provided herein are readily adaptable to closed and automated blood processing systems, where a blood sample and its components that will be reintroduced into the subject, only make contact with disposable, single -use components.
  • Methods for performing adoptive cell therapy typically include 1) methods of transducing lymphocytes, such as T cell(s) or NK cell(s), which in illustrative embodiments are resting T cell(s) and/or NK cell(s), and/or include 2) methods for genetically modifying a lymphocyte such as T cell(s) and/or an NK cell(s), which in illustrative embodiments are resting T cell(s) and/or NK cell(s), both (1 and 2) of which themselves each form distinct aspects of the present disclosure.
  • Such methods can be performed with or without other steps identified herein for performing adoptive cell therapy.
  • neutrophils/granulocytes are separated away from the blood cells before the cells are contacted with replication incompetent recombinant retroviral particles.
  • peripheral blood mononuclear cells PBMCs
  • PBLs peripheral blood lymphocytes
  • T cell and/or NK cells are isolated away from other components of a blood sample using for example, apheresis, and/or density gradient centrifugation.
  • neutrophils are removed before PBMCs and/or T cells and/or NK cells are processed, contacted with a replication incompetent recombinant retroviral particle, transduced, or transfected.
  • the cells may be allogeneic and/or autologous.
  • PBMCs are isolated using a Sepax or Sepax 2 cell processing system (BioSafe).
  • the PBMCs are isolated using a CliniMACS Prodigy cell processor (Miltenyi Biotec).
  • an automated apheresis separator is used which takes blood from the subject, passes the blood through an apparatus that sorts out a particular cell type (such as, for example, PBMCs), and returns the remainder back into the subject. Density gradient centrifugation can be performed after apheresis.
  • the PBMCs are isolated using a leukoreduction filter device.
  • magnetic bead activated cell sorting is then used for purifying a specific cell population from PBMCs, such as, for example, PBLs or a subset thereof, according to a cellular phenotype (i.e. positive selection).
  • Other methods for purification can also be used, such as, for example, substrate adhesion, which utilizes a substrate that mimics the environment that a T cell encounters during recruitment, allowing them to adhere and migrate, or negative selection, in which unwanted cells are targeted for removal with antibody complexes that target the unwanted cells.
  • red blood cell resetting can be used to purify cells.
  • the PBLs include T cells and/or NK cells.
  • the T cells and/or NK cells that are contacted by replication incompetent recombinant retroviral particles of the present disclosure during certain embodiments herein, for example in methods of modifying lymphocytes and methods of performing adoptive cellular therapy, are mainly resting T cells.
  • the T cells and/or NK cells consist of between 95 and 100% resting cells (Ki-67 ).
  • the T cell and/or NK cells that are contacted by replication incompetent recombinant retroviral particles include between 90, 91, 92, 93, 94, and 95% resting cells on the low end of the range and 96, 97, 98, 99, or 100% resting cells on the high end of the range.
  • the T cells and/or NK cells include naive cells.
  • T cells and/or NK cells are contacted ex vivo with replication incompetent recombinant retroviral particles to genetically modify T cells and/or NK cells to illicit a targeted immune response in the subject when reintroduced into the subject.
  • the replication incompetent recombinant retroviral particles identify and bind to T cells and/or NK cells at which point the retroviral and host cell membranes start to fuse.
  • genetic material from the replication incompetent recombinant retroviral particles enters the T cells and/or NK cells and is incorporated into the host cell DNA.
  • Many of the methods provided herein include transduction of T cells and/or NK cells. Methods are known in the art for transducing T cells and/or NK cells ex vivo with replication incompetent recombinant retroviral particles, such as replication incompetent recombinant lentiviral particles. Methods provided herein, in illustrative embodiments, do not require ex vivo stimulation or activation. Thus, this common step in prior methods can be avoided in the present method, although ex vivo stimulatory molecule(s) such as anti-CD3 and/or anti-CD28 beads, can be present during the transduction. However, with illustrative methods provided herein, ex vivo stimulation is not required. In certain exemplary methods, between 3 and 10 multiplicity of infection (MOI), and in some embodiments, between 5 and 10 MOI units of replication incompetent recombinant retroviral particles, for example lentivirus, can be used.
  • MOI multiplicity of infection
  • the transduction reaction can be carried out in a closed system, such as a Sepax system, as discussed herein, wherein the transduction reaction can be carried out in disposable bags loaded on the system.
  • Blood cells such as PBMCs, from the collected blood sample from the subject, can be contacted with replication incompetent recombinant retroviral particles disclosed herein, in a bag as soon as these blood cells are separated, isolated, and/or purified away from granulocytes, including neutrophils, which are typically not present during the contacting step (i.e. the transduction reaction).
  • the replication incompetent recombinant retroviral particles can be introduced into the bag that contains the isolated PBMCs, thereby contacting the PBMCs.
  • the time from blood collection from the subject to the time when blood cells, such as PBMCs are added to the transduction reaction bag can be between 30 minutes and 4 hours, between 30 minutes and 2 hours, or around 1 hour, in some examples.
  • the cells are washed to remove the transduction reaction mixture before being infused back into the subject.
  • the system such as a Sepax instrument
  • the system can be used to wash cells, for example with 10-50 ml of wash solution, before the transduced cells are infused back into the subject.
  • neutrophils are removed before PBMCs and/or T cells and/or NK cells are processed, contacted with replication incompetent recombinant retroviral particles, transduced, or transfected.
  • blood is collected from a subject into a blood bag and the blood bag is attached to a cell processing system such as a Sepax cell processing system.
  • PBMCs isolated using the cell processing system are collected into a bag, contacted with the replication incompetent recombinant retroviral particles in conditions sufficient to transduce T cells and/or NK cells, and incubated.
  • the bag containing the mixture of PBMCs and replication incompetent recombinant retroviral particles is attached to a cell processing system and the PBMCs are washed. The washed PBMCs are collected into a bag and reinfused into the subject.
  • the entire method from collecting blood to reinfusing transduced T and/or NK cells, is performed within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, or 24 hours. In illustrative embodiments, the entire method is performed within 12 hours.
  • the target cells for the replication incompetent recombinant retroviral particles are PBLs.
  • the target cells are T cells and/or NK cells.
  • the T cells are helper T cells and/or killer T cells.
  • the replication incompetent recombinant retroviral particles provided herein have pseudotyping elements on their surface that are capable of binding to T cells and/or NK cells and facilitating membrane fusion of the replication incompetent recombinant retroviral particles thereto.
  • the replication incompetent recombinant retroviral particles have activation elements on their surface that are capable of binding to resting T cells and/or NK cells.
  • the replication incompetent recombinant retroviral particles have membrane -bound cytokines on their surface.
  • the replication incompetent recombinant retroviral particles include a polynucleotide having one or more transcriptional units encoding one or more engineered signaling polypeptides, one or more of which includes one or more lymphoproliferative elements.
  • one when two signaling polypeptides are utilized, one includes at least one lymphoproliferative element and the other is typically a chimeric antigen receptor (CAR) that includes an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain.
  • CAR chimeric antigen receptor
  • an activation element(s) that is typically associated with the surface of a replication incompetent recombinant retroviral particle provided herein is capable of, and as a resulting of contacting resting T cells and/or NK cells for a sufficient period of time and under appropriate conditions, activates resting T cells and/or NK cells. It will be understood that such activation occurs over time during a contacting step of methods herein. Furthermore, it will be understood that in some embodiments where a pseudotyping element is found on the surface of a replication incompetent recombinant retroviral particle, that binds a T cell and/or an NK cell, in methods herein, activation can be induced by binding of the pseudotyping element. An activation element is optional in those
  • pseudotyping element an activation element, a membrane -bound cytokine, an engineered signaling polypeptide, a lymphoproliferative element, and a CAR are provided in other sections herein.
  • the percent of lymphocytes that are transduced is between 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60% on the low end of the range, and 50, 55, 60, 65, 70, 75, 80, 85, and 90% on the high end of the range. In some embodiments, the percent of lymphocytes that are transduced is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%.
  • the genetically modified T cells and/or NK cells are introduced back, reintroduced, or reinfused into the subject without additional ex vivo manipulation, such as stimulation and/or activation of T cells and/or NKs.
  • ex vivo manipulation is used for stimulation/activation of T cells and/or NK cells and for expansion of genetically modified T cells and/or NK cells prior to introducing the genetically modified T cells and/or NK cells into the subject.
  • this generally takes days or weeks and requires a subject to return to a clinic for a blood infusion days or weeks after an initial blood draw.
  • T cells and/or NK cells are not stimulated ex vivo by exposure to anti-CD3/anti-CD28 solid supports such as, for example, beads coated with anti-CD3/anti-CD28, prior to contacting the T cells and/or NK cells with the replication incompetent recombinant retroviral particles.
  • anti-CD3/anti-CD28 solid supports such as, for example, beads coated with anti-CD3/anti-CD28
  • genetically modified T cells and/or NK cells are not expanded ex vivo, or only expanded for a small number of cell divisions (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 rounds of cell division), but are rather expanded, or predominantly expanded, in vivo, i.e. within the subject.
  • no additional media is added to allow for further expansion of the cells.
  • no cell manufacturing of the PBLs occurs while the PBLs are contacted with the replication incompetent recombinant retroviral particles. In illustrative embodiments, no cell manufacturing of the PBLs occurs while the PBLs are ex vivo.
  • subjects were lymphodepleted prior to reinfusion with genetically modified T cells and or NK cells.
  • patients or subjects are not lymphodepleted prior to blood being withdrawn.
  • patients or subjects are not lymphodepleted prior to reinfusion with genetically modified T cells and or NK cells.
  • T cells and/or NK cells can be stimulated ex vivo by exposure to anti-CD3/anti-CD28 solid supports prior to contacting the T cells and/or NK cells with the replication incompetent recombinant retroviral particles.
  • the T cells and/or NK cells can be exposed to anti-CD3/anti-CD28 solid supports for less than 1, 2, 3, 4,
  • the T cells and/or NK cells can be exposed to anti-CD3/anti-CD28 solid supports for less than 1, 2, 3, 4, 6, or 8 hours before the T cells and/or NK cells are contracted the replication incompetent recombinant retroviral particles.
  • the number of T cells and/or NK cells to be reinfused into a subject can be between 1 x 10 3 , 2.5 x 10 3 , 5 x 10 3 , 1 x 10 4 , 2.5 x 10 4 , 5 x 10 4 , 1 x 10 5 , 2.5 x 10 5 , 5 x 10 5 , 1 x 10 6 , 2.5 x 10 6 , 5 x 10 6 , and 1 x 10 7 cells/kg on the low end of the range and 5 x 10 4 , 1 x 10 5 , 2.5 x
  • the number of T cells and/or NK cells to be reinfused into a subject can be between 1 x 10 4 , 2.5 x 10 4 , 5 x 10 4 , and 1 x 10 5 cells/kg on the low end of the range and 2.5 x 10 4 , 5 x 10 4 , 1 x 10 5 , 2.5 x 10 5 , 5 x 10 5 , and 1 x 10 6 cells/kg on the high end of the range.
  • the number of PBLs to be reinfused into a subject can be fewer than 5 x 10 5 , 1 x 10 6 , 2.5 x
  • the number of T cells and/or NK cells available for reinfusion into a 70 kg subject or patient is between 7 x 10 5 and 2.5 x 10 8 cells. In other embodiments, the number of T cells and/or NK cells available for transduction is approximately 7 x 10 6 plus or minus 10%.
  • the entire adoptive cell therapy procedure from withdrawing blood to the reinfusion of genetically modified T cells and/or NK cells, can advantageously be performed in a shorter time than previous methods.
  • the entire adoptive cell therapy procedure can be performed in less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, or 24 hours.
  • the entire adoptive cell therapy procedure can be performed in less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours.
  • the entire adoptive cell therapy procedure can be performed in between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15 hours on the low end of the range and 1, 2, 3, 4, 5, 6,
  • a closed system is used to process PBMCs, for example in methods that include genetically modifying PBMCs, NK cells and in illustrative embodiments T cells, for example by transducing the PBMCs or subset(s) thereof.
  • Such methods can be used to genetically modify lymphocytes to be used in scientific research, commercial production, or therapeutic methods.
  • such methods can include transferring peripheral blood mononuclear cells (PBMCs) including NK cells, T cells, or both, and in some embodiments resting T cells, and or resting NK cells, from a vessel into a transduction reaction mixture within a closed system, which thus is without environmental exposure.
  • PBMCs peripheral blood mononuclear cells
  • the vessel for example, can be a tube, bag, syringe, or other container.
  • the vessel is a vessel that is used in a research facility.
  • the vessel is a vessel used in commercial production.
  • the vessel can be a collection vessel used in a blood collection process.
  • Methods for genetically modifying herein typically involve a contacting step wherein lymphocytes are contacted with a replication incompetent recombinant retroviral particle.
  • the contacting in some embodiments, can be performed in the vessel, for example, within a blood bag.
  • PBMCs or a subtraction thereof can be transferred from the vessel to another vessel (for example from a first vessel to a second vessel) within the closed system for the contacting.
  • the second vessel can be a cell processing compartment of a closed device, such as a G-Rex device.
  • the genetically modified (e.g. transduced) cells can be transferred to a different vessel within the closed system (i.e. without exposure to the environment).
  • the process disclosed in this paragraph from transfer of the PBMCs or a fraction thereof, into the vessel in which the contacting (e.g. transduction) will occur through washing the cells after the contacting is performed within 12 hours. In some embodiments it is performed for between 1, 2, 3, or 4 hours on the low end of the range, and 4, 8, 10, or 12 hours on the high end of the range.
  • the steps of withdrawing a blood sample from a subject, contacting T cells and or NK cells with replication incompetent recombinant retroviral particles, and or introducing genetically modified T cells and or NK cells into the subject occur in a closed system.
  • a closed system is a culture process that is generally closed or fully closed to contamination. As such, such a system or process does not expose cells to an environment.
  • An advantage of the present invention is that provided herein are methods for performing CAR therapy in a closed system.
  • One of the greatest risks to safety and regulatory control in the cell processing procedure is the risk of contamination through frequent exposure to the environment as is found in traditional open cell culture systems.
  • a closed-system process a process that is designed and can be operated such that the product is not exposed to the outside environment. This is important because the outside environment is typically not sterile. Material transfer occurs via sterile connections or tube welding. Air for gas exchange occurs via a gas permeable membrane or like other additions, via 0.2 ⁇ filter to prevent environmental exposure.
  • the closed system includes an ex vivo circulating system connected to the in vivo circulatory system of the subject such that blood is drawn and then circulated to the ex vivo circulatory system before being introduced back into the subject.
  • the ex vivo circulatory system includes a system or apparatus for isolating PBLs and/or a system or apparatus for isolating T cells and/or NK cells, in combination with the system or apparatus for exposing the cells to the replication incompetent recombinant retroviral particles.
  • the closed system does not allow the T cells and/or NK cells to be exposed to air.
  • Such closed system methods can be performed with commercially available devices.
  • the method can be carried out in devices adapted for closed system T cell production.
  • Such devices include a G-RexTM, a WAVE BioreactorTM, an OriGen PermaLifeTM bags, and a VueLife® bags.
  • control element can be a riboswitch and the compound can bind the aptamer domain of the riboswitch.
  • control element can be a molecular chaperone.
  • Molecular chaperones are compounds that directly affect the activity of, typically by binding, a lymphoproliferative element or other component of a first or second engineered signaling polypeptide herein.
  • the compound can be a nucleoside analogue.
  • the nucleoside analogue can be a nucleoside analogue antiviral drug, wherein an antiviral drug is a compound approved by the Food and Drug Administration for antiviral treatment or a compound in an antiviral clinical trial in the United States.
  • the compound can be acyclovir or penciclovir.
  • the compound can be famciclovir, the oral prodrug of penciclovir, or valaciclovir, the oral prodrug of acyclovir. Binding of the compound to the control element affects expression of the introduced genetic material and hence, propagation of genetically modified T cells and/or NK cells.
  • the nucleoside analogue antiviral drug or prodrug for example acyclovir, valaciclovir, penciclovir or famciclovir, is administered to the subject prior to, concurrent with, and/or following PBLs being isolated from the blood of the subject and before T cells and/or NK cells are contacted with replication incompetent recombinant retroviral particles.
  • the nucleoside analogue antiviral drug or prodrug is administered to the subject for between 5, 10, 15, 30, and 60 minutes on the low end of the range and 1.5, 2, 3, 4, 5, 6, 8, 12, or 24 hours on the high end of the range prior to PBLs being isolated from the blood or prior to T cells and/or NK cells being contacted with replication incompetent recombinant retroviral particles.
  • the nucleoside analogue antiviral drug or prodrug is administered to the subject for between 1.5, 2, 3, 4, 5, 6, 8, 12, or 24 hours on the low end of the range and 1 ⁇ 2, 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days on the high end of the range after PBLs are isolated from the blood and T cells and/or NK cells are contacted with replication incompetent recombinant retroviral particles in methods provided herein.
  • the nucleoside analogue antiviral drug or prodrug is administered to the subject for at least 1.5, 2, 3, 4, 5, 6, 8, 12, or 24 hours, or at least 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days after PBLs are isolated from the blood and T cells and/or NK cells are contacted with replication incompetent recombinant retroviral particles in methods provided herein.
  • the nucleoside analogue antiviral drug or prodrug is administered to the subject for at least 1, 2, 3, 4, 5, 7, 10, 14, 21, 28, 30, 60, 90, or 120 days or 5, 6, 9, 12, 24, 36, 48, 60, 72, 84, 96, 120 months or indefinitely after the PBLs have been reinfused into the subject.
  • the nucleoside analogue antiviral drug or prodrug can be administered before and/or during the reinfusion of the PBLs and/or after the PBLs have been reinfused.
  • the compound that binds to the control element is administered once, twice, three times, or four times daily to the subject.
  • daily doses of the compound are provided for 1 week, 2 weeks, 4 weeks, 3 months, 6 months, 1 year, until a subject is disease free, such as cancer free, or indefinitely.
  • the drug in illustrative embodiments is a nucleoside analogue antiviral drug that binds to a nucleoside analog, such as a riboswitch, as disclosed in
  • Methods are known in the art for delivering drugs, whether small molecules or biologies, and can be used in methods provided herein. Any such methods can be used to deliver drugs or candidate compounds or antibodies for use in methods of the present invention.
  • common routes of administration include non-invasive peroral (through the mouth), topical (skin), transmucosal (nasal, buccal/sublingual, vaginal, ocular and rectal) and inhalation routes.
  • Many protein and peptide drugs, such as monoclonal antibodies have to be delivered by injection or a nanoneedle array. For example, many immunizations are based on the delivery of protein drugs and are often done by injection.
  • the replication incompetent recombinant retroviral particles used to contact T cells and/or NK cells have a polynucleotide having one or more transcriptional units that encode one or more engineered signaling polypeptides.
  • an engineered signaling polypeptide includes any combination of an extracellular domain (e.g. an antigen-specific targeting region or ASTR), a stalk and a transmembrane domain, combined with one or more intracellular activating domains, optionally one or more modulatory domains (such as a co-stimulatory domain), and optionally one or more T cell survival motifs.
  • At least one, two, or all of the engineered signaling polypeptides is a chimeric antigen receptor (CAR) or a lymphoproliferative element (LE) such as a chimeric lymphoproliferative element (CLE).
  • CAR chimeric antigen receptor
  • LE lymphoproliferative element
  • CLE chimeric lymphoproliferative element
  • polypeptides are utilized, one encodes a lymphoproliferative element and the other encodes a chimeric antigen receptor (CAR) that includes an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain.
  • CAR chimeric antigen receptor
  • ASTR antigen-specific targeting region
  • an engineered signaling polypeptide includes an extracellular domain that is a member of a specific binding pair.
  • the extracellular domain can be the extracellular domain of a cytokine receptor, or a mutant thereof, or a hormone receptor, or a mutant thereof.
  • Such mutant extracellular domains in some embodiments have been reported to be constitutively active when expressed at least in some cell types.
  • such extracellular and transmembrane domains do not include a ligand binding region. It is believed that such domains do not bind a ligand when present in an engineered signaling polypeptide and expressed in B cells, T cells, and/or NK cells.
  • Mutations in such receptor mutants can occur in the extracellular juxtamembrane region.
  • a mutation in at least some extracellular domains (and some extracellular- transmembrane domains) of engineered signaling polypeptides provided herein are responsible for signaling of the engineered signaling polypeptide in the absence of ligand, by bringing activating chains together that are not normally together.
  • extracellular domains that comprise mutations in extracellular domains can be found, for example, in the Lymphoproliferative Element section herein.
  • the extracellular domain comprises a dimerizing motif.
  • the dimerizing motif comprises a leucine zipper.
  • the leucine zipper is from a jun polypeptide, for example c-jun.
  • extracellular domains that comprise a dimerizing motif can be found, for example, in the Lymphoproliferative Element section herein.
  • the extracellular domain is an antigen-specific targeting region (ASTR), sometimes called an antigen binding domain herein. Specific binding pairs include, but are not limited to, antigen-antibody binding pairs; ligand-receptor binding pairs; and the like.
  • a member of a specific binding pair suitable for use in an engineered signaling polypeptide of the present disclosure includes an ASTR that is an antibody, an antigen, a ligand, a receptor binding domain of a ligand, a receptor, a ligand binding domain of a receptor, and an affibody.
  • An ASTR suitable for use in an engineered signaling polypeptide of the present disclosure can be any antigen-binding polypeptide.
  • the ASTR is an antibody such as a full-length antibody, a single-chain antibody, an Fab fragment, an Fab' fragment, an (Fab')2 fragment, an Fv fragment, and a divalent single-chain antibody or a diabody.
  • the ASTR is a single chain Fv (scFv).
  • the heavy chain is positioned N-terminal of the light chain in the engineered signaling polypeptide.
  • the light chain is positioned N-terminal of the heavy chain in the engineered signaling polypeptide.
  • the heavy and light chains can be separated by a linker as discussed in more detail herein.
  • the heavy or light chain can be at the N-terminus of the engineered signaling polypeptide and is typically C-terminal of another domain, such as a signal sequence or peptide.
  • T cell receptor (TCR) based recognition domains such as single chain TCR (scTv, single chain two-domain TCR containing V ⁇ V ⁇ ) are also suitable for use.
  • the ASTR can be multispecific, e.g. bispecific antibodies.
  • Multispecific antibodies have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for one target antigen and the other is for another target antigen.
  • bispecific antibodies may bind to two different epitopes of ta target antigen. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a target antigen. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.
  • An ASTR suitable for use in an engineered signaling polypeptide of the present disclosure can have a variety of antigen-binding specificities.
  • the antigen-binding domain is specific for an epitope present in an antigen that is expressed by (synthesized by) a target cell.
  • the target cell is a cancer cell associated antigen.
  • the cancer cell associated antigen can be an antigen associated with, e.g., a breast cancer cell, a B cell lymphoma, a Hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell (e.g., a small cell lung cancer cell), a non-Hodgkin B-cell lymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancer cell, a
  • mesothelioma cell a lung cancer cell (e.g., a small cell lung cancer cell), a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a neuroblastoma cell, a glioma, a glioblastoma, a medulloblastoma, a colorectal cancer cell, etc.
  • a cancer cell associated antigen may also be expressed by a non-cancerous cell.
  • Non-limiting examples of antigens to which an ASTR of an engineered signaling polypeptide can bind include, e.g., CD19, CD20, CD38, CD30, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight-melanoma associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, Axl, Ror2, and the like.
  • PSMA prostate-specific membrane antigen
  • CEA carcinoembryonic antigen
  • EGFR epidermal growth factor receptor
  • EGFRvIII vascular endothelial growth factor receptor-2
  • HMW-MAA high molecular weight-melanoma associated antigen
  • MAGE-A1 IL-13
  • a member of a specific binding pair suitable for use in an engineered signaling polypeptide is an ASTR that is a ligand for a receptor.
  • Ligands include, but are not limited to, hormones (e.g. erythropoietin, growth hormone, leptin, etc.); cytokines (e.g., interferons, interleukins, certain hormones, etc.); growth factors (e.g., heregulin; vascular endothelial growth factor (VEGF); and the like); an integrin-binding peptide (e.g., a peptide comprising the sequence Arg-Gly-Asp); and the like.
  • hormones e.g. erythropoietin, growth hormone, leptin, etc.
  • cytokines e.g., interferons, interleukins, certain hormones, etc.
  • growth factors e.g., heregulin; vascular endothelial growth factor (
  • the engineered signaling polypeptide can be activated in the presence of a second member of the specific binding pair, where the second member of the specific binding pair is a receptor for the ligand.
  • the second member of the specific binding pair can be a VEGF receptor, including a soluble VEGF receptor.
  • the member of a specific binding pair that is included in an engineered signaling polypeptide is an ASTR that is a receptor, e.g., a receptor for a ligand, a co-receptor, etc.
  • the receptor can be a ligand-binding fragment of a receptor.
  • Suitable receptors include, but are not limited to, a growth factor receptor (e.g., a VEGF receptor); a killer cell lectin-like receptor subfamily K, member 1 (NKG2D) polypeptide (receptor for MICA, MICB, and ULB6); a cytokine receptor (e.g., an IL-13 receptor; an IL-2 receptor; etc.); CD27; a natural cytotoxicity receptor (NCR) (e.g., NKP30 (NCR3/CD337) polypeptide (receptor for HLA-B-associated transcript 3 (BAT3) and B7-H6); etc.); etc.
  • a growth factor receptor e.g., a VEGF receptor
  • a killer cell lectin-like receptor subfamily K, member 1 (NKG2D) polypeptide receptor for MICA, MICB, and ULB6
  • a cytokine receptor e.g., an IL-13 receptor; an IL-2 receptor; etc.
  • the ASTR can be directed to an intermediate protein that links the ASTR with a target molecule expressed on a target cell.
  • the intermediate protein may be endogenously expressed or introduced exogenously and may be natural, engineered, or chemically modified.
  • the ASTR can be an anti-tag ASTR such that at least one tagged intermediate, typically an antibody-tag conjugate, is included between a tag recognized by the ASTR and a target molecule, typically a protein target, expressed on a target cell. Accordingly, in such embodiments, the ASTR binds a tag and the tag is conjugated to an antibody directed against an antigen on a target cell, such as a cancer cell.
  • Non-limiting examples of tags include fluorescein isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horse radish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase, and maltose binding protein.
  • FITC fluorescein isothiocyanate
  • streptavidin biotin
  • biotin histidine
  • dinitrophenol dinitrophenol
  • peridinin chlorophyll protein complex green fluorescent protein
  • PE phycoerythrin
  • horse radish peroxidase palmitoylation
  • nitrosylation alkaline phosphatase
  • glucose oxidase glucose oxidase
  • maltose binding protein a binds the tag.
  • the engineered signaling polypeptide includes a stalk which is located in the portion of the engineered signaling polypeptide lying outside the cell and interposed between the ASTR and the transmembrane domain.
  • the stalk has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to a wild-type CD 8 stalk region (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG AVHTRGLDFA (SEQ ID NO:79), has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to a wild-type CD28 stalk region (FCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:80)), or has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to a wild-type immunoglobulin heavy chain stalk region.
  • the stalk employed allows the antigen-specific
  • the stalk region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa.
  • the stalk of an engineered signaling polypeptide includes at least one cysteine.
  • the stalk can include the sequence Cys-Pro-Pro-Cys (SEQ ID NO:62). If present, a cysteine in the stalk of a first engineered signaling polypeptide can be available to form a disulfide bond with a stalk in a second engineered signaling polypeptide.
  • Stalks can include immunoglobulin hinge region amino acid sequences that are known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87: 162; and Huck et al. (1986) Nucl. Acids Res. 14: 1779.
  • an immunoglobulin hinge region can include a domain with at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids of any of the following amino acid sequences: DKTHT (SEQ ID NO:63); CPPC (SEQ ID NO:62); CPEPKSCDTPPPCPR (SEQ ID NO:64) (see, e.g., Glaser et al. (2005) J. Biol. Chem.
  • ELKTPLGDTTHT SEQ ID NO:65
  • KSCDKTHTCP SEQ ID NO:66
  • KCCVDCP SEQ ID NO:67
  • KYGPPCP SEQ ID NO:68
  • EPKSCDKTHTCPPCP SEQ ID NO:69
  • ELKTPLGDTTHTCPRCP SEQ ID NO: 71
  • SPNMVPHAHHAQ SEQ ID NO:72
  • the stalk can include a hinge region with an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge region.
  • the stalk can include one or more amino acid substitutions and/or insertions and/or deletions compared to a wild-type (naturally-occurring) hinge region.
  • His229 of human IgG 1 hinge can be substituted with Tyr, so that the stalk includes the sequence EPKSCDKTYTCPPCP (see, e.g., Yan et al. (2012) J. Biol. Chem. 287:5891).
  • the stalk can include an amino acid sequence derived from human CD8; e.g., the stalk can include the amino acid sequence:
  • TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:73), or a variant thereof.
  • An engineered signaling polypeptide of the present disclosure can include transmembrane domains for insertion into a eukaryotic cell membrane.
  • the transmembrane domain can be interposed between the ASTR and the co-stimulatory domain.
  • the transmembrane domain can be interposed between the stalk and the co-stimulatory domain, such that the chimeric antigen receptor includes, in order from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus): an ASTR; a stalk; a transmembrane domain; and an activating domain.
  • TM domain transmembrane domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell is suitable for use in aspects and embodiments disclosed herein.
  • Non-limiting examples of TM domains suitable for any of the aspects or embodiments provided herein, include a domain with at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids of any of the following TM domains or combined stalk and TM domains: a) CD8 alpha TM (SEQ ID NO:46); b) CD 8 beta TM (SEQ ID NO:47); c) CD4 stalk (SEQ ID NO:48); d) CD3Z TM (SEQ ID NO:49); e) CD28 TM (SEQ ID NO:50); f) CD134 (OX40) TM: (SEQ ID NO:51); g) CD7 TM (SEQ ID NO:52); h) CD 8 stalk and TM (SEQ ID NO:75); and i) CD28 stalk and TM (SEQ ID NO:76).
  • a transmembrane domain of an aspect of the invention can have at least 80%, 90%, or 95% or can have 100% sequence identity to the SEQ ID NO: 46 transmembrane domain, or can have 100% sequence identity to any of the transmembrane domains from the following genes respectively: the CD8 beta transmembrane domain, the CD4 transmembrane domain, the CD3 zeta transmembrane domain, the CD28 transmembrane domain, the CD 134 transmembrane domain, or the CD7 transmembrane domain.
  • Intracellular activating domains suitable for use in an engineered signaling polypeptide of the present disclosure when activated typically induce the production of one or more cytokines; increase cell death; and/or increase proliferation of CD8 + T cells, CD4 + T cells, NKT cells, ⁇ T cells, and/or neutrophils.
  • Activating domains can also be referred to as activation domains herein.
  • Activating domains can be used in CARs or in lymphoproliferative elements provided herein.
  • the intracellular activating domain includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motifs as described below.
  • an intracellular activating domain of an aspect of the invention can have at least 80%, 90%, or 95% or can have 100% sequence identity to the CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP 12, FCER1G, FCGR2A, FCGR2C. DAP10/CD28, or ZAP70 domains as described below.
  • Intracellular activating domains suitable for use in an engineered signaling polypeptide of the present disclosure include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides.
  • ITAM immunoreceptor tyrosine-based activation motif
  • An ITAM motif is YX 1 X 2 L/I, where Xi and X 2 are independently any amino acid.
  • the intracellular activating domain of an engineered signaling polypeptide includes 1, 2, 3, 4, or 5 ITAM motifs.
  • an ITAM motif is repeated twice in an intracellular activating domain, where the first and second instances of the ITAM motif are separated from one another by 6 to 8 amino acids, e.g., (YXiX 2 L/I)(X3) n (YXiX 2 L/I), where n is an integer from 6 to 8, and each of the 6-8 X3 can be any amino acid.
  • the intracellular activating domain of an engineered signaling polypeptide includes 3 ITAM motifs.
  • a suitable intracellular activating domain can be an ITAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif.
  • a suitable intracellular activating domain can be an ITAM motif-containing domain from any ITAM motif-containing protein.
  • a suitable intracellular activating domain need not contain the entire sequence of the entire protein from which it is derived.
  • ITAM motif-containing polypeptides include, but are not limited to: CD3Z (CD3 zeta); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD79A (antigen receptor complex-associated protein alpha chain); CD79B (antigen receptor complex-associated protein beta chain)DAP12; and FCER1G (Fc epsilon receptor I gamma chain).
  • the intracellular activating domain is derived from T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.).
  • a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa, of either of the following amino acid sequences (2 isoforms):
  • a suitable intracellular activating domain polypeptide can include ansEp]lTAM motif- containing a portion of the full length CD3 zeta amino acid sequence.
  • a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa, of either of the following amino acid sequences:
  • the intracellular activating domain is derived from T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3 -DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain; etc.).
  • T cell surface glycoprotein CD3 delta chain also known as CD3D; CD3 -DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain; etc.
  • a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa, of eithe _piof the following amino acid sequences:
  • a suitable intracellular activating domain polypeptide can comprise an ITAM motif- containing portion of the full length CD3 delta amino acid sequence.
  • a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequence: DQV[YQPLRDRDDAQYSHL]GGN (SEQ ID NO: 19), where the ITAM motifs are set out in brackets.
  • the intracellular activating domain is derived from T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3/Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.).
  • T cell surface glycoprotein CD3 epsilon chain also known as CD3e, T cell surface antigen T3/Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.
  • a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa, of the following amino acid sequence:
  • a suitable intracellular activating domain polypeptide can comprise an ITAM motif- containing portion of the full length CD3 epsilon amino acid sequence.
  • a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequence: NPD[YEPIRKGQRDLYSGL]NQR (SEQ ID NO:21), where the ITAM motifs are set out in brackets.
  • the intracellular activating domain is derived from T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3 -GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.).
  • a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa, of the following amino acid sequence:
  • a suitable intracellular activating domain polypeptide can comprise an ITAM motif- containing portion of the full length CD3 gamma amino acid sequence.
  • a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequence: DQL[YQPLKDREDDQYSHL]_QGN (SEQ ID NO:23), where the ITAM motifs are set out in brackets.
  • the intracellular activating domain is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; Ig-alpha; membrane -bound immunoglobulin-associated protein; surface IgM-associated protein; etc.).
  • CD79A also known as B-cell antigen receptor complex-associated protein alpha chain
  • CD79a antigen immunoglobulin-associated alpha
  • MB-1 membrane glycoprotein Ig-alpha
  • membrane -bound immunoglobulin-associated protein surface IgM-associated protein; etc.
  • a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa, of either of the following amino acid sequences:
  • a suitable intracellular activating domain polypeptide can comprise an ITAM motif- containing portion of the full length CD79A amino acid sequence.
  • a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequence: ENL[YEGLNLDDCSMYEDI]SRG (SEQ ID NO:26), where the ITAM motifs are set out in brackets.
  • the intracellular activating domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase -binding protein; killer activating receptor associated protein; killer-activating receptor-associated protein; etc.).
  • DAP12 also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase -binding protein; killer activating receptor associated protein; killer-activating receptor-associated protein; etc.
  • a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa, of either of the following amino acid sequences (4 isoforms):
  • a suitable intracellular activating domain polypeptide can comprise an ITAM motif- containing portion of the full length DAP 12 amino acid sequence.
  • a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequence: ESP[YQELQGQRSDVYSDL1NTQ (SEQ ID NO:31), where the ITAM motifs are set out in brackets.
  • the intracellular activating domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon Rl-gamma; fcRgamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.).
  • FCER1G also known as FCRG
  • Fc epsilon receptor I gamma chain Fc receptor gamma-chain
  • fcRgamma fceRI gamma
  • high affinity immunoglobulin epsilon receptor subunit gamma immunoglobulin E receptor, high affinity, gamma chain; etc.
  • a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 50 amino acids to about 60 amino acids (aa), from about 60 aa to about 70 aa, from about 70 aa to about 80 aa, or from about 80 aa to about 88 aa _piof the following amino acid sequence:
  • a suitable intracellular activating domain polypeptide can comprise an ITAM motif- containing portion of the full length FCER1G amino acid sequence.
  • a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequence: DGV[YTGLSTRNQETYETL1KHE (SEQ ID NO:33), where the ITAM motifs are set out in brackets.
  • Intracellular activating domains suitable for use in an engineered signaling polypeptide of the present disclosure include a DAP10/CD28 type signaling chain.
  • An example of a DAP 10 signaling chain is the amino acid SEQ ID NO:34.
  • a suitable intracellular activating domain includes a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in SEQ ID NO:34.
  • a CD28 signaling chain is the amino acid sequence is SEQ ID NO:35.
  • a suitable intracellular domain includes a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids of SEQ ID NO:35.
  • Intracellular activating domains suitable for use in an engineered signaling polypeptide of the present disclosure include a ZAP70 polypeptide
  • a suitable intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following sequences or to a contiguous stretch of from about 300 amino acids to about 400 amino acids, from about 400 amino acids to about 500 amino acids, or from about 500 amino acids to 619 amino acids, of SEQ ID NO:36.
  • Modulatory domains can change the effect of the intracellular activating domain in the engineered signaling polypeptide, including enhancing or dampening the downstream effects of the activating domain or changing the nature of the response.
  • Modulatory domains suitable for use in an engineered signaling polypeptide of the present disclosure include co-stimulatory domains.
  • a modulatory domain suitable for inclusion in the engineered signaling polypeptide can have a length of from about 30 amino acids to about 70 amino acids (aa), e.g., a modulatory domain can have a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
  • modulatory domain can have a length of from about 70 aa to about 100 aa, from about 100 aa to about 200 aa, or greater than 200 aa.
  • Co-stimulatory domains typically enhance and/or change the nature of the response to an activation domain.
  • Co-stimulatory domains suitable for use in an engineered signaling polypeptide of the present disclosure are generally polypeptides derived from receptors. In some embodiments, co- stimulatory domains homodimerize.
  • a subject co-stimulatory domain can be an intracellular portion of a transmembrane protein (i.e., the co-stimulatory domain can be derived from a transmembrane protein).
  • co-stimulatory polypeptides include, but are not limited to, 4-1BB (CD137), CD27, CD28, CD28 deleted for Lck binding (ICA), ICOS, OX40, BTLA, CD27, CD30, GITR, and HVEM.
  • a co-stimulatory domain of an aspect of the invention can have at least 80%, 90%, or 95% sequence identity to the co-stimulatory domain of 4-1BB (CD137), CD27, CD28, CD28 deleted for Lck binding (ICA), ICOS, OX40, BTLA, CD27, CD30, GITR, or HVEM.
  • a co- stimulatory domain of an aspect of the invention can have at least 80%, 90%, or 95% sequence identity to the co-stimulatory domain of non-limiting examples of suitable co-stimulatory polypeptides include, but are not limited to, 4-1BB (CD137), CD27, CD28, CD28 deleted for Lck binding (ICA), ICOS, OX40, BTLA, CD27, CD30, GITR, and HVEM.
  • 4-1BB CD137
  • CD27, CD28, CD28 deleted for Lck binding (ICA), ICOS, OX40, BTLA, CD27, CD30, GITR, and HVEM include, but are not limited to, 4-1BB (CD137), CD27, CD28, CD28 deleted for Lck binding (ICA), ICOS, OX40, BTLA, CD27, CD30, GITR, and HVEM.
  • a co-stimulatory domain of an aspect of the invention can have at least 80%, 90%, or 95% sequence identity to the co-stimulatory domain of 4-1BB (CD137), CD27, CD28, CD28 deleted for Lck binding (ICA), ICOS, OX40, BTLA, CD27, CD30, GITR, or HVEM.
  • a co-stimulatory domain suitable for inclusion in an engineered signaling polypeptide can have a length of from about 30 amino acids to about 70 amino acids (aa), e.g., a co-stimulatory domain can have a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
  • the co-stimulatory domain can have a length of from about 70 aa to about 100 aa, from about 100 aa to about 200 aa, or greater than 200 aa.
  • the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein CD137 (also known as TNFRSF9; CD137; 4-1BB; CDwl37; ILA; etc.).
  • a suitable co-stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 1.
  • the co-stimulatory domain has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
  • the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein CD28 (also known as Tp44).
  • a suitable co-stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:2.
  • the co-stimulatory domain has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
  • the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein CD28 deleted for Lck binding (ICA).
  • ICA transmembrane protein CD28 deleted for Lck binding
  • a suitable co-stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:3.
  • the co-stimulatory domain has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
  • the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1).
  • a suitable co- stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:4.
  • the co-stimulatory domain has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
  • the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein OX40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX-40, TXGPIL).
  • OX40 contains a p85 PI3K binding motif at residues 34-57 and a TRAF binding motif at residues 76-102, each of SEQ ID NO: 504.
  • the costimulatory domain can include the p85 PI3K binding motif of OX40.
  • the costimulatory domain can include the TRAF binding motif of OX40.
  • Lysines corresponding to amino acids 17 and 41 of SEQ ID NO: 504 are potentially negative regulatory sites that function as parts of ubiquitin targeting motifs. In some embodiments, one or both of these Lysines in the costimulatory domain of OX40 are mutated Arginines or another amino acid.
  • a suitable co-stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:5.
  • the co-stimulatory domain has a length of from about 20 aa to about 25 aa, about 25 aa to about 30 aa, 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to about 50 aa.
  • the co-stimulatory domain has a length of from about 20 aa to about 50 aa, for example 20 aa to 45 aa, or 20 aa to 42 aa.
  • the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein CD27 (also known as S 152, T 14, TNFRSF7, and Tp55).
  • a suitable co-stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:6.
  • the co-stimulatory domain has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to about 50 aa.
  • the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein BTLA (also known as BTLAl and CD272).
  • BTLA also known as BTLAl and CD272.
  • a suitable costimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:7.
  • the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1).
  • a suitable costimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, from about 150 aa to about 160 aa, or from about 160 aa to about 185 aa of SEQ ID NO:8.
  • the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D).
  • GITR also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D.
  • a suitable co-stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:9.
  • the co-stimulatory domain has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
  • a suitable co-stimulatory domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 10.
  • the co-stimulatory domain of both the first and the second polypeptide has a length of from about 30 aato about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
  • the engineered signaling polypeptide includes a linker between any two adjacent domains.
  • a linker can be between the transmembrane domain and the first co-stimulatory domain.
  • the ASTR can be an antibody and a linker can be between the heavy chain and the light chain.
  • a linker can be between the ASTR and the transmembrane domain and a co-stimulatory domain.
  • a linker can be between the co-stimulatory domain and the intracellular activating domain of the second polypeptide.
  • the linker can be between the ASTR and the intracellular signaling domain.
  • the linker peptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded.
  • a linker can be a peptide of between about 1 and about 100 amino acids in length, or between about 1 and about 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins. Peptide linkers with a degree of flexibility can be used.
  • the linking peptides may have virtually any amino acid sequence, bearing in mind that suitable linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art.
  • Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.
  • Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n , GSGGS n , GGGS n , and GGGGS n where n is an integer of at least one), glycine-alanine polymers, alanine -serine polymers, and other flexible linkers known in the art.
  • Glycine and glycine -serine polymers are of interest since both of these amino acids are relatively unstructured, and therefore may serve as a neutral tether between components.
  • Glycine polymers are of particular interest since glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).
  • Exemplary flexible linkers include, but are not limited GGGGSGGGGSGGGGS (SEQ ID NO: 53),
  • a polynucleotide provided by the replication incompetent recombinant retroviral particles has one or more transcriptional units that encode certain combinations of the one or more engineered signaling polypeptides.
  • genetically modified T cells include the combinations of the one or more engineered signaling polypeptides after transduction of T cells by the replication incompetent recombinant retroviral particles.
  • the first engineered signaling polypeptide includes an extracellular antigen binding domain, which is capable of binding an antigen, and an intracellular signaling domain. In other embodiments, the first engineered signaling polypeptide also includes a T cell survival motif and/or a transmembrane domain. In some embodiments, the first engineered signaling polypeptide does not include a co-stimulatory domain, while in other embodiments, the first engineered signaling polypeptide does include a co-stimulatory domain.
  • a second engineered signaling polypeptide includes a lymphoproliferative gene product and optionally an extracellular antigen binding domain.
  • the second engineered signaling polypeptide also includes one or more of the following: a T cell survival motif, an intracellular signaling domain, and one or more co-stimulatory domains.
  • at least one is a CAR.
  • the one or more engineered signaling polypeptides are expressed under a T cell specific promoter or a general promoter under the same transcript wherein in the transcript, nucleic acids encoding the engineered signaling polypeptides are separated by nucleic acids that encode one or more internal ribosomal entry sites (IREs) or one or more protease cleavage peptides.
  • IREs internal ribosomal entry sites
  • the polynucleotide encodes two engineered signaling polypeptides wherein the first engineered signaling polypeptide includes a first extracellular antigen binding domain, which is capable of binding to a first antigen, and a first intracellular signaling domain but not a co- stimulatory domain, and the second polypeptide includes a second extracellular antigen binding domain, which is capable of binding VEGF, and a second intracellular signaling domain, such as for example, the signaling domain of a co-stimulatory molecule.
  • the first antigen is PSCA, PSMA, or BCMA.
  • the first extracellular antigen binding domain comprises an antibody or fragment thereof (e.g., scFv), e.g., an antibody or fragment thereof specific to PSCA, PSMA, or BCMA.
  • the second extracellular antigen binding domain that binds VEGF is a receptor for VEGF, i.e., VEGFR.
  • the VEGFR is VEGFR1, VEGFR2, or VEGFR3. In a certain embodiment, the VEGFR is VEGFR2.
  • the polynucleotide encodes two engineered signaling polypeptides wherein the first engineered signaling polypeptide includes an extracellular tumor antigen binding domain and a CD3 ⁇ signaling domain, and the second engineered signaling polypeptide includes an antigen- binding domain, wherein the antigen is an angiogenic or vasculogenic factor, and one or more co- stimulatory molecule signaling domains.
  • the angiogenic factor can be, e.g., VEGF.
  • the one or more co- stimulatory molecule signaling motifs can comprise, e.g., co-stimulatory signaling domains from each of CD27, CD28, OX40, ICOS, and 4-1BB.
  • the polynucleotide encodes two engineered signaling polypeptides wherein the first engineered signaling polypeptide includes an extracellular tumor antigen-binding domain and a CD3 ⁇ signaling domain, the second polypeptide comprises an antigen-binding domain, which is capable of binding to VEGF, and co-stimulatory signaling domains from each of CD27, CD28, OX40, ICOS, and 4-1BB.
  • the first signaling polypeptide or second signaling polypeptide also has a T cell survival motif.
  • the T cell survival motif is, or is derived from, an intracellular signaling domain of IL-7 receptor (IL-7R), an intracellular signaling domain of IL-12 receptor, an intracellular signaling domain of IL-15 receptor, an intracellular signaling domain of IL-21 receptor, or an intracellular signaling domain of transforming growth factor ⁇ (TGF ⁇ ) receptor or the TGF ⁇ decoy receptor (TGF- ⁇ — dominant-negative receptor II (DNRII)).
  • IL-7R an intracellular signaling domain of IL-7 receptor
  • TGF ⁇ transforming growth factor ⁇
  • TGF- ⁇ TGF- ⁇ — dominant-negative receptor II
  • the polynucleotide encodes two engineered signaling polypeptides wherein the first engineered signaling polypeptide includes an extracellular tumor antigen-binding domain and a CD3 ⁇ signaling domain, and the second engineered signaling polypeptide includes an antigen-binding domain, which is capable of binding to VEGF, an IL-7 receptor intracellular T cell survival motif, and co-stimulatory signaling domains from each of CD27, CD28, OX40, ICOS, and 4- 1BB.
  • more than two signaling polypeptides are encoded by the polynucleotide.
  • only one of the engineered signaling polypeptides includes an antigen binding domain that binds to a tumor-associated antigen or a tumor-specific antigen; each of the remainder of the engineered signaling polypeptides comprises an antigen binding domain that binds to an antigen that is not a tumor-associated antigen or a tumor-specific antigen.
  • two or more of the engineered signaling polypeptides include antigen binding domains that bind to one or more tumor- associated antigens or tumor-specific antigens, wherein at least one of the engineered signaling polypeptides comprises an antigen binding domain that does not bind to a tumor-associated antigen or a tumor-specific antigen.
  • the tumor-associated antigen or tumor-specific antigen is Her2, prostate stem cell antigen (PSCA), PSMA (prostate-specific membrane antigen), B cell maturation antigen (BCMA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen- 125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD 117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo- Dl, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (N
  • the first engineered signaling polypeptide includes a first extracellular antigen binding domain that binds a first antigen, and a first intracellular signaling domain; and a second engineered signaling polypeptide includes a second extracellular antigen binding domain that binds a second antigen, or a receptor that binds the second antigen; and a second intracellular signaling domain, wherein the second engineered signaling polypeptide does not comprise a co-stimulatory domain.
  • the first antigen-binding domain and the second antigen-binding domain are independently an antigen-binding portion of a receptor o r an antigen-binding portion of an antibody.
  • first antigen binding domain or the second antigen binding domain are scFv antibody fragments.
  • first engineered signaling polypeptide and/or the second engineered signaling polypeptide additionally comprises a transmembrane domain.
  • first engineered signaling polypeptide or the second engineered signaling polypeptide comprises a T cell survival motif, e.g., any of the T cell survival motifs described herein.
  • the first engineered signaling polypeptide includes a first extracellular antigen binding domain that binds HER2 and the second engineered signaling polypeptide includes a second extracellular antigen binding domain that binds MUC- 1.
  • the second extracellular antigen binding domain of the second engineered signaling polypeptide binds an interleukin.
  • the second extracellular antigen binding domain of the second engineered signaling polypeptide binds a damage associated molecular pattern molecule (DAMP; also known as an alarmin).
  • DAMP is a heat shock protein, chromatin-associated protein high mobility group box 1 (HMGB l), S100A8 (also known as MRP8, or calgranulin A), S 100A9 (also known as MRP14, or calgranulin B), serum amyloid A (SAA), deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.
  • HMGB l chromatin-associated protein high mobility group box 1
  • S100A8 also known as MRP8, or calgranulin A
  • S 100A9 also known as MRP14, or calgranulin B
  • SAA serum amyloid A
  • said second antigen is an antigen on an antibody that binds to an antigen presented by a tumor cell.
  • signal transduction activation through the second engineered signaling polypeptide is non-antigenic, but is associated with hypoxia.
  • hypoxia is induced by activation of hypoxia-inducible factor-la (HIF-la), HIF- ⁇ , HIF-2a, HIF-2 , HIF-3a, or HIF-3 .
  • HIF-la hypoxia-inducible factor-la
  • expression of the one or more engineered signaling polypeptides is regulated by a control element, which is disclosed in more detail herein.
  • the engineered signaling polypeptides can further include one or more additional polypeptide domains, where such domains include, but are not limited to, a signal sequence; an epitope tag; an affinity domain; and a polypeptide whose presence or activity can be detected (detectable marker), for example by an antibody assay or because it is a polypeptide that produces a detectable signal.
  • Non- limiting examples of additional domains for any of the aspects or embodiments provided herein include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the following sequences as described below: a signal sequence, an epitope tag, an affinity domain, or a polypeptide that produces a detectable signal.
  • Signal sequences that are suitable for use in a subject CAR include any eukaryotic signal sequence, including a naturally-occurring signal sequence, a synthetic (e.g., man-made) signal sequence, etc.
  • the signal sequence can be the CD8 signal sequence MALPVTALLLPLALLLHAARP (SEQ ID NO:74).
  • Suitable epitope tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA; SEQ ID NO:37); FLAG (e.g.,DYKDDDDK; SEQ ID NO:38); c-myc (e.g., EQKLISEEDL; SEQ ID NO: 39), and the like.
  • Affinity domains include peptide sequences that can interact with a binding partner, e.g., such as one immobilized on a solid support, useful for identification or purification.
  • DNA sequences encoding multiple consecutive single amino acids, such as histidine, when fused to the expressed protein, may be used for one -step purification of the recombinant protein by high affinity binding to a resin column, such as nickel sepharose.
  • affinity domains include His5 (HHHHH; SEQ ID NO:40), HisX6 (HHHHHH; SEQ ID NO:41), c-myc (EQKLISEEDL; SEQ ID NO: 39), Flag (DYKDDDDK; SEQ ID NO: 38), Strep Tag (WSHPQFEK; SEQ ID NO: 42), hemagglutinin, e.g., HA Tag (YPYDVPDYA; SEQ ID NO:37), GST, thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO:43), Phe-His-His-Thr (SEQ ID NO:44), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-end RNA tag,
  • WEAAAREACCRECCARA SEQ ID NO:45
  • metal binding domains e.g., zinc binding domains or calcium binding domains such as those from calcium-binding proteins, e.g., calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, frequenin, caltractin, calpain large-subunit, SlOOproteins, parvalbumin, calbindin D9K, calbindin D28K, and calretinin, inteins, biotin, streptavidin, MyoD, Id, leucine zipper sequences, and maltose binding protein.
  • Suitable detectable signal-producing proteins include, e.g., fluorescent proteins; enzymes that catalyze a reaction that generates a detectable signal as a product; and the like.
  • Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP
  • Phycobiliproteins and Phycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrin and Allophycocyanin.
  • Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel,!sEpJmRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like.
  • Any of a variety of fluorescent and colored proteins from Anthozoan species as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973, is suitable for use.
  • Suitable enzymes include, but are not limited to, horse radish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N- acetylglucosaminidase, ⁇ -glucuronidase, invertase, Xanthine Oxidase, firefly luciferase, glucose oxidase (GO), and the like.
  • HRP horse radish peroxidase
  • AP alkaline phosphatase
  • GAL beta-galactosidase
  • glucose-6-phosphate dehydrogenase beta-N- acetylglucosaminidase
  • ⁇ -glucuronidase invertase
  • Xanthine Oxidase firefly luciferase
  • glucose oxidase GO
  • any of the replication incompetent recombinant retroviral particles provided herein can include nucleic acids that encode a recognition or elimination domain as part of, or separate from, nucleic acids encoding any of the engineered signaling polypeptides provided herein.
  • any of the engineered signaling polypeptides provided herein can include a recognition or elimination domain.
  • any of the CARs disclosed herein can include a recognition or elimination domain.
  • a recognition or elimination domain can be expressed together with, or even fused with any of the lymphoproliferative elements disclosed herein. The recognition or elimination domains are expressed on the T cell and or NK cell but are not expressed on the replication incompetent recombinant retroviral particles.
  • the recognition or elimination domain can be derived from herpes simplex virus-derived enzyme thymidine kinase (HSV-tk) or inducible caspase-9.
  • the recognition or elimination domain can include a modified endogenous cell-surface molecule, for example as disclosed in U.S. Patent 8,802,374.
  • the modified endogenous cell-surface molecule can be any cell- surface related receptor, ligand, glycoprotein, cell adhesion molecule, antigen, integrin, or cluster of differentiation (CD) that is modified.
  • the modified endogenous cell-surface molecule is a truncated tyrosine kinase receptor.
  • the truncated tyrosine kinase receptor is a member of the epidermal growth factor receptor (EGFR) family (e.g., ErbBl, ErbB2, ErbB3, and ErbB4).
  • the recognition domain can be a polypeptide that is recognized by an antibody that recognizes the extracellular domain of an EGFR member.
  • the recognition domain can be at least 20 contiguous amino acids of an EGFR family member, or for example, between 20 and 50 contiguous amino acids of an EGFR family member.
  • SEQ ID NO:78 is an exemplary polypeptide that is recognized by, and under the appropriate conditions bound by an antibody that recognizes the extracellular domain of an EGFR member.
  • Such extracellular EGFR epitopes are sometimes referred to herein as eTags. In illustrative embodiments, such epitopes are recognized by commercially available anti-EGFR monoclonal antibodies.
  • Epidermal growth factor receptor also known as EGFR, ErbB 1 and FIERI, is a cell-surface receptor for members of the epidermal growth factor family of extracellular ligands. Alterations in EGFR activity have been implicated in certain cancers.
  • a gene encoding an EGFR polypeptide including human epidermal growth factor receptor (EGFR) is constructed by removal of nucleic acid sequences that encode polypeptides including the membrane distal EGF-binding domain and the cytoplasmic signaling tail, but retains the extracellular membrane proximal epitope recognized by an anti-EGFR antibody.
  • the antibody is a known, commercially available anti-EGFR monoclonal antibody, such as cetuximab, matuzumab, necitumumab or panitumumab.
  • EGFR was demonstrated to have suicide gene potential through Erbitux® mediated antibody dependent cellular cytotoxicity (ADCC) pathways.
  • ADCC antibody dependent cellular cytotoxicity
  • the inventors of the present disclosure have successfully expressed eTag in PBMCs using lentiviral vectors, and have found that expression of eTag in vitro by PBMCs exposed to Cetuximab, provided an effective elimination mechanism for PBMCs.
  • EGFR may be used as a non-immunogenic selection tool, tracking marker, and suicide gene for transduced T cells that have immunotherapeutic potential.
  • the EGFR nucleic acid may also be detected by means well known in the art.
  • EGFR is expressed as part of a single polypeptide that also includes the CAR or as part of a single polypeptide that includes the lymphoproliferative element.
  • the amino acid sequence encoding the EGFR recognition domain can be separated from the amino acid sequence encoding the chimeric antigen receptor by a cleavage signal and/or a ribosomal skip sequence.
  • the ribosomal skip and/or cleavage signal can be any ribosomal skip and/or cleavage signal known in the art.
  • the ribosomal skip sequence can be, for example T2A (also referred to as 2A-1 herein) with amino acid sequence
  • GSGEGRGSLLTCGDVEENPGP SEQ ID NO:77.
  • other examples of cleavage signals and ribosomal skip sequences include FMDV 2A (F2A); equine rhinitis A virus 2A (abbreviated as E2A); porcine te scho virus- 1 2 A (P2A); and Thoseaasigna virus 2 A (T2A).
  • the polynucleotide sequence encoding the recognition domain can be on the same transcript as the CAR or lymphoproliferative element but separated from the polynucleotide sequence encoding the CAR or lymphoproliferative element by an internal ribosome entry site.
  • a recognition domain can be expressed as part of a fusion polypeptide, fused to a lymphoproliferative element.
  • Such constructs provide the advantage, especially in combination with other "space saving" elements provided herein, of taking up less genomic space on an RNA genome compared to separate polypeptides.
  • an eTag is expressed as a fusion polypeptide, fused to an IL7Rot mutant, as experimentally demonstrated herein.
  • an engineered signaling polypeptide is a chimeric antigen receptor (CAR) or a polynucleotide encoding a CAR, which, for simplicity, is referred to herein as "CAR.”
  • a CAR of the present disclosure includes: a) at least one antigen-specific targeting region (ASTR); b) a transmembrane domain; and c) an intracellular activating domain.
  • the antigen-specific targeting region of the CAR is an scFv portion of an antibody to the target antigen.
  • the intracellular activating domain is from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP 12, FCER1G, FCGR2A, FCGR2C. DAP10/CD28, or ZAP70, and some further illustrative embodiments, from CD3z.
  • the CAR further comprises a co-stimulatory domain, for example any of the co-stimulatory domains provided above in the Modulatory Domains section, and in further illustrative embodiments the co-stimulatory domain is the intracellular co-stimulatory domain of 4-1BB (CD 137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.
  • the CAR includes any of the transmembrane domains listed in the Transmembrane Domain section above.
  • a CAR of the present disclosure can be present in the plasma membrane of a eukaryotic cell, e.g., a mammalian cell, where suitable mammalian cells include, but are not limited to, a cytotoxic cell, a T lymphocyte, a stem cell, a progeny of a stem cell, a progenitor cell, a progeny of a progenitor cell, and an NK cell, an NK-T cell, and a macrophage.
  • a CAR of the present disclosure is active in the presence of one or more target antigens that, in certain conditions, binds the ASTR.
  • the target antigen is the second member of the specific binding pair.
  • the target antigen of the specific binding pair can be a soluble (e.g., not bound to a cell) factor; a factor present on the surface of a cell such as a target cell; a factor presented on a solid surface; a factor present in a lipid bilayer; and the like.
  • the antigen can be a soluble (e.g., not bound to a cell) antigen; an antigen present on the surface of a cell such as a target cell; an antigen presented on a solid surface; an antigen present in a lipid bilayer; and the like.
  • a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by one or more target antigens, increases expression of at least one nucleic acid in the cell.
  • a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by the one or more target antigens, increases expression of at least one nucleic acid in the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10- fold, compared with the level of transcription of the nucleic acid in the absence of the one or more target antigens.
  • the CAR of the present disclosure can include an immunoreceptor tyrosine-based activation motif (ITAM) -containing intracellular signaling polypeptide.
  • ITAM immunoreceptor tyrosine-based activation motif
  • a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by one or more target antigens, can, in some instances, result in increased production of one or more cytokines by the cell.
  • a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by the one or more target antigens, can increase production of a cytokine by the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10- fold, compared with the amount of cytokine produced by the cell in the absence of the one or more target antigens.
  • Cytokines whose production can be increased include, but are not limited to interferon gamma (IFN ⁇ y), tumor necrosis factor-alpha (TNF-a), IL-2, IL-15, IL-12, IL-4, IL-5, IL-10; a chemokine; a growth factor; and the like.
  • IFN ⁇ y interferon gamma
  • TNF-a tumor necrosis factor-alpha
  • IL-2 tumor necrosis factor-alpha
  • IL-15 IL-12
  • IL-4 IL-5
  • IL-10 a chemokine
  • chemokine a growth factor
  • a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by one or more target antigens, can result in both an increase in transcription of a nucleic acid in the cell and an increase in production of a cytokine by the cell.
  • a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by one or more target antigens, results in cytotoxic activity by the cell toward a target cell that expresses on its cell surface an antigen to which the antigen-binding domain of the first polypeptide of the CAR binds.
  • a CAR of the present disclosure when present in the plasma membrane of the cell, and when activated by the one or more target antigens, increases cytotoxic activity of the cell toward a target cell that expresses on its cell surface the one or more target antigens.
  • a CAR of the present disclosure when present in the plasma membrane of the cell, and when activated by the one or more target antigens, increases cytotoxic activity of the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, compared to the cytotoxic activity of the cell in the absence of the one or more target antigens.
  • a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by one or more target antigens, can result in other CAR activation related events such as proliferation and expansion (either due to increased cellular division or anti- apoptotic responses).
  • a CAR of the present disclosure when present in the plasma membrane of a eukaryotic cell, and when activated by one or more target antigens, can result in other CAR activation related events such as intracellular signaling modulation, cellular differentiation, or cell death.
  • CARs of the present disclosure are microenvironment restricted. This property is typically the result of the microenvironment restricted nature of the ASTR domain of the CAR.
  • CARs of the present disclosure can have a lower binding affinity or, in illustrative embodiments, can have a higher binding affinity to one or more target antigens under a condition(s) in a microenvironment than under a condition in a normal physiological environment. Lymphoproliferative elements
  • Peripheral T lymphocyte numbers are maintained at remarkably stable levels throughout adulthood, despite the continuing addition of cells, due to emigration from the thymus and proliferation in response to antigen encounter, and loss of cells owing to the removal of antigen-specific effectors after antigen clearance (Marrak, P. et al. 2000. Nat Immunol 1 : 107-111; Freitas, A.A. et al. 2000. Annu Rev Immunol 18:83-111).
  • the size of the peripheral T cell compartment is regulated by multiple factors that influence both proliferation and survival. However, in a lymphopenic environment, T lymphocytes divide independently of cognate antigen, due to "acute homeostatic proliferation" mechanisms that maintain the size of the peripheral T cell compartment.
  • lymphodepletion of a subject is not desirable because it can cause serious side effects, including immune dysfunction and death.
  • lymphodepletion removes endogenous lymphocytes functioning as cellular sinks for homeostatic cytokines, thereby freeing cytokines to induce survival and proliferation of adoptively transferred cells.
  • Some cytokines such as for example, IL-7 and IL-15, are known to mediate antigen-independent proliferation of T cells and are thus capable of eliciting homeostatic proliferation in non-lymphopenic environments.
  • these cytokines and their receptors have intrinsic control mechanisms that prevent lymphoproliferative disorders at homeostasis.
  • an engineered signaling polypeptide is a lymphoproliferative element (LE) such as a chimeric lymphoproliferative element (CLE).
  • LE comprises an extracellular domain, a transmembrane domain, and at least one intracellular signaling domain that drives proliferation, and in illustrative embodiments a second intracellular signaling domain.
  • the intracellular domain of an LE or the first intracellular domain in an LE that has two or more intracellular domains, is other than a functional intracellular activating domain from an ITAM-containing intracellular domain, for example, an intracellular domain from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP 12, FCER1G, FCGR2A, FCGR2C. DAP10/CD28, or ZAP70, and in a further illustrative subembodiment, CD3z.
  • a second intracellular domain of an LE is other than a co-stimulatory domain of 4- IBB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.
  • the extracellular domain of an LE does not comprise a single-chain variable fragment (scFv).
  • the extracellular domain of an LE that upon binding to a binding partner activates an LE does not comprise a single-chain variable fragment (scFv).
  • a CLE does not comprise both an ASTR and an activation domain from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP 12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70.
  • the extracellular domain and transmembrane domain are believed to play support roles in LEs, assuring that the intracellular signaling domain(s) is in an effective
  • a lymphoproliferative element includes an intracellular domain that is a signaling polypeptide that is capable of driving proliferation of T cells or NK cells that is associated with a membrane through a membrane-associating motif (e.g. a transmembrane domain) and is oriented in, or capable of being oriented into, an active conformation.
  • the ASTR of an LE in illustrative embodiments, does not include an scFv.
  • a lymphoproliferative element does not include an extracellular domain.
  • the extracellular domains, transmembrane domains, and intracellular domains of LEs can vary in their respective amino acid lengths.
  • the overall length of the LE can be between 3 and 4000 amino acids, for example between 10 and 3000, 10 and 2000, 50 and 2000, 250 and 2000 amino acids, and, in illustrative embodiments between 50 and 1000, 100 and 1000 or 250 and 1000 amino acids.
  • the extracellular domain when present to form an extracellular and transmembrane domain, can be between 1 and 1000 amino acids, and is typically between 4 and 400, between 4 and 200, between 4 and 100, between 4 and 50, between 4 and 25, or between 4 and 20 amino acids.
  • the extracellular region is GGGS for an extracellular and transmembrane domain of this aspect of the invention.
  • the transmembrane domains, or transmembrane regions of extracellular and transmembrane domains, can be between 10 and 250 amino acids, and are more typically at least 15 amino acids in length, and can be, for example, between 15 and 100, 15 and 75, 15 and 50, 15 and 40, or 15 and 30 amino acids in length.
  • the intracellular signaling domains can be, for example, between 10 and 1000, 10 and 750, 10 and 500, 10 and 250, or 10 and 100 amino acids.
  • the intracellular signaling domain can be at least 30, or between 30 and 500, 30 and 250, 30 and 150, 30 and 100, 50 and 500, 50 and 250, 50 and 150, or 50 and 100 amino acids.
  • an intracellular signaling domain for a particular gene is at least 90%, 95%, 98%, 99% or 100% identical to at least 10, 25, 30, 40, or 50 amino acids from a sequence of that intracellular signaling domain, such as a sequence provided herein for that intracellular domain, up to the size of the entire intracellular domain sequence, and can include for example, up to an additional 1, 2, 3, 4, 5, 10, 20, or 25 amino acids, provided that such sequence still is capable of providing any of the properties of LEs disclosed herein.
  • CLEs were identified in Example 10, Example 11, and Example 12 herein, that promoted proliferation in cell culture of PBMCs that were transduced with lentiviral particles encoding the CLEs between day 7 and day 21, 28, 35 and/or 42 after transduction.
  • These Examples provide tests and/or criteria that can be used to identify whether any test polypeptide, including LEs, or test domains of an LE, such as a first intracellular domain, or a second intracellular domain, or both a first and second intracellular domain, are indeed LEs or effective intracellular domains of LEs, or especially effective LEs or intracellular domains of LEs.
  • any aspect or other embodiment provided herein that includes an LE or a polynucleotide or nucleic acid encoding an LE can recite that the LE meets, or provides the property of, or is capable of providing and/or possesses the property of, any one or more of the identified tests or criteria for identifying an LE provided herein, or that a cell genetically modified and/or transduced with a retroviral particle, such as a lentiviral particle encoding the LE, is capable of providing, is adapted for, possesses the property of, and/or is modified for achieving the results of one or more of the recited tests.
  • the LE provides, is capable of providing and/or possesses the property of, (or a cell genetically modified and/or transduced with a retroviral particle encoding the LE is capable of providing, is adapted for, possesses the property of, and/or is modified for) improved expansion to pre-activated PBMCs transduced with a lentivirus comprising a nucleic acid encoding the LE and an anti-CD 19 CAR comprising a CD3 zeta intracellular activating domain but no co- stimulatory domain, between day 7 and day 21, 28, 35, and/or 42 of in vitro culturing post-transduction in the absence of exogenously added cytokines, compared to a control retroviral particle, e.g.
  • a lymphoproliferative element test for improved or enhanced survival, expansion, and/or proliferation of cells transduced with a retroviral particle e.g. lentiviral particle
  • a retroviral particle e.g. lentiviral particle
  • test cells can be performed based on a comparison to control cells, which can be, for example, either untransduced cells or cells transduced with a control retroviral (e.g.
  • control cells are transduced with a retroviral particle (e.g. lentiviral particle) having a genome encoding a lymphoproliferative element or intracellular domain(s) thereof, identified herein as exemplifying a lymphoproliferative element.
  • a retroviral particle e.g. lentiviral particle having a genome encoding a lymphoproliferative element or intracellular domain(s) thereof, identified herein as exemplifying a lymphoproliferative element.
  • the test criteria can include that there is at least as much enrichment, survival and/or expansion, or no statistical difference of enrichment, survival, and/or expansion when the test is performed using a retroviral particle (e.g. lentiviral particle) having a genome encoding a test construct versus encoding the control lymphoproliferative element, typically by analyzing cells transcribed therewith.
  • a retroviral particle e.g. lentiviral particle
  • Exemplary or illustrative embodiments of lymphoproliferative elements herein, in some embodiments, are illustrative embodiments of control lymphoproliferative elements for such a test.
  • this test for an improved property of a putative or test lymphoproliferative element is performed by performing replicates and/or performing a statistical test.
  • a skilled artisan will recognize that many statistical tests can be used for such a lymphoproliferative element test.
  • the statistical test can be a T-test or a Mann-Whitney-Wilcoxon test.
  • the normalized enrichment level of a test construct is significant at a p-value of less than 0.1, or less than 0.05, or less than 0.01.
  • the LE provides, is capable of providing and/or possesses the property of (or a cell genetically modified and/or transduced with the LE is capable of providing, is adapted for, possesses the property of, and/or is modified for) at least a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold expansion, or between 1.5 fold and 25-fold expansion, or between 2-fold and 20-fold expansion, or between 2-fold and 15-fold expansion, or between 5-fold and 25-fold expansion, or between 5-fold and 20-fold expansion, or between 5-fold and 15-fold expansion, of pre- activated PBMCs transduced with a nucleic acid encoding the LE when transduced along with an anti- CD ⁇ CAR comprising a CD3 zeta intracellular activating domain but no co-stimulatory domain, between day 7 and day 21, 28, 35, and/or 42 of in vitro culturing in the absence of exogenously added
  • the test is performed in the presence of PBMCs, for example at a 1 : 1 ratio of transduced cells to PBMCs, which can be for example, from a matched donor, and in some embodiments, the test is performed in the absence of PBMCs.
  • the analysis of expansion for any of these tests is performed as provided in Example 11 or Example 12.
  • the test can include a further statistical test and a cut-off such as a P value below 0.1, 0.05, or 0.01, wherein a test polypeptide or nucleic acid encoding the same, needs to meet one or both thresholds (i.e. fold expansion and statistical cutoff).
  • the number of test cells and the number of control cells can be compared between day 7 and day 14, 21, 28, 35, 42 or 60 post- transduction.
  • the numbers of test and control cells can be determined by sequencing DNA and counting the occurrences of unique identifiers present in each construct.
  • the numbers of test and control cells can be counted directly, for example with a hemocytometer or a cell counter.
  • all the test cells and control cells can be grown within the same vessel, well or flask.
  • test cells can be seeded in one or more wells, flasks or vessels, and the control cells can be seeded in one or more flasks or vessels.
  • test and control cells can be seeded individually into wells or flasks, e.g., one cell per well.
  • the numbers of test cells and control cells can be compared using enrichment levels.
  • the enrichment level for a test or control construct can be calculated by dividing the number of cells at a later time point (day 14, 21, 28, 35, or day 45) by the number of cells at day 7 for each construct.
  • the enrichment level for a test or control construct can be calculated by dividing the number of cells at a time point (day 14, 21, 28, 35, or day 45) by the number of cells at that time point for untransduced cells.
  • the enrichment level of each test construct can be normalized to the enrichment level of the respective control construct to generate a normalized enrichment level.
  • a LE encoded in the test construct provides (or a cell genetically modified and/or transduced with a retroviral particle (e.g.
  • lentiviral particle) having a genome encoding the LE is capable of providing, is adapted for, possesses the property of, and/or is modified for) at least a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold normalized enrichment level, or between 1.5 fold and 25 -fold normalized enrichment level, or between 3 -fold and 20- fold normalized enrichment level, or between 5-fold and 25-fold normalized enrichment level, or between 5 -fold and 20-fold normalized enrichment level, or between 5 -fold and 15 -fold normalized enrichment level.
  • Enrichment can be measured, for example, by direct cell counting.
  • Cutoff values can be based on a single test, or two, three, four, or five repeats, or based on many repeats.
  • the cutoff can be met when a lymphoproliferative element meets one or more repeat tests, or meets or exceeds a cutoff for all repeats.
  • the enrichment is measured as log 2 ((normalized count data on the test day + l)/(normalized count data on day 7 + 1)).
  • CLEs were identified from libraries of constructs that included constructs that encoded test chimeric polypeptides that were designed to comprise an intracellular domain believed to induce proliferation and/or survival of lymphoid or myeloid cells, and an anti-CD 19 CAR that comprised an intracellular activating domain but not a co- stimulatory domain.
  • Preactivation which was performed overnight at 37 °C, was performed in a preactivation reaction mixture comprising PBMCs, a commercial media for lymphocytes (Complete OpTmizer CTS T-Cell Expansion SFM), recombinant human interleukin-2 (lOOIU/ml) and anti-CD3 Ab (OKT3) (50ng/ml).
  • transduction was performed overnight at 37 °C after addition of test and control lentiviral particles to the preactivation reaction mixtures at a multiplicity of infection (MOI) of 5.
  • MOI multiplicity of infection
  • Some control lentiviral particles contained constructs encoding polypeptides with extracellular and transmembrane domains but no intracellular domains.
  • the test lentiviral particles contained constructs encoding polypeptides with extracellular and transmembrane domains and either one or two intracellular domains.
  • Complete OpTmizerTM CTSTM T-Cell Expansion SFM was added to dilute the reaction mixture 5- to 20-fold and the cells were cultured for up to 45 days at 37 °C.
  • Example 10 As shown in Example 10, Example 11, and Example 12, test constructs were identified as CLEs because the CLEs induced proliferation/expansion in these fed or unfed cultures without added cytokines such as IL-2 between days 7 and day 21, 28, 35, and/or 42.
  • Example 10 identified effective CLEs by identifying test CLEs that provided increased expansion of these in vitro cultures, whether fed or unfed with untransduced PBMCs, between day 7 and day 21, 28, 35, and/or 42 post-transduction, compared to control constructs that did not include any intracellular domains.
  • Example 10 discloses that at least one and typically more than one test CLE that included an intracellular domain from a test gene provided more expansion than every control construct that was present at day 7 post-transduction, that did not include an intracellular domain.
  • Example 10 further provides a statistical method that was used to identify exceptionally effective genes with respect to a first intracellular domain, and one or more exemplary intracellular domain(s) from these genes. The method used a Mann-Whitney-Wilcoxon test and a false discovery cutoff rate of less than 0.1 or less than 0.05.
  • Example 12 identified especially effective genes for the first intracellular domain or the second intracellular domain, for example, by analyzing scores for genes calculated as combined score for all constructs with that gene.
  • Such analysis can use a cutoff of greater than 1, or greater than negative control constructs without any intracellular domains, or greater than 2, as shown for some of the tests performed in Example 12.
  • a LE which typically include a CAR
  • the genetically modified cell is modified so as to possess new properties not previously possessed by the cell before genetic modification and/or transduction.
  • Such a property can be provided by genetic modification with a nucleic acid encoding a CAR or a LE, and in illustrative embodiments both a CAR and a LE.
  • the genetically modified and/or transduced cell is capable of, is adapted for, possesses the property of, and/or is modified for survival and/or proliferation in ex vivo culture for at least 7, 14, 21, 28, 35, 42, or 60 days or from between day 7 and day 14, 21, 28, 35, 42 or 60 post-transduction, in the absence of added IL-2 or in the absence of added cytokines such as IL-2, IL-15, or IL-7, and in certain illustrative embodiments, in the presence of the antigen recognized by the CAR where the method comprises genetically modifying and/or transducing using a retroviral particle having a pseudotyping element and optionally a separate or fused activation domain on its surface and typically does not require pre -activation.
  • the genetically modified and/or transduced cell exhibits, is capable of, is adapted for, possesses the property of, and/or is modified for improved survival or expansion in ex vivo or in vitro culture in culture media in the absence of one or more added cytokines such as IL-2, IL-15, or IL-7, or added lymphocyte mitogenic agent, compared to a control cell(s) identical to the genetically modified and/or transduced cell(s) before it was genetically modified and/or transduced or to a control cell that was transduced with a retroviral particle identical to an on-test retroviral particle that comprises an LE or a putative LE, but without the LE or the intracellular domains of the LE, wherein said survival or proliferation of said control cell(s) is promoted by adding said one or more cytokines, such as IL-2, IL-15, or IL-7, or said lymphocyte mitogenic agent to the culture media.
  • cytokines such as IL-2, IL-15, or IL-7
  • cytokine or lymphocyte mitogenic agent By added cytokine or lymphocyte mitogenic agent, it is meant that cytokine or lymphocyte mitogenic agent is added from an exogenous source to a culture media such that the concentration of said cytokine or lymphocyte mitogenic agent is increased in the culture media during culturing of the cell(s) compared to the initial culture media, and in some embodiments can be absent from the initial culture media before said adding.
  • added or exogenously added it is meant that such cytokine or lymphocyte mitogenic agent is added to a lymphocyte media used to culture the genetically modified and/or transduced cell after the genetically modifying and/or transducing, where the culture media may or may not already possess the cytokine or lymphocyte mitogenic agent.
  • All or a portion of the media that includes a mixture of multiple media components is typically stored and in illustrative embodiments has been shipped to a site where the culturing takes place, without the exogenously added cytokine(s) or lymphocyte mitogenic agent(s).
  • the lymphocyte media in some embodiments is purchased from a supplier, and a user such as a technician not employed by the supplier and not located within a supplier facility, adds the exogenously added cytokine or lymphocyte mitogenic agent to the lymphocyte media and then the genetically modified and/or transduced cells are cultured in the presence or absence of such exogenously added cytokine or lymphocyte mitogenic agent.
  • improved or enhanced survival, expansion, and/or proliferation can be shown as an increase in the number of cells determined by sequencing DNA from cells transduced with retroviral particle (e.g. lentiviral particle) having a genome encoding CLEs and counting the occurrences of sequences present in unique identifiers from each CLE.
  • retroviral particle e.g. lentiviral particle
  • improved survival and/or improved expansion can be determined by counting the cells directly, for example with a hemocytometer or a cell counter, at each time point.
  • improved survival and/or improved expansion and/orenrichment can be calculated by dividing the number of cells at the later time point (day 21, 28, 35, and/or day 45) by the number of cells at day 7 for each construct.
  • the cells can be counted by hemocytometer or cell counters.
  • the enrichment level determined using the nucleic acid counts or the cell counts of each specific test construct can be normalized to the enrichment level of the respective control construct, i.e., the construct with the same extracellular domain and transmembrane domain but lacking the intracellular domains present in the test construct.
  • the LE encoded in the construct provides (or a cell genetically modified and/or transduced with a retroviral particle (e.g.
  • lentiviral particle) having a genome encoding the LE is capable of providing, is adapted for, possesses the property of, and/or is modified for) at least a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold normalized enrichment level, or between 1.5 fold and 25-fold normalized enrichment level, or between 3-fold and 20-fold normalized enrichment level, or between 5 -fold and 25 -fold normalized enrichment level, or between 5- fold and 20-fold normalized enrichment level, or between 5-fold and 15-fold normalized enrichment level.
  • one or more lymphoproliferative elements is introduced into a T cell and/or NK cell, typically by genetically modifying and/or transducing the T cell and/or NK cell with replication incompetent recombinant retroviral particles whose genome encodes the
  • lymphoproliferative element as part of an engineered signaling polypeptide.
  • the lymphoproliferative element can include a single intracellular domain or can include more than one intracellular domain.
  • a lymphoproliferative element includes two intracellular domains. Tables 8 to 25 provided herein and discussed in Example 10, Example 11, and Example 12, provide CLEs with one intracellular domain and CLEs with two intracellular domains that were identified using experimental methods provided therein. For CLEs that include two intracellular signaling domains in these Examples, the first intracellular signaling domain was between the transmembrane domain and the second intracellular signaling domain. "Intracellular signaling domain" of LEs are sometimes referred to as "intracellular domain" of LEs herein.
  • a lymphoproliferative element intracellular domain can include any of the intracellular parts listed in Table 7 (S036-S216) that were included as P3 parts in Example 10, Example 11, or Example 12 or any P4 part that was identified in Example 10, Example 11, or Example 12 as being active at inducing expansion between day 7 and day 21 or a later time point, in the absence of a P3 part.
  • the first intracellular signaling domain P3 in Example 10, Example 11, or Example 12 and FIGs. 15-18
  • the second intracellular signaling domain (P4 in Example 10, Example 11, and Example 12 and FIGs. 15-18) was chosen to be a co-stimulatory domain of the first intracellular signaling domain.
  • the extracellular and transmembrane domains (Pl/2 in Example 11 and FIGs. 16-17; and PI and P2, respectively, in Example 10 and Example 12 and FIGs. 15 and 18) were designed to play a role in orienting a first intracellular signaling domain in an active conformation or orientation.
  • the intracellular signaling domain can have at least 80%, 90%, or 95% or can have 100% sequence identity to any of SEQ ID NOs: 404-509.
  • the intracellular signaling domain can have at least 80%, 90%, or 95% or can have 100% sequence identity to an intracellular domain from BTLA, CD2, CD3D, CD3E, CD3G, CD3Z, CD4, CD8A, CD8B, CD27, mutated Delta Lck CD28, CD28, CD40, CD79A, CD79B, CD137, CRLF2, CSF2RB, CSF2RA, CSF3R, DAP10/CD28, DAP 12, EPOR, FCER1G, FCGR2A, FCGR2C, FCGRA2, GHR, ICOS, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL10RB,
  • the intracellular domain can have at least 80%, 90%, or 95% or can have 100% sequence identity to an intracellular domain from CD2, CD4, CD8A, CD8B, CD40, CD79B, CRLF2, CSF2RB, CSF2RA, CSF3R, EPOR, FCGR2C, FCGR2A, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL9R, IL10RA, IL10RB, IL11RA, IL13RA1, IL13RA2, IL17RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL22RA1, IL31RA, LE
  • a lymphoproliferative element can be or can comprise a cytokine or in further illustrative embodiments, a cytokine receptor, or a fragment that includes a signaling domain thereof, that activates a STAT3 pathway, a STAT4 pathway, or in even further illustrative embodiments, a Jak/STAT5 pathway.
  • a lymphoproliferative element can be, in a non-limiting example, a cytokine receptor, or active fragment that includes a signaling domain thereof, such as an interleukin receptor, or an active fragment that includes a signaling domain thereof, that activates STAT5.
  • a lymphoproliferative element is a polypeptide that promotes proliferation, and optionally survival (anti-apoptotic), and optionally provides a co-stimulatory signal that enhances a differentiation state, proliferative potential or resistance to cell death of a lymphocyte.
  • a lymphoproliferative element is a polypeptide that induces proliferation of a T cell and/or NK cell.
  • Illustrative lymphoproliferative elements induce proliferation by activating STAT5.
  • fragments of such lymphoproliferative elements retain the ability to induce proliferation of T cells and/or NK cells, in illustrative embodiments, by activating STAT5.
  • lymphoproliferative elements when present in genetically modified PBMCs, lymphocytes, or genetically modified T cells and/or NK cells are capable of promoting lymphocyte proliferation/expansion and optionally survival ex vivo or in vitro in culture in the absence of exposure of the cells to cytokines such as IL-15, IL-7, or in illustrative embodiments IL-2, in some embodiments further in the absence of the target for an ASTR of a CAR expressed by the cells, or in certain illustrative embodiments, in the presence of the antigen recognized by the CAR where the method comprises genetically modifying and/or transducing using a retroviral particle having a pseudotyping element and optionally a separate or fused activation domain on its surface and typically does not require pre -activation during culturing for 6, 7, 14, 21, or 35 days.
  • a lymphoproliferative element is used to promote proliferation or expansion of genetically modified T cells in vivo without having to lymphodeplete subjects.
  • non-limiting illustrative embodiments of methods provided herein that include inserting a lymphoproliferative element into a resting T cell and/or NK cell of a subject, typically by transducing such T cell and/or NK cell can be performed without lymphodepleting the subject before, during and/or after performing the method, or without lymphodepleting the subject before, during and/or after collecting blood from a subject before performing such method, or without lymphodepleting the subject before, during, and/or after genetically modifying T cells or NK cells ex vivo from the subject, and/or before, during, or after reintroducing the genetically modified T cells and/or NK cells into the subject.
  • Factors that promote proliferation of T cells in vivo include cytokines and their receptors, in which a receptor typically
  • the lymphoproliferative element used in the methods and compositions disclosed herein is a cytokine and/or a cytokine receptor.
  • the cytokine can be an interleukin
  • the cytokine receptor can be an interleukin receptor.
  • the lymphoproliferative element can be a functional fragment of a cytokine and/or a functional fragment of a cytokine receptor, such as a signaling domain thereof, wherein the fragment is capable of promoting proliferation of T cells, for example by activating STAT5.
  • compositions herein include one or more of the following: Interleukin-7 (IL-7) or its receptor (IL-7R), or a signaling domain thereof; Interleukin-12 (IL-12) or its receptor (IL-12R), or a signaling domain thereof; Interleukin-23 (IL-23) or its receptor composed of IL-12R ⁇ and IL-23R, or a signaling domain thereof; Interleukin-27 (IL-27) or its receptor (IL-27R), or a signaling domain thereof; Interleukin- 15 (IL-15) or its receptor (IL-15R), or a signaling domain thereof; Interleukin-21 (IL-21) or its receptor (IL-21R), or a signaling domain thereof; or transforming growth factor ⁇ (TGF ⁇ ) or its receptor (TGF R) or a signaling domain thereof; or the TGF ⁇ decoy receptor (TGF- ⁇ — dominant-negative receptor II (DNRII)).
  • the lymphoproliferative element is the IL-12R or the TGF ⁇ or the
  • IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7R alpha and common gamma chain receptor. Binding results in a cascade of signals important for T cell development within the thymus and survival within the periphery. Binding of IL-7 to the IL-7 receptor is known to activate the
  • IL-12 is involved in the differentiation of naive T cells into Thl cells (Hsieh CS et al. 1993. Science. 260(5107):547-9) and is known as a T cell-stimulating factor.
  • IL-12 binds to the IL-12 receptor, which is a heterodimeric receptor formed by IL-12R- 1 and IL-12R- 2.
  • IL12 can act by activating STAT4, but has been shown to activate STAT5 in T cells as well (Ahn, H., et al. 1998. J. Immun.
  • the IL-12 family is composed of the cytokines IL-12, IL-23, and IL-27.
  • the receptor for IL-23 is composed of IL-12R ⁇ and IL-23R.
  • IL-27 is a heterodimeric cytokine that is composed of two distinct genes, Epstein-Barr virus-induced gene 3(EBI3) and IL-27p28. IL-27 interacts with IL-27 receptor.
  • IL-15 is a T and NK cell stimulatory factor that is similar in structure and function to IL-2. Both cytokines induce proliferation of T cells; and their shared functions are thought to result from both receptors using the IL-2/IL-15Rp and common ⁇ chains.
  • IL-15Ra receptor Upon binding to IL-15Ra receptor, with subsequent presentation to surrounding cells bearing IL-15RJ3yc complex on their cell surface.
  • ⁇ _,-15 ⁇ subunit activates Janus kinase 1 (Jakl) and yc subunit Janus kinase 3 (Jak3), which leads to phosphorylation and activation of STAT3 and STAT5.
  • IL-21 is expressed in activated human CD4+ T cells and in NKT cells, and IL-21 expression is up-regulated in Th2 and Thl7 subsets of T helper cells.
  • the IL-21 receptor (IL-21R) is expressed on the surface of T, B and NK cells and is similar in structure to the receptors for other type I cytokines like IL- 2R or IL-15.
  • IL-21R requires dimerization with the common gamma chain (yc) in order to bind IL-21.
  • the IL-21 receptor acts through the Jak/STAT pathway, activating STAT1, STAT3, and STAT5.
  • TGF ⁇ decoy receptors (TGF- ⁇ — dominant-negative receptor II (DNRII)) block TGF ⁇ signaling by competing with the natural receptors for TGF ⁇ binding.
  • TGF ⁇ -DNRII is a kinase-dead truncated form of RII that contains the extracellular TGF ⁇ binding domain and the transmembrane domain of RII.
  • TGF ⁇ - DNRII binds the ligand but does not phosphorylate and activate RI, which thereby diminishes or eliminates Smad phosphorylation.
  • Gain-of-function mutations in IL-7R ⁇ have been identified in subjects with B and T cell acute lymphoblastic leukemias (B-ALL and T-ALL) (Zenatti PP, et al. 2011. Nat Genet 43:932-939; Snochat, C. et al. 2011. JExpMed 208:901-908; McElroy, C.A. et al. 2012. PNAS 109(7):2503-2508).
  • the cysteine resulted in constitutive activation of the receptor.
  • Some of the mutations in the T-all group activated JAKl.
  • the lymphoproliferative element is a mutated IL-7 receptor.
  • the mutated IL-7 receptor is constitutively active, activating the JAK-STAT5 pathway in the absence of the cytokine ligand.
  • the mutated IL-7 receptor comprises a 1 to 10 amino acid insertion at a position between 237 and 254 that includes a cysteine residue that includes the ability to constitutively activate the STAT5 pathway.
  • the mutated IL-7 receptor is IL-7R ⁇ -insPPCL (represented by SEQ ID NO: 82).
  • the lymphoproliferative element is a chimeric cytokine receptor such as but not limited to a cytokine tethered to its receptor that typically constitutively activates the same STAT pathway as a corresponding activated wild-type cytokine receptor such as STAT3, STAT4, and in illustrative embodiments, STAT5.
  • the chimeric cytokine receptor is an interleukin, or a fragment thereof, tethered to or covalently attached to its cognate receptor, or a fragment thereof, via a linker.
  • the chimeric cytokine receptor is IL-7 tethered to IL-7R ⁇ .
  • the chimeric cytokine receptor is IL-7 tethered to a domain of IL-7R ⁇ , such as for example, the extracellular domain of IL-7R ⁇ and/or the transmembrane domain of IL-7R ⁇ .
  • the lymphoproliferative element is a cytokine receptor that is not tethered to a cytokine, and in fact in illustrative embodiments, provided herein a lymphoproliferative element is a constitutively active cytokine receptor that is not tethered to a cytokine.
  • chimeric IL-7 receptors typically constitutively activate STAT5 when expressed.
  • the lymphoproliferative element can comprise an interleukin polypeptide covalently attached to a portion of its cognate interleukin receptor polypeptide via a linker.
  • this portion of the cognate interleukin receptor includes a functional portion of the extracellular domain capable of binding the interleukin cytokine and the transmembrane domain.
  • the intracellular domain is an intracellular portion of the cognate interleukin receptor.
  • the intracellular domain is an intracellular portion of a different cytokine receptor that is capable of promoting lymphocyte proliferation.
  • the lymphoproliferative element is an interleukin polypeptide covalently attached to its full length cognate interleukin receptor polypeptide via a linker.
  • the intracellular domain can be derived from an intracellular portion of the transmembrane protein CD40.
  • the CD40 protein contains several binding sites for TRAF proteins. Not to be limited by theory, binding sites for TRAFl, TRAF2, and TRAF3 are located at the membrane distal domain of the intracellular portion of CD40 and include the amino acid sequence PXQXT (SEQ ID NO:522) where each X can be any amino acid, (corresponding to amino acids 35-39 of SEQ ID NO:416) (Elgueta et al. Immunol Rev. 2009 May; 229(1): 152-72).
  • TRAF2 has also been shown to bind to the consensus sequence SXXE (SEQ ID NO:523) where each X can be any amino acid, (corresponding to amino acids 57-60 of SEQ ID NO:416) (Elgueta et al. Immunol Rev. 2009 May; 229(1): 152-72).
  • a distinct binding site for TRAF6 is situated at the membrane proximal domain of intracellular portion of CD40 and includes the consensus sequence QXPXEX (SEQ ID NO:524) where each X can be any amino acid (corresponding to amino acids 16-21 of SEQ ID NO:416) (Lu et al. J Biol Chem. 2003 Nov 14; 278(46):45414-8).
  • the intracellular portion of the transmembrane protein CD40 can include all the binding sites for the TRAF proteins.
  • the TRAF binding sites are known in the art and a skilled artisan will be able to identify corresponding TRAF binding sites in similar CD40 polypeptides.
  • a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:416 or SEQ ID NO:417.
  • the intracellular domain derived from CD40 has a length of from about 30 amino acids (aa) to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, or from about 60 aa to about 65 aa.
  • the intracellular domain derived from CD40 has a length of from about 30 aa to about 66 aa, for example, 30 aa to 65 aa, or 50 aa to 66 aa.
  • the second intracellular domain can be other than an intracellular domain derived from MyD88, a CD28 family member (e.g. CD28, ICOS), Pattern Recognition Receptor, a C-reactive protein receptor (i.e., Nodi , Nod2, PtX3-R), a TNF receptor (i.e., CD40, RANK/TRANCE-R, OX40, 4- IBB), an HSP receptor (Lox- 1 and CD91), or CD28.
  • a CD28 family member e.g. CD28, ICOS
  • Pattern Recognition Receptor e.e., a C-reactive protein receptor (i.e., Nodi , Nod2, PtX3-R), a TNF receptor (i.e., CD40, RANK/TRANCE-R, OX40, 4- IBB), an HSP receptor (Lox- 1 and CD91), or CD28.
  • CD28 family member e.g. CD28, ICOS
  • Pattern Recognition Receptors include, but are not limited to endocytic pattern- recognition receptors (i.e., mannose receptors, scavenger receptors (i.e., Mac-1 , LRP, peptidoglycan, techoic acids, toxins, CD 1 1 c/CR4)); external signal pattern-recognition receptors (Toll-like receptors (TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10), peptidoglycan recognition protein, (PGRPs bind bacterial peptidoglycan, and CD 14); internal signal pattern-recognition receptors (i.e., NOD-receptors 1 & 2), and RIG1
  • endocytic pattern- recognition receptors i.e., mannose receptors, scavenger receptors (i.e., Mac-1 , LRP, peptidoglycan, techoic acids, toxins, CD 1 1 c
  • the intracellular domain can be derived from an intracellular portion of CD27.
  • the serine at amino acid 219 of full-length CD27 (corresponding to the serine at amino acid 6 of SEQ ID NO:413) has been shown to be phosphorylated.
  • a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:413.
  • the intracellular domain derived from CD27 has a length of from about 30 amino acids (aa) to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to about 50 aa.
  • the intracellular domain can be derived from an intracellular portion of CSF2RB.
  • Full-length CSF2RB contains a Box 1 motif at amino acids 474-482 (corresponding to amino acids 14-22 of SEQ ID NO:421).
  • the tyrosine at amino acid 766 of full-length CSF2RB (corresponding to the tyrosine at amino acid 306 of SEQ ID NO:421) has been shown to be phosphorylated.
  • a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 421.
  • the intracellular domain derived from CSF2RB has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, from about 200 aa to about 250 aa, from about 250 aa to 300 aa, from about 300 aa to 350 aa, from about 350 aa to about 400 aa, or from about 400 aa to about 450 aa.
  • the intracellular domain can be derived from an intracellular portion of IL2RB.
  • Full-length IL2RB contains a Box 1 motif at amino acids 278-286 (corresponding to amino acids 13-21 of SEQ ID NO:448).
  • a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:448.
  • the intracellular domain derived from IL2RB has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, from about 200 aa to about 250 aa, or from about 250 aa to 300 aa.
  • the intracellular domain can be derived from an intracellular portion of IL6ST.
  • Full-length IL6ST contains a box 1 motif at amino acids 651-659 (corresponding to amino acids 10-18 of SEQ ID NO:455).
  • the serines at amino acids 661, 667, 782, 789, 829, and 839 of full-length IL6ST (corresponding to serines at amino acids 20, 26, 141, 148, 188, and 198, respectively, of SEQ ID NO:455) have been shown to be phosphorylated.
  • a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:454 or SEQ ID NO:455.
  • the intracellular domain derived from IL6ST has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, from about 200 aa to about 250 aa, or from about 250 aa to 300 aa.
  • the intracellular domain can be derived from an intracellular portion of IL17RE.
  • Full-length IL17RE contains a TIR domain at amino acids 372-495 (corresponding to amino acids 13-136 of SEQ ID NO:473).
  • a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 473.
  • the intracellular domain derived from IL17RE has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, or from about 175 aa to about 200 aa.
  • the intracellular domain can be derived from an intracellular portion of IL2RG.
  • Full-length IL2RG contains a box 1 motif at amino acids 286-294 (corresponding to amino acids 3-11 of SEQ ID NO: 449).
  • a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:449.
  • the intracellular domain derived from IL2RG has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, or from about 70 aa to about 100 aa.
  • the intracellular domain can be derived from an intracellular portion of IL18R1.
  • Full-length IL18R1 contains a TIR domain at amino acids 222-364 (corresponding to amino acids 28-170 of SEQ ID NO:474).
  • a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 474.
  • the intracellular domain derived from IL18R l has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, or from about 70 aa to about 100 aa.
  • the intracellular domain can be derived from an intracellular portion of IL27RA.
  • Full-length IL27RA contains a box 1 motif at amino acids 554-562 (corresponding to amino acids 17-25 of SEQ ID NO:481).
  • a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:481 or SEQ ID NO:482.
  • the intracellular domain derived from IL27RA has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, or from about 70 aa to about 100 aa.
  • the intracellular domain can be derived from an intracellular portion of IFNGR2.
  • Full-length IFNGR2 contains a dileucine internalization motif at amino acids 276-277
  • a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:438.
  • the intracellular domain derived from IFNGR2 l has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.
  • the intracellular domain can be derived from a portion of the protein MyD88.
  • the MyD88 protein has an N-terminal death domain that mediates interactions with other death domain-containing proteins (corresponding to amino acids 29-106 of SEQ ID NO: 492), an intermediate domain that interacts with IL-1R associated kinase (corresponding to amino acids 107-156 of SEQ ID NO:492), and a C-terminal TIR domain (corresponding to amino acids 160-304 of SEQ ID NO:492) that associates with the TLR-TIR domain (Biol Res. 2007; 40(2):97-l 12).
  • MyD88 also has canonical nuclear localization and export motifs. Point mutations have been identified in MyD88 and include the loss-of- function mutations L93P and R193C (corresponding to L93P and R196C in SEQ ID NO:492), and the gain-of-function mutation L265P (corresponding to L260P in SEQ ID NO:492) (Deguine and Barton. FlOOOPrime Rep. 2014 Nov 4;6:97).
  • the portion of the protein MyD88 can include all the domains disclosed herein. The domains, motifs, and point mutations of MyD88 are known in the art and a skilled artisan will be able to identify corresponding domains, motifs, and point mutations in similar MyD88 polypeptides.
  • a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:492-501.
  • the intracellular domain derived from MyD88 has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, from about 200 aa to about 250 aa, from about 250 aa to 300 aa, or from about 300 aa to 350 aa.
  • the intracellular domain derived from MyD88 has a length of from about 30 aa to about 350 aa, for example, 50 aa to 350 aa, or 100 aa to 350 aa, 100 aa to 304 aa, 100 aa to 296 aa, 100 aa to 251 aa, 100 aa to 191 aa, 100 aato 172 aa, 100 aa to 146 aa, or 100 aa to 127 aa.
  • the second intracellular domain can be other than an intracellular domain derived from a CD28 family member (e.g. CD28, ICOS), Pattern Recognition Receptor, a C-reactive protein receptor (i.e., Nodi , Nod2, PtX3-R), a TNF receptor (i.e., CD40, RANK/TRANCE-R, OX40, 4- IBB), an HSP receptor (Lox-1 and CD91), or CD28.
  • a CD28 family member e.g. CD28, ICOS
  • Pattern Recognition Receptor e.e., a C-reactive protein receptor (i.e., Nodi , Nod2, PtX3-R), a TNF receptor (i.e., CD40, RANK/TRANCE-R, OX40, 4- IBB), an HSP receptor (Lox-1 and CD91), or CD28.
  • CD28 family member e.g. CD28, ICOS
  • Pattern Recognition Receptor e.
  • the intracellular domain can be derived from a portion of the intracellular domain
  • the transmembrane MPL protein contains the Boxl motif PXXP (SEQ ID NO:525) where each X can be any amino acid (corresponding to amino acids 17-20 in SEQ ID NO:491) and the Box2 motif, a region with increased serine and glutamic acid content (corresponding to amino acids 46-64 in SEQ ID NO:491) (Drachman and Kaushansky. Proc Natl Acad Sci U S A. 1997 Mar 18; 94(6):2350-5).
  • the Boxl and Box2 motifs are involved in binding to JAKs and signal transduction, although the Box2 motif presence is not always required for a proliferative signal (Murakami et al. Proc Natl Acad Sci U S A.
  • cytokine receptors have hydrophobic residues at positions -1, -2, and -6 relative to the Boxl motif (corresponding to amino acids 16, 15, and 11, respectively, of SEQ ID NO:491), that form a "switch motif," which is required for cytokine-induced JAK2 activation but not for JAK2 binding (Constantinescu et al. Mol Cell. 2001 Feb; 7(2):377-85; and Huang et al. Mol Cell.
  • a MPL intracellular signaling domain does not comprise the region comprising amino acids 70-95 in SEQ ID NO:491.
  • the lysines K553 corresponding to K40 of SEQ ID NO:491
  • K573 corresponding to K60 of SEQ ID NO:491
  • a MPL intracellular signaling domain does not comprise these ubiquitination targeting motif residues.
  • the tyrosines Y521 (corresponding to Y8 of SEQ ID NO:491), Y542 (corresponding to Y29 of SEQ ID NO:491), Y591 (corresponding to Y78 of SEQ ID NO:491), Y626 (corresponding to Yl 13 of SEQ ID NO:491), and Y631 (corresponding to Yl 18 of SEQ ID NO:491) have been shown to be phosphorylated (Varghese et al. Front Endocrinol (Lausanne). 2017 Mar 31; 8:59).
  • Y521 and Y591 of full-length MPL are negative regulatory sites that function either as part of a lysosomal targeting motif (Y521) or via an interaction with adaptor protein AP2 (Y591) (Drachman and Kaushansky. Proc Natl Acad Sci U S A. 1997 Mar 18; 94(6):2350-5; and Hitchcock et al. Blood. 2008 Sep 15; 112(6):2222-31).
  • Y626 and Y631 of full-length MPL are positive regulatory sites (Drachman and Kaushansky. Proc Natl Acad Sci U S A.
  • MPL contains the She phosphotyrosine-binding binding motif NXXY (SEQ ID NO:526) where each X can be any amino acid (corresponding to amino acids 110-113 of SEQ ID NO:491), and this tyrosine is phosphorylated and important for the TPO- dependent phosphorylation of She, SHIP, and STAT3 (Laminet et al. J Biol Chem. 1996 Jan 5;
  • MPL also contains the STAT3 consensus binding sequence YXXQ (SEQ ID NO:527) where each X can be any amino acid (corresponding to amino acids 118-121 of SEQ ID NO:491) (Stahl et al. Science. 1995 Mar 3; 267(5202): 1349-53).
  • the tyrosine of this sequence can be phosphorylated and MPL is capable of partial STAT3 recruitment (Drachman and Kaushansky. Proc Natl Acad Sci U S A. 1997 Mar 18; 94(6):2350-5).
  • MPL also contains the sequence YLPL (SEQ ID NO: 528) (corresponding to amino acid 113-116 of SEQ ID NO:491), which is similar to the consensus binding site for STAT5 recruitment pYLXL (SEQ ID NO:529) where pY is phosphotyrosine and X can be any amino acid (March et al. FEBS Lett. 1996 Sep 30; 394(2): 221-6). Using computer simulations, Lee et al.
  • the intracellular portion of MPL can include all the domains and motifs described herein that are present in SEQ ID NO:491.
  • a transmembrane portion of MPL can include all the domains and motifs described herein that are present in SEQ ID NO: 395.
  • the domains, motifs, and point mutations of MPL provided herein are known in the art and a skilled artisan would recognize that MPL intracellular signaling domains herein in illustrative embodiments would include corresponding domains, motifs, and point mutations in that have been shown to promote proliferative activity and would not include that that have been shown to inhibit MPLs proliferative activity..
  • a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:491.
  • the intracellular domain derived from MPL has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about 175 aa, from about 175 aato about 200 aa, from about 200 aa to about 250 aa, from about 250 aa to 300 aa, from about 300 aa to 350 aa, from about 350 aa to about 400 aa, from about 400 aa to about 450 aa, from about
  • the intracellular domain derived from MPL has a length of from about 30 aa to about 200 aa, for example, 30 aa to 150 aa, 30 aa to 119 aa, 30 aa to 121 aa, 30 aa to 122 aa, or 50 aa to 125 aa.
  • the intracellular domain can be derived from a portion of the intracellular domain
  • transmembrane protein CD79B also known as B29; IGB; AGM6.
  • CD79B contains an ITAM motif at residues 193-212 (corresponding to amino acids 16-30 of SEQ ID NO:419).
  • CD79B has two tyrosines that are known to be phosphorylated, Y196 and Y207 (corresponding to Y16 and Y27 of SEQ ID NO:419).
  • the intracellular portion of the transmembrane protein CD79B can include the ITAM motif and the known phosphorylation sites disclosed herein.
  • a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:419.
  • the intracellular domain derived from CD79B has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to about 50 aa.). In illustrative embodiments, the intracellular domain derived from CD79B has a length of from about 30 aa to about 50 aa.
  • a suitable CD79B intracellular activating domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids of the following sequence:
  • the intracellular domain can be derived from a portion of the intracellular domain
  • OSMR transmembrane protein
  • OSMR contains a Box 1 motif at amino acids 771-779 of isoform 3 (corresponding to amino acids 16-30 of SEQ ID NO:502).
  • OSMR has two serines at amino acids 829 and 890 of isoform 3 that are known to be phosphorylated (serines at amino acids 65 and 128 of SEQ ID NO:502).
  • the intracellular portion of the protein OSMR can include the box 1 motif and the known phosphorylation sites disclosed herein.
  • the motif and phosphorylatable serines of OSMR are known in the art and a skilled artisan will be able to identify corresponding motifs and phosphorylatable serines in similar OSMR polypeptides.
  • a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:502.
  • the intracellular domain derived from OSMR has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa., from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aato 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, or from about 200 aa to about 250 aa.
  • the intracellular domain can be derived from a portion of the intracellular domain
  • transmembrane protein PRLR PRLR contains a growth hormone receptor binding domain at amino acids 185-261 of isoform 6 (corresponding to amino acids 28-104 of SEQ ID NO:503).
  • the growth hormone receptor binding domain of PRLR is known in the art and a skilled artisan will be able to identify corresponding domain in similar PRLR polypeptides.
  • a suitable intracellular domain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 503.
  • the intracellular domain derived from PRLR has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aato 150 aa, from about 150 to about 175 aa, from about 175 aa to about 200 aa, from about 200 aa to about 250 aa, from about 250 aa to 300 aa, from about 300 aa to 350 aa, or from about 350 aa to about 400 aa.
  • the intracellular domain can be, the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from CSF2RB, CRLF2, CSF2RA, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, ILIRLI, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL5RA, IL6R, IL6ST, IL7RA, IL9R, ILIORA, IL10RB, ILl lRA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL17RC, IL17RD, IL18R1, IL18RAP, IL20RA
  • the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from CSF2RB, CRLF2, CSF2RA, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, ILIRAP, ILIRLI, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL5RA, IL6R, IL6ST, IL9R, ILIORA, IL10RB, ILl lRA, IL13RA1, IL13RA2, IL17RB, IL17RC, IL17RD, IL18R1, IL18RAP, IL20RA, IL20RB, IL22RA1, IL31RA, LEPR, LIFR
  • the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from CSF2RB, CSF2RA, CSF3R, EPOR, IFNGR1, IFNGR2, IL1R1, ILIRAP, ILIRLI, IL2RA, IL2RG, IL5RA, IL6R, IL9R, IL10RB, ILl lRA, IL12RB1, IL12RB2, IL13RA2, IL15RA, IL17RD, IL21R, IL23R, IL27RA, IL3 IRA, LEPR, MPL, MyD88, or OSMR.
  • the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from CSF2RB, CSF2RA, CSF3R, EPOR, IFNGR1, IFNGR2, IL1R1, ILIRAP, ILIRLI, IL2RA, IL2RG, IL5RA, IL6R, IL9R, IL10RB, ILl lRA, IL13RA2, IL17RD, IL31RA, LEPR, MPL, MyD88, or OSMR.
  • the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from CSF2RB, CSF3R, IFNAR1, IFNGRl, IL2RB, IL2RG, IL6ST, ILIORA, IL12RB2, IL17RC, IL17RE, IL18R1, IL22RA1, IL27RA, IL3 IRA, MPL, MyD88, OSMR, or PRLR (Table 1). Each of these genes had an intracellular domain that was significantly enriched (p ⁇ 0.1) at the P3 position across the replicates of Library 3.1.
  • the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from CSF2RB, CSF3R, IFNAR1, IFNGRl, IL2RB, IL2RG, IL6ST, ILIORA, IL17RC, IL17RE, IL18R1, IL22RA1, IL31RA, MPL, MyD88, OSMR, or PRLR.
  • the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from CSF2RB, CSF3R, IFNGRl, IL2RB, IL2RG, IL6ST, ILIORA, IL12RB2, IL17RE, IL18R1, IL22RA1, IL27RA, IL31RA, MPL, MyD88, OSMR, or PRLR (Table 1).
  • Each of these genes had an intracellular domain that was significantly enriched (p ⁇ 0.05) at the P3 position across the replicates of Library 3.1.
  • the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from CSF2RB, CSF3R, IFNGRl, IL2RB, IL2RG, IL6ST, ILIORA, IL17RE, IL18R1, IL22RA1, IL31RA, MPL, MyD88, OSMR, or PRLR.
  • the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from CSF2RB, CSF3R, IL2RB, IL2RG, IL6ST, IL27RA, IL31RA, MPL, or MyD88.
  • the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from CSF2RB, CSF3R, IL2RB, IL2RG, IL6ST, IL31RA, MPL, or MyD88.
  • the lymphoproliferative element can include the sequences of S054, S057, S058, S059, S062, S063, S064, S069, S072, S077, S081, S082, S083, S084, S085, S086, S087, S098, S099, S 100, S 101, S102, S 103, S104, S 105, S106, S109, S115, S116, S117, S 120, S121, S126, S129, S 130, S 135, S 136, S137, S 138, S141, S 142, S143, S145, S147, S148, S154, S 155, S156, S157, S158, S 161, S 165, S 168, S169, S 170, S171, S 174, S175, S176, S177, S180, S183, S 186, S189, S190, S191, S 192, S 193, S 194, S195, S 196,
  • the lymphoproliferative element can include the sequences of S057, S058, S059, S064, S069, S072, S084, S085, S099, S 100, S101, S 102, S104, S106, S115, S116, S126, S130, S135, S 137, S138, S142, S143, S 148, S 158, S 165, S168, S 169, S170, S171, S174, S175, S176, S177, S186, S 190, S191, S192, S193, SI 97, SI 98, or S I 99 as shown in Table 7, or any fragments thereof that include a signaling domain.
  • the lymphoproliferative element can include the sequences as shown in Table 7 of S057, S062, S063, S064, S081, S084, S 105, S106, S117, S129, S138, S149, S 161, S168, S169, S170, S 186, S 190, S 191, S 192, S 194, S 196, S I 97, SI 98, SI 99, or S202 (Table 1), or any fragments thereof that include a signaling domain.
  • Each of these parts was significantly enriched at the P3 position across the replicates of Library 3.1.
  • the intracellular domain can include an intracellular domain or a fragment thereof that includes a signaling domain from CD3D, CD3E, CD3G, CD27, CD28, CD40, CD79A, CD79B, FCER1G, FCGR2C, FCGRA2, ICOS, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18.
  • Each of these genes had an intracellular domain that was present at the P4 position on at least one construct in the top 100 hits of at least one of the replicates of Library 3.1.
  • the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from CD40, CD79B, FCGR2C, or FCGRA2.
  • the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from CD3D, CD3G, CD40, CD79A, ICOS, TNFRSF8, or TNFRSF9.
  • Each of these genes had an intracellular domain that was present at the P4 position on at least one construct that did not have an intracellular domain in the P3 position and was in the top 100 hits of at least one of the replicates of Library 3.1.
  • the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from CD40. In some embodiments, the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from a TNF receptor family member, and in illustrative embodiments as a second intracellular signaling domain, for example a TNF receptor family member shown in Table 2. In some embodiments, the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from CD27, CD40, CD79B, TNFRSF4, TNFRSF8, TNFRSF9, or TNFRSF18 (Table 2). Each of these genes had an intracellular domain that was significantly enriched at the P4 position across the replicates of Library 3.1 (p ⁇ 0.1). In some embodiments, the
  • lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from CD40 or CD79B.
  • the lymphoproliferative element can include the sequences of S037, S038, S039, S047, S048, S049, S050, S051, S052, S053, S074, S075, S076, S080, S211, S212, S213, S214, S215, or S216 as shown in Table 7, or any fragments thereof that include a signaling domain. Each of these parts was present at the P4 position on at least one construct in the top 100 hits of at least one of the replicates of Library 3.1. In some embodiments, the
  • lymphoproliferative element can include the sequences of S037, S039, S050, S051, S052, S080, S212, or S213 as shown in Table 7, or any fragments thereof that include a signaling domain. Each of these parts was present at the P4 position on at least one construct that did not have an intracellular domain in the P3 position and was in the top 100 hits of at least one of the replicates of Library 3.1.
  • the lymphoproliferative element can include the sequences as shown in Table 7 of S047, S050, S051, S053, S211, S212, S213, or S215 (Table 2), or any fragments thereof that include a signaling domain. Each of these parts was significantly enriched at the P4 position across the replicates of Library 3.1.
  • the lymphoproliferative element can include a cytokine receptor or a fragment that includes a signaling domain thereof.
  • the cytokine receptor can be CD27, CD40, CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2R, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL7R, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL13R, IL13RA1, IL13RA2, IL15R, IL15RA, IL17RA, IL17RB, IL17RC, IL17RE, IL18R1,
  • the cytokine receptor can be CD27, CD40, CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL13RA1, IL13RA2, IL15RA, IL17RA, IL17RB, IL17RC, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL22RA1, IL27RA, IL31RA, LEPR, LIFR, MPL, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9, TNFR
  • chimeric cytokine receptors that include functional intracellular domains of the above- listed cytokine receptors were tested in Example 11 and Example 12 to confirm that chimeric cytokine receptors that included these functional intracellular domains could function as lymphoproliferative elements.
  • the lymphoproliferative element can include a first intracellular domain, or a variant or fragment thereof, that includes a signaling domain from the following genes or the respective parts in parentheses, which were the most effective by this statistical analysis (P ⁇ 0.1): CSF2RB (S057), CSF3R (S062, S063, and S064), IFNAR1 (S081), IFNGR1 (S084), IL2RB (S105), IL2RG (S106), IL6ST (SI 17), IL10RA (S129), IL12RB2 (S138), IL17RE (S149), IL22RA1 (S161), IL27RA (S168 and S169), IL31RA (S170), MPL (S186), MyD88 (S190, S
  • lymphoproliferative element can include an include a first intracellular domain, or a variant or fragment thereof, that includes a signaling domain from the following genes or the respective parts in parentheses, which were the most effective by this statistical analysis (P ⁇ 0.1) across at least 2 libraries: CSF2RB (S057), CSF3R (S062, S063, and S064), IL2RB (S105), IL2RG (S 106), IL6ST (SI 17), IL27RA (S168 and S169), IL31RA (S170), MPL (S186), and MyD88 (S190, S191, S192, S194, S 196, S197, S198).
  • the lymphoproliferative element can include an intracellular domain, or a variant or fragment thereof, that includes a signaling domain from the following genes or the respective parts in parentheses: CSF2RB (S057), CSF3R (S062, S063, and S064), IL2RB (S 105), IL6ST (SI 17), IL27RA (S 168 and S169), IL31RA (S 170), MPL (S 186), and MyD88 (S190, S191, S192, S194, S196, S197, S198).
  • CSF2RB S057)
  • CSF3R S062, S063, and S064
  • IL2RB S 105
  • IL6ST SI 17
  • IL27RA S 168 and S169
  • IL31RA S 170
  • MPL S 186
  • MyD88 S190, S191, S192, S194, S196, S197, S198.
  • the lymphoproliferative element can include an intracellular domain or a fragment thereof that includes a signaling domain from CSF3R, IL6ST, IL27RA, MPL, and MyD88.
  • Each of these genes was represented in a construct that were most effective by this statistical analysis across at least 2 libraries of Example 10, even at a statistical cutoff of P less than 0.05 where the libraries were not fed/unfed pairs from the same replicate.
  • some genes and parts at P3 were significant when an intracellular domain was present at P4, some genes and parts at P3 were significant when a stop codon was present at P4, and some genes and parts at P3 were significant when either an intracellular domain or a stop codon was present at P4.
  • the lymphoproliferative element can include a first intracellular domain, or a variant or fragment thereof, that includes a signaling domain from the following genes or the respective parts in parentheses, which were significant at a p-value less than 0.1 when the P4 part was a second intracellular domain: CSFS2RB (S057), CSF3R (S062 and S064), IL2RB (S105), IL2RG (S 106), IL6ST (S117), IL10RA (S 129), IL17RC, IL17RE (S149), IL27RA (S168 and S 169), IL31RA (S 170), MPL (S 186), MyD88 (S 190, S 192, S 194, S 196, and S197), OSMR (S199), and PRLR (S202).
  • CSFS2RB S057)
  • CSF3R S062 and S064
  • IL2RB S105
  • IL2RG S IL6ST
  • S117 IL
  • the lymphoproliferative element can include a first intracellular domain, or a variant or fragment thereof, that includes a signaling domain from the following genes or the respective parts in parentheses, which were significant at a p-value less than 0.05 when the P4 part was a second intracellular domain: CSFS2RB (S057), CSF3R (S062 and S064), IL2RB (S105), IL2RG (S106), IL6ST (S 117), IL10RA (S129), IL17RE (S 149), IL27RA (S168 and S169), IL31RA (S170), MPL (S186), MyD88 (S 190, S194, and S197), OSMR (S 199), and PRLR (S202).
  • the lymphoproliferative element can include a first intracellular domain, or a variant or fragment thereof, that includes a signaling domain from the following genes or the respective parts in parentheses, which were significant at a p-value less than 0.05 when
  • lymphoproliferative element can include a first intracellular domain, or a variant or fragment thereof, that includes a signaling domain from the following genes or the respective parts in parentheses, which were significant at a p-value less than 0.1 when the P4 part was the stop codon X002: CSF3R (S063), IFNAR1 (S081), IFNGR1 (S084), IL2RB (S 105), IL2RG (S106), IL6ST (S 117), IL12RB2 (S138), IL17RE (S 149), IL18R1, IL22RA1 (S 161), IL27RA (S169), IL31RA (S170), MyD88 (S191, S196, and S198), and PRLR (S202).
  • CSF3R S063R
  • IFNAR1 S081
  • IFNGR1 IFNGR1
  • IL2RB S 105
  • IL2RG S106
  • IL6ST S 117
  • the lymphoproliferative element can include a first intracellular domain, or a variant or fragment thereof, that includes a signaling domain from the following genes or the respective parts in parentheses, which were significant at a p-value less than 0.05 when the P4 part was the stop codon X002: CSF3R (S063), IFNGR1 (S084), IL2RB (S105), IL2RG (S106), IL6ST (SI 17), IL12RB2 (S138), IL17RE (S149), IL18R1, IL22RA1 (S161), IL27RA (S169), IL31RA (S170), MyD88 (S191 and S198), and PRLR (S202).
  • Table 2 provides the identity of the most effective second intracellular signaling domain (P4) parts and corresponding genes when the analysis was performed on all constructs irrespective of the P3 part.
  • the following genes were represented by second intracellular signaling domain parts that were identified in constructs that showed up as most effective by this statistical analysis (P ⁇ 0.1 or p ⁇ 0.05) with part numbers in parenthesis: CD27 (S047), CD40 (S050 and S051), CD79B (S053), TNFRSF4 (S211), TNFRSF8 (S212), TNFRSF9 (S213), and TNFRSF18 (S215).
  • second intracellular domain genes were represented by second intracellular signaling domain parts that were most effective by this statistical analysis (P ⁇ 0.1) across at least 2 libraries with part numbers in parenthesis: CD40 (S050 and S051), TNFRSF8 (S212), and TNFRSF18 (S215).
  • the following second intracellular domain genes were represented by second intracellular signaling domain parts that were most effective by this statistical analysis across at least 2 libraries even at a statistical cutoff of P less than 0.05: CSF2RB (S057), CSF3R (S062, S063, and S064), IL2RB (S105), IL6ST (S117), IL27RA (S168 and S169), IL31RA (S170), MPL (S186), and MyD88 (S190, S191, S192, S194, S196, S197, S198).
  • the lymphoproliferative element can include a second intracellular domain, or a variant or fragment thereof, that includes a signaling domain from the following genes or the respective parts in parentheses, which were significant at a p-value less than 0.1 when the P3 part was a first intracellular domain: CD27 (S047), CD40 (S050 and S051), CD79B (S053), TNFRSF4 (S211), TNFRSF8 (S212), and TNFRSF18 (S215).
  • the lymphoproliferative element can include a second intracellular domain, or a variant or fragment thereof, that includes a signaling domain from the following genes or the respective parts in parentheses, which were significant at a p-value less than 0.05 when the P3 part was a first intracellular domain: CD27 (S047), CD40 (S050 and S051, CD79B (S053), TNFRSF4 (S211), TNFRSF8 (S212), and TNFRSF18 (S215).
  • the lymphoproliferative element can include a second intracellular domain, or a variant or fragment thereof, that includes a signaling domain from the following genes or the respective parts in parentheses, which were significant at a p-value less than 0.1 when the P3 part was the linker X001: CD27 (S047), CD40 (S050), TNFRSF9 (S213), and TNFRSF18.
  • the linker X001 CD27 (S047), CD40 (S050), TNFRSF9 (S213), and TNFRSF18.
  • lymphoproliferative element can include a second intracellular domain, or a variant or fragment thereof, that includes a signaling domain from the following genes or the respective parts in parentheses, which were significant at a p-value less than 0.05 when the P3 part was the linker X001: CD27 (S047), CD40 (S050), TNFRSF9 (S213), and TNFRSF18.
  • Example 10 the constructs were statistically analyzed to determine which P4 parts were the most effective with specific P3 parts, and the results are listed in Table 3.
  • cells transduced with polynucleotides encoding lymphoproliferative elements containing intracellular signaling domains derived from the intracellular signaling domains of the following pairs of genes were enriched at a p-value less than 0.1: CSF2RA and TNFRSF4; CSF2RA and CD28; CSF2RA and TNFRSF8; CSF2RA and CD27; CSFR3 and CD79B; IFNAR2 and TNFRSF14; IL1RAP and CD79A; IL3RA and CD40; IL10RA and CD79B; IL1 IRA and FCGRA2; IL13RA2 and TNFRSF14; IL18RAP and CD3G; IL27RA and FCGRA2; LEPR and CD3G; LIFR and TNFRSF18; MPL and CD40;
  • the lymphoproliferative element comprises intracellular signaling domains derived from the following pairs of genes: CSF2RA and TNFRSF4; CSF2RA and CD28;
  • the polynucleotide can encode intracellular signaling domains derived from the following pairs of genes: CSF2RA and TNFRSF4; CSF2RA and CD28; CSF2RA and TNFRSF8; CSF2RA and CD27; CSFR3 and CD79B; IFNAR2 and TNFRSF14; ILIRAP and CD79A; IL3RA and CD40; ILIORA and CD79B; IL1 IRA and FCGRA2; IL13RA2 and TNFRSF14; IL18RAP and CD3G; IL27RA and FCGRA2; LEPR and CD3G; LIFR and TNFRSF18; MPL and CD40; MPL and CD79B; MPL and TNFRSF4; MPL and CD79A;
  • cells transduced with polynucleotides encoding lymphoproliferative elements containing intracellular signaling domains derived from the intracellular signaling domains of the following pairs of genes were enriched at a p-value less than 0.05: CSF2RA and TNFRSF4; CSF2RA and CD28; CSFR3 and CD79B; IFNAR2 and TNFRSF14; ILIORA and CD79B; IL1 IRA and FCGRA2; IL27RA and FCGRA2; LIFR and TNFRSF18; MPL and CD40; MPL and CD79B; MPL and TNFRSF4; MPL and CD79A; MPL and CD3G; and MyD88 and CD79B, where the intracellular signaling domain from the first gene in each pair is the P3 part and the intracellular signaling domain from the second gene in each pair is the P4 part.
  • the lymphoproliferative element comprises intracellular signaling domains derived from the following pairs of genes: CSF2RA and TNFRSF4; CSF2RA and CD28; CSFR3 and CD79B; IFNAR2 and TNFRSF14; IL10RA and CD79B; IL1 IRA and FCGRA2; IL27RA and FCGRA2; LIFR and TNFRSF18; MPL and CD40; MPL and CD79B; MPL and TNFRSF4; MPL and CD79A; MPL and CD3G; and MyD88 and CD79B.
  • the polynucleotide can encode intracellular signaling domains derived from the following pairs of genes: CSF2RA and TNFRSF4; CSF2RA and CD28; CSFR3 and CD79B; IFNAR2 and TNFRSF14; IL10RA and CD79B; IL1 IRA and FCGRA2; IL27RA and FCGRA2; LIFR and TNFRSF18; MPL and CD40; MPL and CD79B; MPL and TNFRSF4; MPL and CD79A; MPL and CD3G; and MyD88 and CD79B.
  • Example 10 cells transduced with polynucleotides encoding lymphoproliferative elements containing the following pairs of intracellular signaling domains, the amino acid sequences of which are shown in Table 7, were enriched at a p-value less than 0.1 : S058 and S211; S058 and S049; S059 and S212; S059 and S047; S064 and S053; S083 and S214; S101 and S052; S109 and S050; S 129 and S053; S135 and S076; S142 and S214; S 155 and S039; S169 and S076; S177 and S039; S 180 and S216; S186 and S050; S 186 and S051; S186 and S053; S 186 and S211; S186 and S039; S 192 and S053; S 194 and S037; and S195 and S053.
  • the lymphoproliferative element comprises the following pairs of intracellular signaling domains: S058 and S211; S058 and S049; S059 and S212; S059 and S047; S064 and S053; S083 and S214; S101 and S052; S 109 and S050; S129 and S053; S 135 and S076; S142 and S214; S 155 and S039; S 169 and S076; S 177 and S039; S180 and S216; S186 and S050; S186 and S051; S186 and S053; S 186 and S211; S186 and S039; S192 and S053; S194 and S037; and S195 and S053.
  • the polynucleotide comprises the following pairs of intracellular signaling domains: S058 and S211; S058 and S049; S059 and S212; S059 and S047; S064 and S053; S083 and S214; S101 and S052; S109 and S050; S129 and S053; S135 and S076; S142 and S214; S155 and S039; S 169 and S076; S177 and S039; S180 and S216; S186 and S050; S186 and S051; S 186 and S053; S 186 and S211; S186 and S039; S192 and S053; S 194 and S037; and S195 and S053.
  • Example 10 cells transduced with polynucleotides encoding lymphoproliferative elements containing the following pairs of intracellular signaling domains, the amino acid sequences of which are shown in Table 7, were enriched at a p-value less than 0.05: S058 and S211; S058 and S049; S064 and S053; S083 and S214; S129 and S053; S135 and S076; S 169 and S076; S180 and S216; S186 and S050; S 186 and S051; S186 and S053; S186 and S211; S 186 and S039; and S195 and S053.
  • the lymphoproliferative element comprises the following pairs of intracellular signaling domains: S058 and S211; S058 and S049; S064 and S053; S083 and S214; S129 and S053; S 135 and S076; S169 and S076; S 180 and S216; S186 and S050; S 186 and S051; S186 and S053; S 186 and S211; S 186 and S039; and S195 and S053.
  • the polynucleotide comprises the following pairs of intracellular signaling domains: S058 and S211; S058 and S049; S064 and S053; S083 and S214; S 129 and S053; S 135 and S076; S 169 and S076; S180 and S216; S186 and S050; S186 and S051; S186 and S053; S 186 and S211; S186 and S039; and S195 and S053.
  • the lymphoproliferative element comprises a cytokine receptor, or a fragment thereof that includes a signaling domain, that activates a Jak/STAT5 pathway.
  • such lymphoproliferative element can include an intracellular domain of IL21R, IL27R, IL3 IRA, LIFR, and OSMR.
  • chimeric lymphoproliferative elements that include intracellular domains of these genes were found among the candidate chimeric polypeptides that induced the highest magnitude of proliferation in PBMCs cultured in the absence of exogenous cytokines such as IL-2.
  • the lymphoproliferative element can comprise an intracellular domain that is an interleukin or an interleukin receptor and that was a part of a Top Construct identified in Libraries 1A, 1.1 A, 2B, 2. IB, 3A, 3.1 A, 3B, 3. IB, 4B, and/or 4. IB of Example 11 and Example 12 (Tables 8 to 18).
  • the lymphoproliferative element can comprise an intracellular domain that is a cytokine receptor and that was a part of a Top Construct identified in Libraries 1 A, 1.1 A, 2B, 2. IB, 3A, 3.1 A, 3B, 3. IB, 4B, and/or 4.
  • the lymphoproliferative element can comprise an intracellular domain that includes at least one ITAM motif and that was a part of a Top Construct identified in Libraries 1 A, 1.1 A, 2B, 2. IB, 3A, 3.1 A, 3B, 3. IB, 4B, and/or 4. IB of Example 11 and Example 12 (Tables 8 to 18).
  • the lymphoproliferative element can comprise one of the following intracellular domains that were a part of a Top Construct in at least one of Libraries 3A, 3.1A, 3B, 3. IB, 4B, or 4.
  • the lymphoproliferative element can comprise an intracellular domain from IL7R, IL12RB1, IL15RA, or IL27RA, which were present in constructs that showed particularly noteworthy enrichments (i.e. elicited highest magnitude of proliferation) in an initial screen and a repeated screen as detailed in Example 11 and Example 12 (Tables 20 to 24).
  • the lymphoproliferative element can comprise an intracellular domain from the cytokine receptors CD27, CD40, CRLF2, CSF2RA, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR2, IL1R1, IL1RL1, IL2RA, IL2RG, IL3RA, IL5RA, IL6R, IL7R, IL9R, IL10RB, IL11RA, IL12RB1, IL13RA1, IL13RA2, IL15RA, IL17RB, IL18R1, IL18RAP, IL20RB, IL22RA1, IL27RA, IL31RA, LEPR, MPL, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18, which were present in constructs that showed particularly noteworthy enrichments in an initial screen and a repeated screen as detailed in Example 11 and Example 12 (
  • the lymphoproliferative element comprises an intracellular domain from CD3D, CD3E, CD3G, CD79A, CD79B, FCER1G, FCGR2A, or FCGR2C, which include at least one ITAM motif and were present in constructs that showed particularly noteworthy enrichments in an initial screen and a repeated screen as detailed in Example 11 and Example 12 (Tables 20 to 24).
  • the lymphoproliferative element in this paragraph which were shown in Example 11 and Example 12 to be active in constructs with only a single intracellular domain, can be the intracellular domain of a lymphoproliferative element with either two or more intracellular domains, or in illustrative embodiments a single intracellular domain, i.e. the lymphoproliferative element does not comprise two or more intracellular domains.
  • the intracellular domain in a lymphoproliferative element comprises a domain from CD40, CRLF2, CSF2RA, CSF3R, EPOR, FCGR2A, IFNAR2, IFNGR2, IL1R1, IL3RA, IL7R, IL10RB, IL1 IRA, IL12RB1, IL13RA2, IL18RAP, IL3 IRA, MPL, MYD88, TNFRSF14, or TNFRSF18, which were present in constructs that showed particularly noteworthy enrichments in an initial screen and a repeated screen as detailed in Example 12 as single intracellular signaling domains (Tables 23 and 24).
  • the intracellular domain in a lymphoproliferative element comprises a domain from IL7R or IL12RB1, which were present in constructs that showed particularly noteworthy enrichments in an initial screen and a repeated screen as detailed in Example 12 (Tables 23 and 24).
  • the intracellular domain in a lymphoproliferative element with a single intracellular domain can be a cytokine receptor.
  • the cytokine receptor in a lymphoproliferative element with a single intracellular domain comprises a domain from CD40, CRLF2, CSF2RA, CSF3R, EPOR, IFNAR2, IFNGR2, IL1R1, IL3RA, IL7R, IL10RB, IL11RA, IL12RB1, IL13RA2, IL18RAP, IL31RA, MPL, TNFRSF14, or TNFRSF18, which were present in constructs that showed particularly noteworthy enrichments in an initial screen and a repeated screen as detailed in Example 12 (Tables 23 and 24).
  • the intracellular domain in a lymphoproliferative element can include at least one ITAM motif.
  • the intracellular domain in a lymphoproliferative element that includes at least one ITAM motif comprises a domain from FCGR2A, which was present in constructs that showed particularly noteworthy enrichments in an initial screen and a repeated screen as detailed in Example 12 (Table 23).
  • the lymphoproliferative element can comprise a costimulatory domain from CD27, CD28, OX40 (also referred to as TNFRSF4), GITR (also referred to as TNFRSF18), or HVEM (also referred to as TNFRSF14), which were present in constructs that showed particularly noteworthy enrichments in an initial screen and a repeated screen as detailed in Example 11 and Example 12 (Tables 20 to 24).
  • a lymphoproliferative element comprising a costimulatory domain from OX40 does not comprise an intracellular domain from CD3Z, CD28, 4-1BB, ICOS, CD27, BTLA, CD30, GITR, or HVEM.
  • a lymphoproliferative element comprising a costimulatory domain from GITR does not comprise an intracellular domain from CD3Z, CD28, 4- IBB, ICOS, CD27, BTLA, CD30, or HVEM.
  • a lymphoproliferative element comprising a costimulatory domain from CD28 does not comprise an intracellular domain from CD3Z, 4-1BB, ICOS, CD27, BTLA, CD30, or HVEM.
  • a lymphoproliferative element comprising a costimulatory domain from OX40, CD3Z, CD28, 4-1BB, ICOS, CD27, BTLA, CD30, GITR, or HVEM does not comprise a coiled-coil spacer domain N-terminal of the transmembrane domain.
  • a lymphoproliferative element comprising a costimulatory domain from GITR does not comprise an intracellular domain from CD3Z that is N-terminal of the costimulatory domain of GITR.
  • the lymphoproliferative element can be one of the constructs M024-S 190- S047, M025-S050-S197, M036-S170-S047, M012-S045-S048, M049-S 194-S064, M025-S190-S050, M025-S190-S05, E013-T041-S186-S051, E013-T028-S 186-S051, E014-T015-S186-S051, E011-T016- S 186-S050, E011-T073-S186-S050, or E013-T011-S186-S211, all of which stimulated proliferation of resting lymphocytes after transduction as shown in Example 13 (FIGs.
  • lymphoproliferative elements comprise an extracellular domain from CSF3R, IL3RA, ICOS, CRLF, CSF2RA, LIFR, or CD40; a first intracellular domain from MyD88, CD40, or MPL, and/or a second intracellular domain from CD27 or MyD88.
  • the lymphoproliferative element can be the construct E013-T041-S186- S051, which stimulated proliferation of resting lymphocytes after transduction with a replication incompetent recombinant retroviral particle displaying UCHTlscFvFc-GPI as shown and analyzed in Example 16 ( FIGs. 22A and 22B).
  • the lymphoproliferative element is IL7- IL7RA-IL2RB, as shown and analyzed in Example 16.
  • TNFRSF4, TNFRSF9, or TNFRSF18 showed particularly noteworthy enrichments in both Libraries 4B and 4. IB (Table 24).
  • the lymphoproliferative element comprises an intracellular domain of CD40, MPL and IL2Rb, which are demonstrated in the Examples herein to promote PBMC proliferation.
  • the lymphoproliferative element can be other than a cytokine receptor.
  • the lymphoproliferative element other than a cytokine receptor can include an intracellular signaling domain from CD2, CD3D, CD3G, CD3Z, CD4, CD8RA, CD8RB, CD28, CD79A, CD79B, FCER1G, FCGR2A, FCGR2C, or ICOS.
  • lymphoproliferative elements that include these recited genes are provided in Example 11 and Example 12, and the tables cited therein.
  • CLE is other than IL-15 tethered to the IL-2/IL-15 receptor.
  • expression of the lymphoproliferative element is induced by and can even dependent on binding of a compound to a control element (as discussed in WO2017/165245A2, WO2018/009923A1, and WO2018/161064A1), which in non-limiting embodiments is a riboswitch.
  • a control element as discussed in WO2017/165245A2, WO2018/009923A1, and WO2018/161064A1
  • the lymphoproliferative element is expressed from a promoter active in a T cell and/or an NK cell.
  • promoters are known that are active in T cells and/or NK cells and can be used to express a first engineered signaling polypeptide or a second engineered signaling polypeptide, or any component thereof.
  • a promoter is not active in a packaging cell line, such as the packaging lines disclosed herein.
  • the promoter is the EFla promoter or the murine stem cell virus (MSCV) promoter (Jones et al., Human Gene Therapy (2009) 20: 630-40).
  • the promoter is the T cell specific CD3 zeta promoter.
  • the lymphoproliferative element is microenvironment restricted.
  • the lymphoproliferative element can be a mutated receptor that binds its respective cytokine differentially in aberrant versus physiological conditions.
  • an IL-7R that can bind IL7 more strongly in a tumor environment than in a normal physiological environment can be used.
  • the lymphoproliferative element is fused to a recognition or elimination domain.
  • recognition or elimination domains are disclosed in more detail herein.
  • Such fusion provides the advantage, especially when a truncated or other mutated lymphoproliferative element is used, of requiring less polynucleotides in the retroviral genome. This is important in illustrative embodiments provided herein, because it helps to permit more nucleic acids encoding functional elements to be included in the retroviral genome and because it adds a mechanism by which cells expressing the lymphoproliferative element can be killed if their proliferation is no longer wanted or is detrimental to an organism.
  • a lymphoproliferative element including a CLE, comprises an intracellular activating domain as disclosed hereinabove.
  • a lymphoproliferative element including a CLE, comprises an intracellular activating domain as disclosed hereinabove.
  • lymphoproliferative element is a CLE comprising an intracellular activating domain comprising an ITAM-containing domain
  • the CLE can comprise an intracellular activating domain having at least 80%, 90%, 95%, 98%, or 100% sequence identity to the CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP 12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70 domains provided herein wherein the CLE does not comprise an ASTR.
  • the intracellular activating domain is an ITAM-containing domain from CD3D, CD3G, CD3Z, CD79A, CD79B,
  • FCER1G FCGR2A, or FCGR2C.
  • CLEs comprising these intracellular activating domains are demonstrated in Example 11 and Example 12 herein, and associated Tables 19 to 24, as being effective at promoting proliferation of PBMCs ex vivo in cultures in the absence of exogenous cytokines such as exogenous IL-2.
  • CLEs comprising an intracellular domain from CD3D, CD3G, CD3Z, CD79A, FCER1G are provided herein.
  • one or more domains of a lymphoproliferative element is fused to a modulatory domain, such as a co-stimulatory domain, and/or an intracellular activating domain of a CAR.
  • a modulatory domain such as a co-stimulatory domain
  • one or more intracellular domains of a lymphoproliferative element can be part of the same polypeptide as a CAR or can be fused and optionally functionally connected to some components of CARs.
  • an engineered signaling polypeptide can include an ASTR, an intracellular activation domain (such as a CD3 zeta signaling domain), a co-stimulatory domain, and a lymphoproliferative domain. Further details regarding co-stimulatory domains, intracellular activating domains, ASTRs and other CAR domains, are disclosed elsewhere herein.
  • a T cell and/or NK cell survival element is introduced into a resting T cell and/or resting NK cell, typically by transducing the resting T cell and/or resting NK cell with a replication incompetent recombinant retroviral particle whose genome encodes the T cell and/or NK cell survival element as part of an engineered signaling polypeptide.
  • a lymphoproliferative element is also a T cell and/or NK cell survival element. As discussed above, some of the lymphoproliferative elements not only promote proliferation, but they promote cell survival as well. In some embodiments, the T cell and/or NK survival cell motif is not a lymphoproliferative element.
  • the T cell and/or NK cell survival motif can be a CD28 T cell survival motif or a CD 137 cell survival motif.
  • T cell survival motifs can be found on engineered signaling polypeptides that include an ASTR, such as an scFv.
  • the T cell survival motif is a CD28 T cell survival motif or a CD137 motif connected to an scFv through a CD8a transmembrane domain or a CD28 transmembrane domain.
  • said intracellular signaling domain comprises a polypeptide sequence comprising an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • said polypeptide sequence is a CD3 ⁇ signaling domain.
  • the lymphoproliferative element is not a polypeptide, but rather comprises an inhibitory RNA.
  • methods, uses, compositions, and products of processes according to any aspect herein include both a lymphoproliferative element comprising an inhibitory RNA and a lymphoproliferative element that is an engineered signaling polypeptide.
  • a lymphoproliferative element is an inhibitory RNA
  • that inhibitory RNA can be a miRNA that stimulates the STAT5 pathway typically by potentiating activation of STAT5 by degrading or causing down- regulation of a negative regulator in the SOCS pathway.
  • the miRNA is directed to mRNA encoding proteins that affect proliferation such as but not limited to ABCG1, SOCS 1, TGFbR2, SMAD2, cCBL, and PD1.
  • inhibitory RNA e.g. miRNAs
  • inclusion of introns in transcription units are believed to result in higher expression and/or stability of transcripts.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNAs in illustrative embodiments between 2 and 5, for example 4 miRNAs, one or more of which each bind nucleic acids encoding one or more of ABCG1, SOCS1, TGFbR2, SMAD2, cCBL, and PD1, can be included in the recombinant retroviral genome and delivered to a target cell, for example T cells and or NK cells, using methods provided herein.
  • 1, 2, 3, or 4 miRNAs can be delivered in a single intron such as the EFla intron.
  • ABCG1 is an ATP-binding cassette transporter that negatively regulates thymocyte and peripheral lymphocyte proliferation (Armstrong et al. 2010. J Immunol 184(1): 173-183).
  • SOCS 1 is a member of the SOCS (Suppressor of cytokine signaling) family of negative regulators of cytokine signal transduction that inhibit the Jak/Stat pathway such as STAT5.
  • SOCS 1 is also known as JAB (Janus Kinase binding protein), SSI-1 (Stat-induced Stat inhibitor- 1), and ⁇ 3 (Tec- interacting protein).
  • TGFbR2 is a member of the serine/threonine protein kinase family that binds TGF- ⁇ , forming a complex that phosphorylates proteins that then enter the nucleus and regulate transcription of genes related to proliferation.
  • SMAD2 mediates the signal of the transforming growth factor (TGF)- and regulates multiple cellular processes, such as cell proliferation, apoptosis, and differentiation.
  • TGF transforming growth factor
  • cCBL is an E3 ubiquitin ligase that inhibits TCR signaling by dephosphorylation and inactivation of ZAP-70 and through internalization of the TCR.
  • PD1 (CD279) is a cell surface receptor expressed on T cells and ProB cells. PD-1 binds two ligands, PD-L1 and PD-L2. Signaling through PD-1 functions to prevent activation of cells.
  • the lymphoproliferative element is a polypeptide comprising the intracellular region of any of the genes of Table 19.
  • the lymphoproliferative element comprises or is an intracellular domain identified in the chimeric polypeptides in Table 19.
  • the lymphoproliferative element can be a polypeptide that is a chimeric polypeptide (See CLEs below), or is not a CLE but comprises or is an intracellular domain of a gene identified in the P3 (first intracellular domain) position of Table 19.
  • Table 19 identifies CLE constructs that promoted cell proliferation of PBMCs between day 7 and the last day where a second intracellular domain was not present on the construct.
  • the lymphoproliferative element is a chimeric polypeptide (i.e. a CLE - see below) that includes a combination of transmembrane domain and intracellular domain, with or without an extracellular domain comprising a dimerizing motif.
  • the lymphoproliferative element comprises MPL, or is MPL, or a variant and/or fragment thereof, including a variant and/or fragment that includes at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of the intracellular domain of MPL, with or without a transmembrane and/or extracellular domain of MPL, and/or has at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to the intracellular domain of MPL, with or without a transmembrane and/or extracellular domain of MPL, wherein the variant and/or fragment retains the ability to promote cell proliferation of PBMCs, and in some embodiments T cells.
  • the lymphoproliferative element comprises an intracellular domain of MPL, or a variant or fragment thereof that includes at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of the intracellular domain of MPL, and the lymphoproliferative element does not comprise a transmembrane domain of MPL.
  • the lymphoproliferative element comprises an intracellular domain of MPL, or a variant or fragment thereof that includes at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of the intracellular domain of MPL, and the lymphoproliferative element does not comprise a transmembrane domain of MPL.
  • the lymphoproliferative element comprises an intracellular domain of MPL, or a variant or fragment thereof that includes at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of the intracellular domain of MPL, and the lymphoproliferative element does not comprise a transmembrane domain of M
  • lymphoproliferative element comprises an intracellular domain of MPL, or a variant or fragment thereof that includes at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of the intracellular domain of MPL, and the lymphoproliferative element comprises a transmembrane domain of MPL.
  • an MPL fragment included in the compositions and methods herein has and/or retains a JAK-2 binding domain.
  • an MPL fragment included herein has or retains the ability to activate a STAT.
  • the full intracellular domain of MPL is SEQ ID NO:491 (part S186 in Tables 8 to 19).
  • MPL is the receptor for tbrom.bopoi.etin.
  • cytokines such as thrombopoietii and EPO are referred to in the literature and herein as either a hormone or a cytokine.
  • chimeric polypeptides that are chimeric lymphoproliferative elements (CLEs), as well as isolated polynucleotides and nucleic acid sequences that encode the same.
  • CLEs can include any of the domains and/or domains derived from specific genes discussed in the section.
  • isolated polynucleotides and nucleic acid sequences encoding CLEs can encode as part of the CLE any of the domains and/or domains derived from specific genes discussed in this section. Exemplary CLEs are illustrated in FIGs. ISIS.
  • CLEs herein promote cell proliferation of T cells and/or NK cells and can optionally also promote survival of T cells and/or NK cells. Some CLEs promote proliferation and optionally also survival of other types of PBMCs, for example B cells.
  • CLE CAR polynucleotide embodiments for drafting convenience.
  • CLEs can include a transmembrane domain and a first intracellular domain.
  • CLEs include an extracellular domain and/or a second intracellular domain (and in further embodiments, third, fourth, etc. intracellular domains).
  • Chimeric polypeptides herein are chimeric because at least one domain is from a different polypeptide than at least one of the other domains and such chimera are not found naturally in an organism without human intervention.
  • Some CLEs include a ligand for a receptor and some CLEs do not include a ligand for a receptor.
  • such CLEs were designed to promote proliferation and optionally cell survival in B cells, NK cells, and/or T cells in a constitutive manner (i.e. without the requirement of ligand binding for activation). None of the CLEs are found in nature, and many of the CLEs have components that are not usually expressed in B cells, T cells, and/or NK cells in vivo and/or some candidate CLEs are not generally known to specifically promote cell proliferation and/or cell survival signaling in B cells, T cells, and/or NK cells. For example, MPL is usually not expressed in B cells, T cells and/or NK cells.
  • CLEs were identified by screening a large number of candidate chimeric polypeptides as set out in Example 11 and Example 12, that promoted PBMC cell proliferation.
  • Such CLEs help to meet the long-felt need of identifying mechanisms to stimulate proliferation and optionally survival as well, of B cells, T cells, and/or NK cells, such as would be beneficial for important clinically-relevant technologies, such as CAR-T and T-cells that are genetically engineered to express a defined TCR.
  • CAR-T and T-cells that are genetically engineered to express a defined TCR.
  • it is believed that some CLEs will provide a T cell and/or NK cell the ability to expand in vivo without the need for lymphodepleting the host.
  • Chimeric lymphoproliferative elements provided herein, and isolated polynucleotides and nucleic acids encoding the same can be included in any aspect provided herein that includes a
  • a first engineered signaling polypeptide can be, or can include a CLE in aspects provided herein that include one or more transcriptional units that encode a first engineered signaling polypeptide regulated by a control element.
  • a CLE in aspects provided herein that include one or more transcriptional units that encode a first engineered signaling polypeptide regulated by a control element.
  • separate aspects of the invention are provided that specifically include a CLE.
  • such aspects include isolated chimeric lymphoproliferative polypeptides, isolated polynucleotides and nucleic acid sequences encoding the same, and vectors, including plasmids, viral, and retroviral vectors including such nucleic acid sequences or isolated polynucleotides.
  • Such aspects further include, methods for transducing or transfecting PBMCs, such as B cells, or especially T cells and NK cells, with the isolated polynucleotides and vectors comprising the same.
  • PBMCs such as B cells, or especially T cells and NK cells
  • Such cells can be isolated, unaltered cells, or they can be cells that have been modified such as cells that are genetically modified, such as to express a defined TCR, or CAR-T cells.
  • Lymphoproliferative elements typically include a transmembrane domain.
  • the transmembrane domain can have 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to any one of the transmembrane domains from the following genes and representative sequences: CD8 beta (SEQ ID NO:47), CD4 (SEQ ID NO:48), CD3 zeta (SEQ ID NO:49), CD28 (SEQ ID NO:50), CD134 (SEQ ID NO:51), CD7 (SEQ ID NO:51), CD2 (SEQ ID NO:322), CD3D (SEQ ID NO:323), CD3E (SEQ ID NO:324), CD3G (SEQ ID NO:325), CD3Z (SEQ ID NO:326), CD4 (SEQ ID NO:327), CD8A (SEQ ID NO:328), CD8B (SEQ ID NO:329), CD27 (SEQ ID NO:330), CD28 (SEQ ID NO:47), CD4
  • TM domains suitable for use in any engineered signaling polypeptide include, but are not limited to, constitutively active cytokine receptors, the TM domain from LMP1, and TM domains from type 1 TM proteins comprising a dimerizing motif, as discussed in more detail herein.
  • the transmembrane domain can be a Type I growth factor receptor, a hormone receptor, a T cell receptor, or a TNF-family receptor.
  • CLEs include both an extracellular portion and a transmembrane portion that is from the same protein, in illustrative embodiments the same receptor, either of which in illustrative embodiments is a mutant, thus forming an extracellular and transmembrane domain.
  • These domains can be from a cytokine receptor, or a mutant thereof, or a hormone receptor, or a mutant thereof in some embodiments that have been reported to be constitutively active when expressed at least in some cell types.
  • such extracellular and transmembrane domains do not include a ligand binding region.
  • a mutation in at least some extracellular - transmembrane domains of CLEs provided herein are responsible for signaling of the CLE in the absence of ligand, by bringing activating chains together that are not normally together, or by changing the confirmation of a linked transmembrane and/or intracellular domain.
  • One aspect that utilizes such transmembrane domains of receptor mutants is an isolated polynucleotide comprising one or more nucleic acid sequences, wherein:
  • a first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising in amino to carboxy orientation
  • a first intracellular domain selected from an intracellular domain of a gene having a first intracellular domain and optionally a second domain of a selected polypeptide identified in Tables 8 to 12, wherein said chimeric polypeptide promotes cell proliferation of B cells, T cells, and/or NK cells.
  • the extracellular region of such extracellular and transmembrane domains in these embodiments is typically long enough to form a linker, in illustrative embodiments a flexible linker between a transmembrane domain and another functional peptide region, such as a clearance domain, that in some embodiments, is linked to the amino terminus of the extracellular region.
  • the extracellular region when present to form an extracellular and transmembrane domain can be between 1 and 1000 amino acids, and is typically between 4 and 400 amino acids, between 4 and 200 amino acids, between 4 and 100 amino acids, between 4 and 50 amino acids, between 4 and 25 amino acids or between 4 and 20 amino acids in length.
  • the extracellular region is GGGS for an extracellular and transmembrane domain of this aspect of the invention.
  • transmembrane domain whether as part of embodiments that include an extracellular and transmembrane domain as one part, for example as shown in Libraries 1A, 1.1 A, 1.1B, 2B and 2.
  • IB of Example 11 or as part of embodiments that include an extracellular dimerizing motif, as shown in Libraries 3A, 3B, 3.1 A, 3. IB, 4B, and 4.
  • IB of Example 12 are typically at least long enough to cross a plasma membrane.
  • transmembrane domains or transmembrane regions of extracellular and transmembrane domains can be between 10 and 250 amino acids, and are more typically at least 15 amino acids in length, and can be for example between 15 and 100, 15 and 75, 15 and 50, 15 and 40, or 15 and 30 amino acids.
  • Exemplary extracellular and transmembrane domains for CLEs of embodiments that include such domains are extracellular regions, typically less than 30 amino acids of the membrane-proximal extracellular domains along with transmembrane domains from mutant receptors that have been reported to be constitutive, that is not require ligand binding for activation of an associated intracellular domain.
  • extracellular and transmembrane domains include IL7RA Ins PPCL, CRLF2 F232C, CSF2RB V449E, CSF3R T640N, EPOR L251C I252C, GHR E260C I270C, IL27RA F523C, and MPL S505N. Further non-limiting examples of such extracellular and transmembrane domains are provided in Table 7 and exemplified in Example 11 and the corresponding tables.
  • the extracellular and transmembrane domain does not comprise more than 10, 20, 25 30 or 50 consecutive amino acids that are identical in sequence to a portion of the extracellular and/or transmembrane domain of IL7RA, or a mutant thereof. In some embodiments, the extracellular and transmembrane domain is other than IL7RA Ins PPCL. In some embodiments, the extracellular and transmembrane does not comprise more than 10, 20, 25, 30, or 50 consecutive amino acids that are identical in sequence to a portion of the extracellular and/or transmembrane domain of IL15R.
  • transmembrane domains in illustrative embodiments are from type I transmembrane proteins.
  • an isolated polynucleotide comprising one or more nucleic acid sequences, wherein:
  • a first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising in amino to carboxy orientation
  • transmembrane domain from a type I transmembrane protein
  • said chimeric polypeptide promotes cell proliferation of PBMCs, for example B cells and/or NK cells, and/or in illustrative embodiments T cells.
  • PBMCs for example B cells and/or NK cells, and/or in illustrative embodiments T cells.
  • such chimeric polypeptides are capable of promoting cell proliferation of PBMCs in the absence of exposure of the PBMCs to exogenous cytokines such as IL-2, IL-15, or IL-7 during culturing, for example in the absence of adding IL-2 to culture media of PBMCs (e.g. B cells, T cells, or NK cells) that express a nucleic acid encoding the chimeric polypeptide.
  • cytokines such as IL-2, IL-15, or IL-7
  • IL-2 can be added during transduction of PBMCs, but not in subsequent culturing.
  • chimeric polypeptides disclosed herein are lymphoproliferative elements because they are capable of promoting cell proliferation and optionally cell survival of PBMCs, and in illustrative embodiments, T cells, without adding IL-2 during culturing of PBMCs (e.g. T cells) after day 1, 2, 3, 4, 5, or 7 of culturing optionally after transduction of the PBMCs with a nucleic acid encoding the chimeric lymphoproliferative element.
  • Example 13 provides another example of a method that can be used to identify and analyze lymphoproliferative elements and that sets out a property or capability of lymphoproliferative elements.
  • Such analysis can include, for example, measuring the relative lymphoproliferative activity of such lymphoproliferative elements, for example by analyzing the magnitude of enrichment provided by genetically modified lymphocytes expressing such lymphoproliferative elements compared to control lymphocytes that do not express such lymphoproliferative elements.
  • the first nucleic acid sequence further encodes an extracellular domain, and in illustrative embodiments, the extracellular domain comprises a dimerizing motif.
  • the extracellular domain comprises a leucine zipper.
  • the leucine zipper is from a jun polypeptide, for example c-jun.
  • the c-jun polypeptide is the c-jun polypeptide region of ECD-11.
  • the transmembrane domain is a type I transmembrane protein
  • the transmembrane domain can be a Type I growth factor receptor, a hormone receptor, a T cell receptor, or a TNF-family receptor.
  • the chimeric polypeptide comprises an extracellular domain and wherein the extracellular domain comprises a dimerizing motif
  • the transmembrane domain can be a Type I cytokine receptor, a hormone receptor, a T cell receptor, or a TNF-family receptor.
  • transmembrane domains include any transmembrane domain that was used in Example 12.
  • the transmembrane domain is from CD4, CD8RB, CD40, CRLF2, CSF2RA, CSF3R, EPOR, FCGR2C, GHR, ICOS, IFNAR1, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL2RG, IL3RA, IL5RA, IL6ST, IL7RA, IL10RB, IL1 IRA, IL13RA2, IL17RA, IL17RB, IL17RC, IL17RE, IL18R1, IL18RAP, IL20RA, IL22RA1, IL3 IRA, LEPR, PRLR, and TNFRSF8, or mutants thereof that are known to promote signaling activity in certain cell types if such mutants are present in the constructs provided in Example 12.
  • the transmembrane domain is from CD40, ICOS, FCGR2C, PRLR, IL3RA, or IL6ST.
  • the transmembrane is the specific transmembrane domain provided for these genes in the constructs of any one or more of Tables 13 to 18, or a transmembrane domain from any of the constructs in the first 50, 25 or 10 listed constructs of those tables.
  • the transmembrane domain is from CD40 or ICOS, as non-limiting examples the transmembrane domains for these genes provided in Table 7, or a fragment thereof that retains the ability to be transmembrane, and/or mutants thereof that are known to promote constitutive signaling activity in certain cell types.
  • transmembrane domains from this aspect are shown as P2 in Tables 13 to 18.
  • Example 11 and Example 12 cells were transduced with replication incompetent recombinant retroviral particles containing a library and then either fed PBMCs, or not fed PBMCs, as discussed in Example 11 and Example 12.
  • Screens where the cells were fed PBMCs are identified with an "A" after the library number, e.g. Library 1A and screens where the cells were not fed PBMCs are identified with a "B" after the library number, e.g. Library 1.1B.
  • screens identified as libraries containing ".1", e.g. Library 1.1A were performed in an identical manner to screens of the corresponding library without the ".1" e.g. Library 1.1A was a repeat screen of Library 1A.
  • a transmembrane domain in a CLE provided herein is a transmembrane domain from CD40 and ICOS.
  • Examples of transmembrane domains or portions and/or mutants thereof, that were present in constructs that promoted cell proliferation for Libraries 3 A, 3B, 3. 1A, 3. IB, 4B, and 4. IB are provided in the tables provided in Example 12.
  • a chimeric polypeptide comprises a transmembrane domain identified in any of the constructs shown in Tables 13 to 18 other than a transmembrane domain mutant V449E of CSF2RB.
  • the extracellular and transmembrane domain is the viral protein LMP1, or a mutant and/or fragment thereof.
  • LMP 1 is a multispan transmembrane protein that is known to activate cell signaling independent of ligand when targeted to lipid rafts or when fused to CD40 (Kaykas et al. EMBO J. 20: 2641 (2001)).
  • a fragment of LMP1 is typically long enough to span a plasma membrane and to activate a linked intracellular domain(s).
  • the LMP1 can be between 15 and 386, 15 and 200, 15 and 150, 15 and 100, 18 and 50, 18 and 30, 20 and 200, 20 and 150, 20 and 50, 20 and 30, 20 and 100, 20 and 40, or 20 and 25 amino acids.
  • the extracellular domain includes at least 1, but typically at least 4 amino acids and is typically linked to another functional polypeptide, such as a clearance domain, for example, an eTag.
  • the lymphoproliferative element comprises an LMP1 transmembrane domain.
  • the lymphoproliferative element comprises an LMP 1 transmembrane domain and the one or more intracellular domains do not comprise an intracellular domain from TNFRSF proteins (i.e. CD40, 4- IBB, RANK, TACI, OX40, CD27, GITR, LTR, and BAFFR), TLR1 to TLR13, integrins, FcyRIII, Dectinl, Dectin2, NODI, NOD2, CD 16, IL-2R, Type I II interferon receptor, chemokine receptors such as CCR5 and CCR7, G-protein coupled receptors, TREM1, CD79A, CD79B, Ig-alpha, IPS-1, MyD88, RIG- 1, MDA5, CD3Z, MyD88ATIR, TRIF, TRAM, TIRAP, MAL, BTK, RTK, RAC1, SYK, NALP3 (NLRP3), NALP1, CARD9, DAI, IPAG, STING, Zap70, or L
  • the extracellular domain includes a dimerizing moiety.
  • dimerizing moieties are capable of homodimerizing.
  • dimerizing moieties can provide an activating function on intracellular domains connected thereto via transmembrane domains. Such activation can be provided, for example, upon dimerization of a dimerizing moiety, which can cause a change in orientation of intracellular domains connected thereto via a transmembrane domain, or which can cause intracellular domains to come into proximity.
  • An extracellular domain with a dimerizing moiety can also serve a function of connecting a recognition tag to a cell expressing a CLE.
  • the dimerizing agent can be located intracellularly rather than extracellularly. In some embodiments, more than one or multiples of dimerizing domains can be used.
  • Extracellular domains for embodiments where extracellular domains have a dimerizing motif are long enough to form dimers, such as leucine zipper dimers.
  • extracellular domains that include a dimerizing moiety can be from 15 to 100, 20 to 50, 30 to 45, or 35 to 40 amino acids, of in illustrative embodiments is a c-Jun portion of a c-Jun extracellular domain provided in Table 7.
  • Extracellular domains of polypeptides that include a dimerizing moiety may not retain other functionalities.
  • such leucine zippers are capable of forming dimers because they retain a motif of leucines spaced 7 residues apart along an alpha helix.
  • leucine zipper moieties of certain embodiments of CLEs provided herein may or may not retain their DNA binding function.
  • One exemplary extracellular domain of this type is a leucine zipper domain from a Jun protein, such as c-Jun.
  • an extracellular domain can be a variant of c-Jun found in NM_002228_3 (Table 7). Such an extracellular domain was used in the constructs discussed in Example 12.
  • a spacer of between 1 and 4 alanine residues can be included in CLEs between the extracellular domain that has a dimerizing moiety, and the transmembrane domain. Not to be limited by theory, it is believed that the alanine spacer affects signaling of intracellular domains connected to the leucine zipper extracellular region via the transmembrane domain, by changing the orientation of the intracellular domains.
  • the first and second intracellular domains of CLEs provided herein are intracellular signaling domains of genes that are known in at least some cell types, to promote proliferation, survival (anti- apoptotic), and/or provide a co-stimulatory signal that enhances a differentiation state, proliferative potential or resistance to cell death.
  • these intracellular domains can be intracellular domains from lymphoproliferative elements and co-stimulatory domains provided herein.
  • Some of the intracellular domains of candidate chimeric polypeptides are known to activate JAK1/JAK2 signaling, JAK3, STATl, STAT2, STAT3, STAT4, STAT5, and STAT6.
  • Intracellular domains from IFNAR1, IFNGR1, IFNLR1, IL2RB, IL4R, IL5RB, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL21R, IL27R, IL31RA, LIFR, and OSMR are known in the art to activate JAK1 signaling.
  • Intracellular domains from CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNGR2, IL3RA, IL5RA, IL6ST, IL20RA, IL20RB, IL23R, IL27R, LEPR, MPL, and PRLR are known in the art to activate JAK2.
  • Intracellular domains from IL2RG are known in the art to activate JAK3.
  • Intracellular domains from GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IL2RB, IL2RG, IL4R, IL5RA, IL5RB, IL7RA, IL9R, IL21R, IL22RA1, IL3 IRA, LIFR, MPL, and OSMR are known in the art to activate STATl .
  • Intracellular domains from IFNAR1 and IFNAR2 are known in the art to activate STAT2.
  • Intracellular domains from GHR, IL2RB, IL2RG, IL6R, IL7RA, IL9R, IL10RA, ILIORB, IL21R, IL22RA1, IL23R, IL27R, IL31RA, LEPR, LIFR, MPL, and OSMR are known in the art to activate STAT3.
  • Intracellular domains from IL12RBl are known in the art to activate STAT4.
  • Intracellular domains from CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL5RB, IL7RA, IL9R, IL15RA, IL20RA, IL20RB, IL21R, IL22RA1, IL31RA, LIFR, MPL, OSMR, and PRLR are known in the art to activate STAT5.
  • Intracellular domains from IL4R and OSMR are known in the art to activate STAT6.
  • genes and intracellular domains thereof that are found in a first intracellular domain are the same as the second intracellular domain, except that if the first and second intracellular domain are identical, then at least one, and typically both the transmembrane domain and the extracellular domain are not from the same gene.
  • Exemplary intracellular domains include those from the constructs listed in Tables 8 to 12.
  • Exemplary intracellular domains from these genes that were empirically determined to be active in the experiment of Example 11, are provided in the first intracellular domain (P3) and second intracellular domain (P4) locations of tables provided in Example 11 and as further listed in this section below.
  • Illustrative intracellular domains identified in the top hits for the Example 11 screen included CD40, CSF2RA, IFNAR1, IL1RAP, IL4R, IL6ST, IL11RA, IL12RB2, IL17RA, IL17RD, IL17RE, IL18R1, IL21R, IL23R, MPL, and MyD88.
  • Illustrative intracellular domains identified in the top hits for the Example 12 screen included CD40, LEPR, MyD88, IFNAR2, MPL, IL18R1, IL13RA2, IL10RB, IL23R, or CSF2RA.
  • the first intracellular domain is MPL, LEPR, MyD88, or IFNAR2.
  • non-limiting exemplary second intracellular domain (P4) domains are those of genes that are linked to the corresponding MPL, LEPR, MyD88, IFNAR2, CD40, CD79B, or CD27 first intracellular domain (P3) provided in Tables 13 to 18, including in some non-limiting examples the first and/or second intracellular domains for those genes provided in these tables.
  • non-limiting exemplary second intracellular (P4) domains are those of genes that are linked to the corresponding MPL, LEPR, MYD88, IFNAR2, CD40, CD79B, or CD27 first intracellular domain in tables provided in Example 12, including as non-limiting examples, the first and/or second intracellular domains of those genes provided in these tables.
  • the chimeric lymphoproliferative element can be any of the polypeptides in Tables 19 to 23 of Example 11 and Example 12.
  • the second intracellular domain is from CD3D, CD3G, CD27, CD40, CD79A, CD79B, FCER1G, FCGRA2, ICOS, TNFRSF4, and TNFRSF8, or mutants thereof that are known to promote signaling activity in certain cell types if such mutants are present in the constructs provided in Example 12,
  • the second intracellular domain is CD40, CD79B, TNFRSF4, TNFRSF9, TNFRSF14, FCGRA2, CD3G, or CD27, including in illustrative examples an intracellular domain of CD40, CD79B, and CD27.
  • non-limiting exemplary first intracellular domains are those of genes that are linked to CD40, CD79B, TNFRSF4, TNFRSF9, TNFRSF14, FCGRA2, CD3G, or CD27, including in illustrative examples an intracellular domain of CD40, CD79B, or CD27 in Tables 13 to 18, including as non- limiting examples, the first and/or second intracellular domains of those genes provided in these tables.
  • first intracellular (P3) domains are those of genes that are linked to CD40, CD79B, TNFRSF4, TNFRSF9, TNFRSF14, FCGRA2, CD3G, or CD27, including in illustrative examples an intracellular domain of CD40, CD79B, and CD27 second intracellular domains in tables for these genes as P3 provided in Example 12, including as non- limiting examples, the first and/or second intracellular domains of those genes provided in these tables.
  • the first intracellular domain is from MPL and the second intracellular domain is from OX40, CD40, CD79A, or CD79B.
  • Intracellular domains from these genes were shown to significantly increase proliferation in at least one of Libraries 3.1 or 4.1 when included as the second intracellular domain of chimeric lymphoproliferative elements containing MPL as the first intracellular domain at a false discovery rate of 0.05 as shown in Example 10.
  • the first intracellular domain is from MPL and the second intracellular is from OX40, CD40, or CD79B.
  • the first intracellular domain contains the sequence from S I 86 (SEQ ID NO: 491), or a variant or fragment thereof that promotes signaling
  • the second intracellular domain contains one of the sequences from S050, S051, S052, S053, or S211 (SEQ ID NOs:416-419 and 504, respectively), or variants or fragments thereof that promote signaling.
  • the first intracellular domain is from CSF2RA and the second intracellular domain is from OX40, CD27, CD28, or CD30.
  • Intracellular domains from these genes were shown to significantly increase proliferation in at least one of Libraries 3.1 or 4.1 when included as the second intracellular domain of chimeric lymphoproliferative elements containing CSF2RA as the first intracellular domain at a false discovery rate of 0.1 as shown in Example 10.
  • the first intracellular domain is from CSF2RA and the second intracellular is from OX40 or CD28.
  • Intracellular domains from these genes were shown to significantly increase proliferation in at least one of Libraries 3.1 or 4.1 when included as the second intracellular domain of chimeric lymphoproliferative elements containing CSF2RA as the first intracellular domain at a false discovery rate of 0.05 as shown in Example 10.
  • the first intracellular domain contains the sequence from S059 (SEQ ID NO:423), or a variant or fragment thereof that promotes signaling
  • the second intracellular domain contains the sequence from S047 (SEQ ID NO:413) or S212 (SEQ ID NO:505), or variants or fragments thereof that promote signaling.
  • the first intracellular domain contains the sequence from S058 (SEQ ID NO: 422), or a variant or fragment thereof that promotes signaling
  • the second intracellular domain contains the sequence from S211 (SEQ ID NO:504), or a variant or fragment thereof that promotes signaling.
  • the first intracellular domain is from CSF3R and the second intracellular domain is from CD79B.
  • Intracellular domains from CD79B were shown to significantly increase proliferation in at least one of Libraries 3.1 or 4.1 when included as the second intracellular domain of chimeric lymphoproliferative elements containing CSF3R as the first intracellular domain at a false discovery rate of 0.05 as shown in Example 10.
  • the first intracellular domain contains the sequence from S064 (SEQ ID NO:426), or a variant or fragment thereof that promotes signaling
  • the second intracellular domain contains the sequence from S053 (SEQ ID NO:419), or a variant or fragment thereof that promotes signaling.
  • the first intracellular domain is from IL1 IRA and the second intracellular domain is from FCGRA2.
  • Intracellular domains from FCGRA2 were shown to significantly increase proliferation in at least one of Libraries 3.1 or 4.1 when included as the second intracellular domain of chimeric lymphoproliferative elements containing IL1 IRA as the first intracellular domain at a false discovery rate of 0.05 as shown in Example 10.
  • the first intracellular domain contains the sequence from SI 35 (SEQ ID NO:461), or a variant or fragment thereof that promotes signaling
  • the second intracellular domain contains the sequence from S076 (SEQ ID NO:431), or a variant or fragment thereof that promotes signaling.
  • the first intracellular domain is from LIFR and the second intracellular domain is from GITR.
  • Intracellular domains from GITR were shown to significantly increase proliferation in at least one of Libraries 3.1 or 4.1 when included as the second intracellular domain of chimeric lymphoproliferative elements containing LIFR as the first intracellular domain at a false discovery rate of 0.05 as shown in Example 10.
  • the first intracellular domain contains the sequence from SI 80 (SEQ ID NO:489), or a variant or fragment thereof that promotes signaling
  • the second intracellular domain contains the sequence from S216 (SEQ ID NO: 509), or a variant or fragment thereof that promotes signaling.
  • the first intracellular domain is from MyD88 and the second intracellular domain is from CD3D or CD79B.
  • Intracellular domains from these genes were shown to significantly increase proliferation in at least one of Libraries 3.1 or 4.1 when included as the second intracellular domain of chimeric lymphoproliferative elements containing MyD88 as the first intracellular domain at a false discovery rate of 0.1 as shown in Example 10.
  • the first intracellular domain contains the sequence from SI 92 (SEQ ID NO:495), or a variant or fragment thereof that promotes signaling
  • the second intracellular domain contains the sequence from S053 (SEQ ID NO:419), or a variant or fragment thereof that promotes signaling.
  • the first intracellular domain contains the sequence from SI 94 (SEQ ID NO:497), or a variant or fragment thereof that promotes signaling
  • the second intracellular domain contains the sequence from S037 (SEQ ID NO:405), or a variant or fragment thereof that promotes signaling
  • the first intracellular domain contains the sequence from SI 95 (SEQ ID NO:498), or a variant or fragment thereof that promotes signaling
  • the second intracellular domain contains the sequence from S053 (SEQ ID NO:419), or a variant or fragment thereof that promotes signaling.
  • Intracellular domains that can be included in CLE embodiments provided herein include mutants and/or fragments of the intracellular domains of the recited genes provided that such mutants and/or fragments retain the ability to promote proliferation, survival (anti-apoptotic), and/or provide a co-stimulatory signal that enhances a differentiation state, proliferative potential or resistance to cell death.
  • the intracellular domains can be, for example, between 10 and 1000, 10 and 750, 10 and 500, 10 and 250, or 10 and 100 amino acids.
  • the intracellular domains can be at least 30, or between 30 and 500, 30 and 250, 50 and 500, or 50 and 250 amino acids.
  • the first intracellular domain is from IFNAR2 and the second intracellular domain is from HVEM.
  • Intracellular domains from HVEM were shown to significantly increase proliferation in at least one of Libraries 3.1 or 4.1 when included as the second intracellular domain of chimeric lymphoproliferative elements containing IFNAR2 as the first intracellular domain at a false discovery rate of 0.05 as shown in Example 10.
  • the first intracellular domain contains the sequence from S083 (SEQ ID NO:436), or a variant or fragment thereof that promotes signaling
  • the second intracellular domain contains the sequence from S214 (SEQ ID NO: 507), or a variant or fragment thereof that promotes signaling.
  • the first intracellular domain is from IL1RAP and the second intracellular domain is from CD79A.
  • Intracellular domains from CD79A were shown to significantly increase proliferation in at least one of Libraries 3.1 or 4.1 when included as the second intracellular domain of chimeric lymphoproliferative elements containing IL1RAP as the first intracellular domain at a false discovery rate of 0.1 as shown in Example 10.
  • the first intracellular domain contains the sequence from S101 (SEQ ID NO:444), or a variant or fragment thereof that promotes signaling
  • the second intracellular domain contains the sequence from S052 (SEQ ID NO:418), or a variant or fragment thereof that promotes signaling.
  • the first intracellular domain is from IL3RA and the second intracellular domain is from CD40.
  • Intracellular domains from CD40 were shown to significantly increase proliferation in at least one of Libraries 3.1 or 4.1 when included as the second intracellular domain of chimeric lymphoproliferative elements containing IL3RA as the first intracellular domain at a false discovery rate of 0.1 as shown in Example 10.
  • the first intracellular domain contains the sequence from S109 (SEQ ID NO:450), or a variant or fragment thereof that promotes signaling
  • the second intracellular domain contains the sequence from S050 (SEQ ID NO:416), or a variant or fragment thereof that promotes signaling.
  • the first intracellular domain is from IL10RA and the second intracellular domain is from CD79B.
  • Intracellular domains from CD79B were shown to significantly increase proliferation in at least one of Libraries 3.1 or 4.1 when included as the second intracellular domain of chimeric lymphoproliferative elements containing IL10RA as the first intracellular domain at a false discovery rate of 0.05 as shown in Example 10.
  • the first intracellular domain contains the sequence from S129 (SEQ ID NO: 450), or a variant or fragment thereof that promotes signaling
  • the second intracellular domain contains the sequence from S050 (SEQ ID NO:416), or a variant or fragment thereof that promotes signaling.
  • the first intracellular domain is from IL13RA2 and the second intracellular domain is from HVEM.
  • Intracellular domains from HVEM were shown to significantly increase proliferation in at least one of Libraries 3.1 or 4.1 when included as the second intracellular domain of chimeric lymphoproliferative elements containing IL13RA2 as the first intracellular domain at a false discovery rate of 0.1 as shown in Example 10.
  • the first intracellular domain contains the sequence from S142 (SEQ ID NO:466), or a variant or fragment thereof that promotes signaling
  • the second intracellular domain contains the sequence from S214 (SEQ ID NO: 507), or a variant or fragment thereof that promotes signaling.
  • the first intracellular domain is from IL18RAP and the second intracellular domain is from CD3G.
  • Intracellular domains from CD3G were shown to significantly increase proliferation in at least one of Libraries 3.1 or 4.1 when included as the second intracellular domain of chimeric lymphoproliferative elements containing
  • the first intracellular domain contains the sequence from S 155 (SEQ ID NO:475), or a variant or fragment thereof that promotes signaling
  • the second intracellular domain contains the sequence from S039 (SEQ ID NO:407), or a variant or fragment thereof that promotes signaling.
  • the first intracellular domain is from IL27RA and the second intracellular domain is from FCGRA2.
  • Intracellular domains from FCGRA2 were shown to significantly increase proliferation in at least one of Libraries 3.1 or 4.1 when included as the second intracellular domain of chimeric lymphoproliferative elements containing IL27RA as the first intracellular domain at a false discovery rate of 0.05 as shown in Example 10.
  • the first intracellular domain contains the sequence from SI 69 (SEQ ID NO:482), or a variant or fragment thereof that promotes signaling
  • the second intracellular domain contains the sequence from S076 (SEQ ID NO:431), or a variant or fragment thereof that promotes signaling.
  • the first intracellular domain is from LEPR and the second intracellular domain is from CD3G.
  • Intracellular domains from CD3G were shown to significantly increase proliferation in at least one of Libraries 3.1 or 4.1 when included as the second intracellular domain of chimeric lymphoproliferative elements containing LEPR as the first intracellular domain at a false discovery rate of 0.1 as shown in Example 10.
  • the first intracellular domain contains the sequence from S177 (SEQ ID NO:488), or a variant or fragment thereof that promotes signaling
  • the second intracellular domain contains the sequence from S039 (SEQ ID NO:407), or a variant or fragment thereof that promotes signaling.
  • all domains of a CLE are other than an IL-7 receptor, or a mutant thereof, and/or a fragment thereof that has at least 10, 15, 20, or 25 contiguous amino acids of IL-7 receptor, or other than an IL-15 receptor, or a mutant thereof, and/or a fragment thereof that has at least 10, 15, 20, or 25 contiguous amino acids of IL-15 receptor.
  • a CLE does not comprise a combination of first intracellular domain and second intracellular domain of CD40 and MyD88.
  • CLEs include a recognition and/or elimination domain. Details regarding recognition and/or elimination domains are provided in other sections herein. Any of the recognition and/or elimination domains provided herein can be part of a CLE. Typically the recognition domain is linked to the N terminus of the extracellular domain. Not to be limited by theory, in some embodiments, the extracellular domain includes the function of providing a linker, in illustrative embodiments a flexible linker, linking a recognition domain to a cell that expresses the CLE. [0365] In illustrative embodiments, as illustrated in FIG. 15 and FIG. 16, a CLE provided herein is co- expressed with a CAR which can be designed according to any of the CAR embodiments provided herein, or otherwise known in the art.
  • Such isolated polynucleotides can include any elements known in the art for providing expression of a transcript, such as a transcript encoding a CLE.
  • the isolated polynucleotide can include a promoter element that is active in B cells, T cells, and/or NK cells. Such promoters that are appropriate for these embodiments are known in the art, some of which are identified in other sections of this disclosure.
  • a Kozak sequence is provided within 10 nucleotides upstream of an ATG start site encoding the amino terminal amine acid of a CLE, or a CAR located upstream a CLE on the same transcription unit.
  • the polynucleotide can be a translation-initiating sequence.
  • the polynucleotide can be translation-initiating sequence can be AGAGGATCCATG (SEQ ID NO:533).
  • the translation-initiating sequence can be a Kozak-type sequence, also referred to herein as a Kozak.
  • the Kozak-type sequence can be CCACCAT/UG(G) (SEQ ID NO:515), CCGCCAT/UG(G) (SEQ ID NO:516),
  • an internal ribosomal binding site can be used instead of a Kozak-type sequence.
  • the polynucleotide can further comprise one or more of a Kozak-related sequence, a WPRE element, and a multiple stop sequence, for example a double stop sequence or a triple stop sequence.
  • a triple stop sequence in a nucleic acid encoding a CLE can be upstream of the promoter for the CLE. In illustrative embodiments, the triple stop sequence can be included upstream of the 5' end of the promoter within 10, 25, 50, or 100 nucleotides. In some embodiments, a triple stop sequence in a nucleic acid encoding a CLE can be included after the promoter and before the associated Kozak-related sequence. In illustrative embodiments, the triple stop sequence can be included downstream of the 3' end of the promoter within 10, 25, 50, or 100 nucleotides and or upstream of the 5' end of the Kozak-related sequence within 10, 25, 50, or 100 nucleotides.
  • a triple stop sequence in a nucleic acid encoding a CLE can be included after the coding sequence of the CLE.
  • the triple stop sequence can be included downstream of the 3' end of the stop codon of the CLE within 10, 25, 50, or 100 nucleotides.
  • the triple stop sequence can be at the 3' end of the coding sequence for the CLE.
  • a nucleic acid encoding a CLE can include one, two, three, or more triple stop sequences.
  • a nucleic acid encoding a CLE can include a triple stop sequence upstream of the promoter, a triple stop sequence downstream of the promoter and upstream of the Kozak-related sequence, and a triple stop sequence downstream of the coding sequence for the CLE.
  • the triple stop sequence can include three stop codons in the same reading frame.
  • the triple stop sequence can be SEQ ID NO:520.
  • the triple stop sequence can include three stop codons in different reading frames.
  • the triple stop sequence can be SEQ ID NO:534.
  • polynucleotides that include a nucleic acid sequence encoding a CLE provided herein also typically comprise a signal sequence to direct expression to the plasma membrane.
  • Exemplary signal sequences are provided herein in other sections.
  • Elements can be provided on the transcript such that both a CAR and CLE are expressed from the same transcript.
  • Such a construct is advantageous in requiring less nucleic acids than if these elements were expressed from different transcripts, which is an important feature that allows such constructs to be present in vectors, such as retroviral genomes, used to transfect or transduce B cells, T cells, and/or NK cells, for example.
  • chimeric polypeptide candidates were designed and identified as lymphoproliferative elements in Example 11 and Example 12 herein.
  • Library 1A which included constructs that encoded the chimeric polypeptides and a CAR, 172 top candidate chimeric polypeptides were identified (See Table 8) that promoted PBMC proliferation between day 7 and the last day when cultured in the absence of IL-2.
  • Library 2B which included constructs that encoded the chimeric polypeptides but did not include a CAR, 167 top candidate chimeric polypeptides were identified (See Table 9) that promoted PBMC proliferation between day 7 and the last day when cultured in the absence of IL-2.
  • Certain illustrative CLEs, as well as polynucleotides, and nucleic acid sequences encoding the same, and methods that include any of these, include intracellular domains from genes having matched domains (e.g.
  • transmembrane gene and first intracellular gene and optionally second intracellular gene on constructs provided in Tables 8 to 12, as well as, in non-limiting examples, the specific matched domains provided on these tables, some of which are set out in the Exemplary Embodiments section herein.
  • the intracellular domains from CLEs having matched domains e.g. transmembrane gene and first intracellular gene and optionally second intracellular gene
  • constructs that had particularly noteworthy enrichments in a first screen and a repeated screen with the same P1-P2, P3 and P4 domains e.g. Libraries 1A and 1.1 A, can be used, as shown in Tables 8 to 12.
  • first and second intracellular domains are derived from, whether the first and/or second intracellular domain are cytokine receptors, and whether the first and/or second intracellular domain have at least one ITAM motif.
  • the intracellular domain of a lymphoproliferative element is from CD40, IL22RA1, IL13RA2, IL17RA, IL17RB, IFNGR2, and FCGR2C.
  • 154 top candidate chimeric polypeptides were identified (See Table 17) that promoted PBMC proliferation between day 7 and the last day in transduced PBMCs that were not stimulated with untransduced PBMCs as set out in Example 12, when cultured in the absence of IL-2.
  • Certain illustrative CLEs, as well as polynucleotides, and nucleic acid sequences encoding the same, and methods that include any of these, include intracellular domains from genes having matched domains (e.g. transmembrane gene and first intracellular gene and optionally second intracellular gene) on constructs provided in Tables 13 to 18, or mutants thereof that retain signaling activity, as well as, in non-limiting examples, the specific matched domains provided on these tables, some of which are set out in the Exemplary Embodiments section herein.
  • Intracellular domains that are identified in the data provided in Example 11 and Example 12, from either the first intracellular domain or the second domain position, are envisioned in exemplary CLE embodiments, interchangeably as either the first or second intracellular domain.
  • the intracellular domains from genes having matched domains e.g. transmembrane gene and first intracellular gene and optionally second intracellular gene
  • constructs that had particularly noteworthy enrichments in a first screen and a repeated screen with the same PI, P2, P3, and P4 domains, e.g. Libraries 3A and 3.1A, can be used.
  • E007/E012-T001-S 186-S047 had particularly noteworthy enrichments in both screens, where the PI parts separated by a slash here included different tags for Libraries 3A and 3.1A as shown in Tables 7, 13, and 15.
  • constructs with particularly noteworthy enrichments were those that had a log2((normalized count data on the last day + l)/(normalized count data on day 7 + 1)) value above 2.
  • E007/E012-T032-S142-S037 E007/E012-T065-S120-S215, E009/E014-T077-S 186-S047, E009/E014-T001-S126-S051, E006/E011-T030-S121-S039, E008/E013-T006-S176-S213, E009/E014- T032-S 130-S215, E008/E013-T041-S 186-S039, E009/E014-T021-S 186-S047, E008/E013-T026-S137- S214, E007/E012-T029-S116-S075, E008/E013-T026-S106-S049, and E007/E012-T032-S168-S075 had particularly noteworthy enrichments in both screens, where the PI parts separated
  • TNFRSF9, FCGR2C, ICOS, TNFRSF18, TNFRSF4, CD28, FCER1G, FCGR2A, CD3D, TNFRSF8, or CD3E when present at the second intracellular domain (P4), showed particularly noteworthy enrichments in both Libraries 3B and 3. IB (Table 23).
  • IB as shown in Tables 7, 17, and 18.
  • constructs with particularly noteworthy enrichments were those that had a log2((normalized count data on the last day + l)/(normalized count data on day 7 + 1)) value above 2.
  • Further information about the first and second intracellular domains in constructs with particularly noteworthy enrichments in screens of both Library 4B and Library 4. IB is provided in Table 24, including the gene(s) the first and second intracellular domain are derived from, whether the first and/or second intracellular domain are cytokine receptors, and whether the first and/or second intracellular domain have at least one ITAM motif.
  • the CLE comprises an intracellular domain from a cytokine receptor in any of the constructs of Tables 19 to 23. In some illustrative embodiments, the CLE comprises an intracellular domain from any of the constructs of Tables 19 to 23 with only a single intracellular domain (other P3 or P4 was a linker or stop).
  • Table 7 Information about various exemplary CLE domains provided herein, is found in Table 7.
  • the first CLE identified in Table 8 is M008-S212-S075.
  • the extracellular and transmembrane domain (PI -2) is M008, the first intracellular domain (P3) is S212, and the second intracellular domain (P4) is S075.
  • PI -2 extracellular and transmembrane domain
  • P3 is S212
  • P4 is S075.
  • Table 7 discloses that M008 is ECDTM-8 and more specifically that this is an interleukin 7 receptor alpha (IL7RA) mutant with a PPCL insert and that the construct includes an eTag.
  • IL7RA interleukin 7 receptor alpha
  • intracellular domains from CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1, IL2RA, IL3RA, IL13RA2, TNFRSF8, and TNFRSF9, or mutants thereof that are known to have signaling activity when found at the first intracellular domain position (P3) of candidate chimeric polypeptides promoted proliferation of PBMCs between day 7 and the last day, when data was considered in a combined manner for all constructs with first intracellular domains (P3) derived from that gene.
  • exemplary embodiments of CLEs provided herein have intracellular domains from CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1, IL2RA, IL3RA, IL13RA2, TNFRSF8, and TNFRSF9, including mutants thereof that retain signaling activity, as either the second intracellular domain, or in illustrative embodiments, the first intracellular domain.
  • P4 second intracellular domain position
  • second intracellular domains or portions and/or mutants thereof, that were present in constructs that promoted cell proliferation are provided in tables in Example 11.
  • exemplary embodiments of CLEs provided herein have intracellular domains from CD3D, CD3G, CD8A, CD8B, CD27, CD40, CD79B, CRLF2, FCGR2C, ICOS , IL2RA, IL13RA1, IL13RA2, IL15RA, TNFRSF9, and TNFRSF18, including mutants thereof that retain signaling activity, as either the first intracellular domain, or in illustrative embodiments, the second intracellular domain.
  • first intracellular domains from IL17RD, IL17RE, IL1RAP, IL23R, and MPL, or mutants thereof that are known to promote signaling activity in certain cell types when found at the first intracellular domain (P3) of candidate chimeric polypeptides elements herein, promoted proliferation of PBMCs between day 7 and day 21, when data is considered in a combined manner for all constructs with a first intracellular domain derived from that gene.
  • first intracellular domains from IL21R, MPL, and OSMR, or mutants thereof that are known to promote signaling activity in certain cell types were the best performers when analyzed in this way.
  • first intracellular domains or portions and/or mutants thereof that were present in constructs that promoted cell proliferation for Libraries 3A and 3B are provided in tables in Example 12. Accordingly, exemplary embodiments of CLEs provided herein have intracellular domains from IL17RD, IL17RE, IL1RAP, IL23R, and MPL F9, including mutants thereof that retain signaling activity, as either the second intracellular domain, or in illustrative embodiments, the first intracellular domain.
  • second intracellular (P4) domains from CD27, CD3G, CD40, and CE79B, or mutants thereof that are known to promote signaling activity in certain cell types when found at the second intracellular domain (P4) of candidate chimeric polypeptides elements herein, promoted proliferation of PBMCs between day 7 and the last day indicated on the table, when data is considered in a combined manner for all constructs with a second intracellular domain derived from that gene.
  • second intracellular domains from CD40, or mutants thereof that are known to promote signaling activity in certain cell types were the best performance when analyzed in this way.
  • exemplary embodiments of CLEs provided herein have intracellular domains from CD27, CD3G, CD40, and CE79B, including mutants thereof that retain signaling activity, as either the first intracellular domain, or in illustrative embodiments, the second intracellular domain.
  • first intracellular (P3) domains from MPL, LEPR, MYD88, IFNAR2, or in some cases mutants thereof that retain signaling activity when found at the first intracellular domain (P3) of candidate chimeric polypeptides herein, promoted proliferation of PBMCs between day 7 and the end of the experiment, when considering the first intracellular domains that occur most frequently in top candidate constructs.
  • Examples of first intracellular domains or portions and/or mutants thereof, that were present in constructs that promoted cell proliferation for Libraries 3A, 3B, 3.1 A, 3, IB, 4B, and 4. IB are provided in tables in Example 12.
  • exemplary embodiments of CLEs provided herein have intracellular domains from MPL, LEPR, MYD88, IFNAR2, including mutants thereof that retain signaling activity, as either the second intracellular domain, or in illustrative embodiments, the first intracellular domain.
  • second intracellular (P4) domains from CD40, CD79B, CD27, or in some cases mutants thereof that retain signaling activity when found at the second intracellular domain (P4) of candidate chimeric polypeptides herein, promoted proliferation of PBMCs between day 7 and the last day indicated on the table, when considering the first intracellular domains that occur most frequently in top candidate constructs.
  • Examples of first intracellular domains or portions and/or mutants thereof, that were present in constructs that promoted cell proliferation for Libraries 3A, 3B, 3.1A, 3, IB, 4B, and 4. IB are provided in tables in Example 12.
  • exemplary embodiments of CLEs provided herein have intracellular domains from CD40, CD79B, CD27, including mutants thereof that retain signaling activity, as either the first intracellular domain, or in illustrative embodiments, the second intracellular domain.
  • a construct for a CLE encodes an intracellular domain from MPL at P3 and an intracellular domain from OX40, CD40, CD79A, or CD79B at P4. Intracellular domains from these genes were shown to significantly increase proliferation at a false discovery rate of 0.05 in at least one of Libraries 3.1 or 4.1 when included at P4 of chimeric lymphoproliferative elements containing MPL at P3 as shown in Example 10.
  • the construct encodes an intracellular domain from MPL at P3 and an intracellular domain from OX40, CD40, or CD79B at P4.
  • the construct encodes the sequence of SI 86 (SEQ ID NO:491) at P3, or a variant or fragment thereof that promotes signaling, and the construct encodes the sequence of S050, S051, S052, S053, or S211 (SEQ ID NOs:416-419 and 504, respectively) at P4, or variants or fragments thereof that promote signaling.
  • a construct for a CLE encodes an intracellular domain from CSF2RA at P3 and an intracellular domain from OX40, CD27, or CD30 at P4.
  • Intracellular domains from these genes were shown to significantly increase proliferation at a false discovery rate of 0.1 in at least one of Libraries 3.1 or 4.1 when included at P4 in chimeric lymphoproliferative elements containing CSF2RA at P3 as shown in Example 10.
  • the construct encodes an intracellular domain from CSF2RA at P3 and an intracellular domain from OX40 at P4.
  • Intracellular domains from OX40 were shown to significantly increase proliferation at a false discovery rate of 0.05 in at least one of Libraries 3.1 or 4.1 when included at P4 of chimeric lymphoproliferative elements containing CSF2RA at P3 as shown in Example 10.
  • the construct encodes the sequence of S059 (SEQ ID NO:423) at P3, or a variant or fragment thereof that promotes signaling, and the construct encodes the sequence of S047 (SEQ ID NO:413) or S212 (SEQ ID NO:505) at P4, or variants or fragments thereof that promote signaling.
  • the construct encodes the sequence of S058 (SEQ ID NO:422) at P3, or a variant or fragment thereof that promotes signaling, and the construct encodes the sequence of S211 (SEQ ID NO:504) at P4, or a variant or fragment thereof that promotes signaling.
  • a construct for a CLE encodes an intracellular domain from CSF3R at P3 and an intracellular domain from CD79B at P4. Intracellular domains from CD79B were shown to significantly increase proliferation at a false discovery rate of 0.05 in at least one of Libraries 3.1 or 4.1 when included at P4 of chimeric lymphoproliferative elements containing CSF3R at P3 as shown in Example 10.
  • the construct encodes the sequence of S064 (SEQ ID NO:426) at P3, or a variant or fragment thereof that promotes signaling
  • the construct encodes the sequence of S053 (SEQ ID NO:419) at P4, or a variant or fragment thereof that promotes signaling.
  • a construct for a CLE encodes an intracellular domain from IL1RL1 at P3 and an intracellular domain from GITR at P4. Intracellular domains from GITR were shown to significantly increase proliferation at a false discovery rate of 0.1 in at least one of Libraries 3.1 or 4.1 when included at P4 of chimeric lymphoproliferative elements containing IL1RL1 at P3 as shown in Example 10.
  • the construct encodes the sequence of S102 (SEQ ID NO:445) at P3, or a variant or fragment thereof that promotes signaling
  • the construct encodes the sequence of S216 (SEQ ID NO:509) at P4, or a variant or fragment thereof that promotes signaling.
  • a construct for a CLE encodes an intracellular domain from IL5RA at P3 and an intracellular domain from mutated delta Lck CD28 at P4. Intracellular domains from mutated delta Lck CD28 were shown to significantly increase proliferation at a false discovery rate of 0.05 in at least one of Libraries 3.1 or 4.1 when included at P4 of chimeric lymphoproliferative elements containing IL5RA at P3 as shown in Example 10.
  • the construct encodes the sequence of SI 15 (SEQ ID NO:453) at P3, or a variant or fragment thereof that promotes signaling
  • the construct encodes the sequence of S048 (SEQ ID NO:414) at P4, or a variant or fragment thereof that promotes signaling.
  • a construct for a CLE encodes an intracellular domain from IL1 IRA at P3 and an intracellular domain from FCGRA2 at P4. Intracellular domains from FCGRA2 were shown to significantly increase proliferation at a false discovery rate of 0.05 in at least one of Libraries 3.1 or 4.1 when included at P4 of chimeric lymphoproliferative elements containing IL1 IRA at P3 as shown in Example 10.
  • the construct encodes the sequence of SI 35 (SEQ ID NO: 461) at P3, or a variant or fragment thereof that promotes signaling
  • the construct encodes the sequence of S076 (SEQ ID NO: 431) at P4, or a variant or fragment thereof that promotes signaling.
  • a construct for a CLE encodes an intracellular domain from LIFR at P3 and an intracellular domain from GITR at P4. Intracellular domains from GITR were shown to significantly increase proliferation at a false discovery rate of 0.05 in at least one of Libraries 3.1 or 4.1 when included at P4 of chimeric lymphoproliferative elements containing LIFR at P3 as shown in Example 10.
  • the construct encodes the sequence of SI 80 (SEQ ID NO:489) at P3, or a variant or fragment thereof that promotes signaling
  • the construct encodes the sequence of S216 (SEQ ID NO:509) at P4, or a variant or fragment thereof that promotes signaling.
  • a construct for a CLE encodes an intracellular domain from MyD88 at P3 and an intracellular domain from CD3D at P4. Intracellular domains from CD3D were shown to significantly increase proliferation at a false discovery rate of 0.1 in at least one of Libraries 3.1 or 4.1 when included at P4 of chimeric lymphoproliferative elements containing MyD88 at P3 as shown in Example 10.
  • the construct encodes the sequence of S194 (SEQ ID NO:497) at P3, or a variant or fragment thereof that promotes signaling, and the construct encodes the sequence of S037 (SEQ ID NO:405) at P4, or a variant or fragment thereof that promotes signaling.
  • a construct for a CLE encodes intracellular domains, or variants or fragments thereof, from both genes in the following pairs of genes: CSF2RA and TNFRSF4; CSF2RA and CD28; CSF2RA and TNFRSF8; CSF2RA and CD27; CSFR3 and CD79B; IFNAR2 and TNFRSF14; ILIRAP and CD79A; IL3RA and CD40; ILIORA and CD79B; IL1 IRA and FCGRA2; IL13RA2 and TNFRSF14; IL18RAP and CD3G; IL27RA and FCGRA2; LEPR and CD3G; LIFR and TNFRSF18; MPL and CD40; MPL and CD79B; MPL and TNFRSF4; MPL and CD3G; MyD88 and CD79B; and MyD88 and CD3D.
  • a construct for a CLE encodes intracellular domains, or variants or fragments thereof, from both genes in the following pairs of genes: CSF2RA and TNFRSF4; CSF2RA and CD28; CSFR3 and CD79B; IFNAR2 and TNFRSF14; ILIORA and CD79B; IL1 IRA and FCGRA2; IL27RA and FCGRA2; LIFR and TNFRSF18; MPL and CD40; MPL and CD40; MPL and CD79B; MPL and TNFRSF4; MPL and CD3G; and MyD88 and CD79B.
  • a construct for a CLE encodes the sequences (as shown in Table 7), or variants or fragments thereof, from both parts in the following pairs of parts: S058 and S211; S058 and S049; S059 and S212; S059 and S047; S064 and S053; S083 and S214; S101 and S052; S109 and S050; S129 and S053; S135 and S076; S142 and S214; S155 and S039; S169 and S076; S177 and S039; S180 and S216; S186 and S050; S186 and S051; S186 and S053; S186 and S211; S186 and S039; S192 and S053; S194 and S037; and S195 and S053.
  • a construct for a CLE encodes the sequences (as shown in Table 7), or variants or fragments thereof, from both parts in the following pairs of parts: S058 and S211; S058 and S049; S064 and S053; S083 and S214; S129 and S053; S135 and S076; S169 and S076; S180 and S216; S 186 and S050; S186 and S051; S186 and S053; S186 and S211; S186 and S039; and S 195 and S053. These pairs of parts were the most effective pairs in Example 10 at a p less than 0.05 (Table 3).
  • the first nucleic acid sequence and the second nucleic acid sequence can be separated by a ribosomal skip sequence.
  • the ribosomal skip sequence is F2A, E2A, P2A, or T2A.
  • lymphoproliferative elements as exemplified herein with CLEs, in some illustrative embodiments can include a dimerizing motif to help enhance and/or regulate activity thereof, for example to affect signals in a cell, such as signals that affect protein activity or gene expression.
  • dimerizing motif is typically a portion of, or the entire extracellular domain.
  • a dimerizing moiety can be attached to a recognition and clearance sequence in the extracellular domain of a CLE herein.
  • a lymphoproliferative element can include an entire lymphoproliferative element from one protein except that the extracellular domain includes a dimerizing moiety.
  • the dimerizing motif can include an amino acid sequence from transmembrane homodimeric polypeptides that naturally exist as homodimers.
  • the dimerizing motif can be a leucine zipper polypeptide, for example a Jun polypeptide as exemplified in Example 12 herein. In some embodiments, these transmembrane homodimeric
  • polypeptides can include early activation antigen CD69 (CD69), Transferrin receptor protein 1 (CD71), B-cell differentiation antigen (CD72), T-cell surface protein tactile (CD96), Endoglin (Cdl05), Killer cell lectin-like receptor subfamily B member 1 (Cdl61), P-selectin glycoprotein ligand 1 (Cdl62), Glutamyl aminopeptidase (Cd249), Tumor necrosis factor receptor superfamily member 16 (CD271), Cadherin-1 (E-Cadherin) (Cd324), or active fragments thereof.
  • CD69 early activation antigen CD69
  • CD71 Transferrin receptor protein 1
  • CD72 B-cell differentiation antigen
  • T-cell surface protein tactile CD96
  • Endoglin Cdl05
  • Killer cell lectin-like receptor subfamily B member 1 Cdl61
  • P-selectin glycoprotein ligand 1 Cdl62
  • Glutamyl aminopeptidase CD27
  • the dimerizing motif can include an amino acid sequence from transmembrane proteins that dimerize upon ligand (also referred to herein as a dimerizer or dimerizing agent) binding.
  • the dimerizing motif and dimerizer can include (where the dimerizer is in parentheses following the dimerizer-binding pair): FKBP and FKBP (rapamycin); GyrB and GyrB (coumermycin); DHFR and DHFR (methotrexate); or DmrB and DmrB (AP20187).
  • FKBP and FKBP rapamycin
  • GyrB and GyrB coumermycin
  • DHFR and DHFR metalhotrexate
  • DmrB and DmrB AP20187
  • rapamycin derivative or analog can be used (see, e.g., W096/41865; WO 99/36553; WO 01/14387; and Ye et al (1999) Science 283:88-91).
  • analogs, homologs, derivatives, and other compounds related structurally to rapamycin include, among others, variants of rapamycin having one or more of the following modifications relative to rapamycin: demethylation, elimination or replacement of the methoxy at C7, C42 and/or C29; elimination, derivatization or replacement of the hydroxy at C13, C43 and/or C28; reduction, elimination or derivatization of the ketone at C14, C24 and/or C30; replacement of the 6-membered pipecolate ring with a 5-membered prolyl ring; and alternative substitution on the cyclohexyl ring or replacement of the cyclohexyl ring with a substituted cyclopentyl
  • the dimerizing agent is methotrexate, e.g., a non-cytotoxic, homo-bifunctional methotrexate dimer (see, e.g., U.S. Pat. No. 8,236,925).
  • a eukaryotic cell when present in the plasma membrane of a eukaryotic cell, a
  • lymphoproliferative element an in illustrative embodiments a CLE, including a dimerizing motif can be active in the absence of a dimerizing agent.
  • lymphoproliferative elements that include a leucine zipper-containing dimerization motif, or that include a dimerizing motif from transmembrane homodimeric polypeptides including CD69, CD71, CD72, CD96, Cdl05, Cdl61, Cdl62, Cd249, CD271, Cd324, active mutants thereof, and/or active fragments thereof can be active in the absence a dimerizing agent.
  • a dimerizing motif from transmembrane homodimeric polypeptides including CD69, CD71, CD72, CD96, Cdl05, Cdl61, Cdl62, Cd249, CD271, Cd324, active mutants thereof, and/or active fragments thereof can be active in the absence a dimerizing agent.
  • a dimerizing motif from transmembran
  • lymphoproliferative element including a dimerizing motif can be active in the presence of a dimerizing agent.
  • lymphoproliferative elements including a dimerizing motif from FKBP, GyrB, DHFR, or DmrB can be active in the presence of the respective dimerizing agents or analogs thereof, e.g. rapamycin, coumermycin, methotrexate, and AP20187, respectively.
  • a dimerizer in method embodiments including lymphoproliferative elements, e.g. CLE, that include a dimerizer for dimerizing a polypeptide comprising a dimerizing motif, can be administered to the host (subject) using any convenient means capable of resulting in the desired effect.
  • the dimerizer can be incorporated into a variety of formulations for administration.
  • a dimerizer can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semisolid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.
  • dose levels can vary as a function of the specific dimerizer, the severity of the symptoms, and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
  • a dimerizer is administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.
  • the dimerizing motif can be selected from the group consisting of: a leucine zipper motif-containing polypeptide, CD69, CD71, CD72, CD96, Cdl05, Cdl61, Cdl62, Cd249, CD271, and Cd324, as well as mutants and/or active fragments thereof that retain the ability to dimerize.
  • the dimerizing motif can require a dimerizing agent, and the dimerizing motif and associated dimerizing agent can be selected from the group consisting of: FKBP and rapamycin or analogs thereof, GyrB and coumermycin or analogs thereof, DHFR and methotrexate or analogs thereof, or DmrB and AP20187 or analogs thereof, as well as mutants and/or active fragments of the recited dimerizing proteins that retain the ability to dimerize.
  • the extracellular domain of a CLE comprises a dimerizing motif
  • the extracellular domain can comprise a leucine zipper motif.
  • the leucine zipper motif is from a jun polypeptide, for example c-jun.
  • the c-jun polypeptide is the c-jun polypeptide region of ECD-11.
  • the lymphoproliferative element can be a polypeptide comprising a membrane targeting region, a dimerizing (or multimerizing) domain, a first intracellular domain, and optionally a second intracellular domain, and further optionally a third intracellular domain, and optionally a fourth intracellular domain, wherein at least one of the first and optional second, third, and fourth intracellular domains are from any gene with an intracellular domain identified in Tables 8 to 18, or selected from any of the first or second intracellular domains identified in Tables 8 to 18, and wherein said internally dimerizing and/or multimerizing lymphoproliferative element promotes cell proliferation of PBMCs, and in illustrative embodiments, of B cells, T cells, and/or NK cells.
  • Such polypeptide can be referred to herein as an internally dimerizing and/or multimerizing lymphoproliferative element.
  • linkers can be added between any of the domains.
  • the first and second intracellular domains are MyD88 and CD40. In certain embodiments, the first and second intracellular domains are other than MyD88 and CD40.
  • the intracellular domain is MPL, or an intracellular fragment thereof that retains the ability to promote PBMC cell proliferation. In some embodiments, the intracellular domain is other than MPL or an intracellular fragment thereof that retains the ability to promote PBMC cell proliferation. In some embodiments, the intracellular domain is IL2Rb, or an intracellular fragment thereof that retains the ability to promote PBMC cell proliferation.
  • the intracellular domain is other than IL2Rb or an intracellular fragment thereof that retains the ability to promote PBMC cell proliferation. In some embodiments, the intracellular domain is IL2Ra, or an intracellular fragment thereof that retains the ability to promote PBMC cell proliferation. In some embodiments, the intracellular domain is other than IL2Ra or an intracellular fragment thereof that retains the ability to promote PBMC cell proliferation. In some embodiments, the intracellular domain is MyD88, or an intracellular fragment thereof that retains the ability to promote PBMC cell proliferation In some embodiments, the intracellular domain is other than MyD88 or an intracellular fragment thereof that retains the ability to promote PBMC cell proliferation.
  • the dimerizing domain of such internally dimerizing and/or multimerizing polypeptide lymphoproliferative elements in illustrative embodiments can be located between the membrane targeting region and the first intracellular domain or after the intracellular domain (see for example Spencer et. al J Clin Invest. 2011 Apr; 121(4): 1524-34) or between intracellular domains.
  • the dimerizing (or multimerizing) domain comprises a multimerizing or dimerizing ligand binding sites, such as, for example, an FKBP region, for example FKBP12.
  • the polypeptide can comprise an extracellular domain, such as any of the extracellular domains provided herein, for example in Tables 8 to 12 of Example 11 or in Tables 13 to 18 of Example 12, with or without the recognition and clearance domain.
  • the membrane targeting region is selected from the group consisting of a myristoylation region, palmitoylation region, prenylation region, and transmembrane sequences of receptors.
  • the membrane targeting region is a myristoylation region.
  • the internally dimerizing and/or multimerizing lymphoproliferative element is typically tethered to the plasma membrane via the membrane targeting region, for example tethered to the membrane with an N- terminal myristate.
  • the FKBP region binds FK506.
  • Internally dimerizing and/or multimerizing lymphoproliferative elements in one embodiment are an integral part of a system that uses a dimeric analog of the lipid permeable immunosuppressant drug, FK506, which loses its normal bioactivity while gaining the ability to crosslink molecules genetically fused to the FK506-binding protein, FKBP 12.
  • FKBP lipid permeable immunosuppressant drug
  • FKBP12 permits specific activation of the recombinant receptor in vivo without the induction of non-specific side effects through endogenous FKBP12.
  • FKBP12 variants having amino acid substitutions and deletions, such as FKBP12V36, that bind to a dimerizer drug, may also be used.
  • the synthetic ligands are resistant to protease degradation, making them more efficient at activating receptors in vivo than most delivered protein agents.
  • pseudotyping elements include a "binding polypeptide” that includes one or more polypeptides, typically glycoproteins, that identify and bind the target host cell, and one or more "fusogenic polypeptides” that mediate fusion of the retroviral and target host cell membranes, thereby allowing a retroviral genome to enter the target host cell.
  • pseudotyping elements are provided as polypeptide(s)/protein(s), or as nucleic acid sequences encoding the polypeptide (s)/protein(s).
  • the pseudotyping element is the feline endogenous virus (RD114) envelope protein, the oncoretroviral amphotropic envelope protein, the oncoretroviral ecotropic envelope protein, the vesicular stomatitis virus envelope protein (VSV-G), the baboon retroviral envelope glycoprotein (BaEV), the murine leukemia envelope protein (MuLV), and/or the paramyxovirus Measles envelope proteins H and F.
  • RD114 feline endogenous virus
  • VSV-G vesicular stomatitis virus envelope protein
  • BaEV baboon retroviral envelope glycoprotein
  • MuLV murine leukemia envelope protein
  • paramyxovirus Measles envelope proteins H and F the paramyxovirus Measles envelope proteins H and F.
  • the pseudotyping element can be wild-type BaEV.
  • BaEV contains an R peptide that has been shown to inhibit transduction.
  • the BaEV can contain a deletion of the R peptide.
  • the BaEV can contain a deletion of the inhibitory R peptide after the nucleotides encoding the amino acid sequence HA, referred to herein as BaEVAR (HA).
  • the BaEV can contain a deletion of the inhibitory R peptide after the nucleotides encoding the amino acid sequence HAM, referred to herein as BaEVAR (HAM).
  • the pseudotyping elements include a binding polypeptide and a fusogenic polypeptide derived from different proteins.
  • the replication incompetent recombinant retroviral particles of the methods and compositions disclosed herein can be pseudotyped with the fusion (F) and hemagglutinin (H) polypeptides of the measles virus (MV), as non-limiting examples, clinical wildtype strains of MV, and vaccine strains including the Edmonston strain (MV-Edm) or fragments thereof.
  • both hemagglutinin (H) and fusion (F) polypeptides are believed to play a role in entry into host cells wherein the H protein binds MV to receptors CD46, SLAM, and Nectin-4 on target cells and F mediates fusion of the retroviral and host cell membranes.
  • the binding polypeptide is a Measles Virus H polypeptide and the fusogenic polypeptide is a Measles Virus F polypeptide.
  • optimal truncation occurred when 18 or 19 residues were deleted (MV(Ed)-HA18 (SEQ ID NO: 106) or MV(Ed)-HA19), although variants with a truncation of 24 residues with and without replacement of deleted residues with alanine (MV(Ed)-HA24 (SEQ ID NO:235) and MV(Ed)-HA24+A) also resulted in optimal titers.
  • the replication incompetent recombinant retroviral particles of the methods and compositions disclosed herein are pseudotyped with mutated or variant versions of the measles virus fusion (F) and hemagglutinin (H) polypeptides, in illustrative examples, cytoplasmic domain deletion variants of measles virus F and H polypeptides.
  • the mutated F and H polypeptides are "truncated H" or "truncated F" polypeptides, whose cytoplasmic portion has been truncated, i.e.
  • amino acid residues (or coding nucleic acids of the corresponding nucleic acid molecule encoding the protein) have been deleted.
  • " ⁇ ” and “FAX” designate such truncated H and F polypeptide, respectively, wherein “Y” refers to 1-34 residues that have been deleted from the amino termini and “X” refers to 1-35 residues that have been deleted from the carboxy termini of the cytoplasmic domains.
  • the "truncated F polypeptide" is FA24 or FA30 and/or the "truncated H protein” is selected from the group consisting of ⁇ 14, ⁇ 15, ⁇ 16, ⁇ 17, ⁇ 18, ⁇ 19, ⁇ 20, ⁇ 21+ ⁇ , ⁇ 24 and ⁇ 24+4 ⁇ , more preferably ⁇ 18 or ⁇ 24.
  • the truncated F polypeptide is MV(Ed)-FA30 and the truncated H polypeptide is MV(Ed)-HA18.
  • the fusogenic polypeptide includes multiple elements expressed as one polypeptide.
  • the binding polypeptide and fusogenic polypeptide are translated from the same transcript but from separate ribosome binding sites; in other embodiments, the binding polypeptide and fusogenic polypeptide are separated by a cleavage peptide site, which not to be bound by theory, is cleaved after translation, as is common in the literature, or a ribosomal skip sequence.
  • the translation of the binding polypeptide and fusogenic polypeptide from separate ribosome binding sites results in a higher amount of the fusogenic polypeptide as compared to the binding polypeptide.
  • the ratio of the fusogenic polypeptide to the binding polypeptide is at least 2: 1, at least 3: 1, at least 4: 1, at least 5: 1, at least 6: 1, at least 7: 1, or at least 8: 1.
  • the ratio of the fusogenic polypeptide to the binding polypeptide is between 1.5: 1, 2: 1, or 3: 1, on the low end of the range, and 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1. 9: 1 or 10: 1 on the high end of the range.
  • an activation element also referred to herein as a T cell activation element, or a nucleic acid encoding an activation element.
  • lentiviral (LV) transduction into resting T cells are attributed to a series of pre-entry and post-entry barriers as well as cellular restrictive factors (Strebel et al 2009. BMC Medicine 7:48).
  • One restriction is the inability for the envelope pseudotyped-LV particles to recognize potential receptors and mediate fusion with the cellular membrane.
  • TCR T cell receptor
  • T lymphocytes recognize and interact with specific antigens through receptors or receptor complexes which, upon recognition or an interaction with such antigens, cause activation of the cell and expansion in the body.
  • An example of such a receptor is the antigen- specific T lymphocyte receptor complex (TCR/CD3).
  • TCR T cell receptor
  • CD3 One component, CD3, is responsible for intracellular signaling following occupancy of the TCR by ligand.
  • T lymphocyte receptor for antigen-CD3 complex recognizes antigenic peptides that are presented to it by the proteins of the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • TCR/CD3 complexes of MHC and peptide are expressed on the surface of antigen presenting cells and other T lymphocyte targets. Stimulation of the TCR/CD3 complex results in activation of the T lymphocyte and a consequent antigen-specific immune response.
  • the TCR/CD3 complex plays a central role in the effector function and regulation of the immune system.
  • activation elements provided herein, activate T cells by binding to one or more components of the T cell receptor associated complex, for example by binding to CD3. In some cases, the activation element can activate alone. In other cases, the activation requires activation through the TCR receptor complex in order to further activate cells.
  • T lymphocytes also require a second, co-stimulatory signal to become fully active in vivo.
  • T lymphocytes are either non-responsive to antigen binding to the TCR, or become anergic.
  • the second, co-stimulatory signal is not required for the transduction and expansion of T cells.
  • a co-stimulatory signal for example, is provided by CD28, a T lymphocyte protein, which interacts with CD80 and CD86 on antigen-producing cells.
  • CD80 a T lymphocyte protein
  • CD86 a functional extracellular fragment of CD80 retains its ability to interact with CD28.
  • OX40, 4-1BB, and ICOS Inducible
  • T cell receptor (TCR) CD3 complex and co-stimulation with CD28 can occur by ex vivo exposure to solid surfaces (e.g. beads) coated with anti-CD3 and anti-CD28.
  • solid surfaces e.g. beads coated with anti-CD3 and anti-CD28.
  • resting T cells are activated by exposure to solid surfaces coated with anti-CD3 and anti-CD28 ex vivo.
  • resting T cells or NK cells are activated by exposure to soluble anti-CD3 antibodies (e.g.
  • activation and/or contacting can be carried out, for example, for 8 hours or less, 4 hours or less or between 2 and 8 hours, 2 and 4 hours, or between 2 and 3.
  • polypeptides that are capable of binding CD3 and/or CD28 are presented as "activation elements" on the surface of replication incompetent recombinant retroviral particles of the methods and compositions disclosed herein, which are also aspects of the invention.
  • the activation elements on the surfaces of the replication incompetent recombinant retroviral particles can include one or more polypeptides capable of binding OX40, 4-1BB, or ICOS.
  • the activation elements can be T cell surface proteins agonists.
  • the activation element can include a co-stimulatory polypeptide that acts as a ligand for a T cell surface protein.
  • the co-stimulatory polypeptide can include one or more of OX40L, 4-1BBL, or ICOSLG.
  • one or typically more copies one or more of these activation elements can be expressed on the surfaces of the replication incompetent recombinant retroviral particles as polypeptides separate and distinct from the pseudotyping elements.
  • the activation elements can be expressed on the surfaces of the replication incompetent recombinant retroviral particles as fusion polypeptides.
  • the fusion polypeptides include one or more activation elements and one or more pseudotyping elements.
  • the fusion polypeptide includes anti-CD3 and a viral envelope protein, for example OKT-3scFv fused to the amino terminal end of the MuLV envelope protein, as shown in Maurice et al. (2002).
  • Polypeptides that bind CD3, CD28, OX40, 4-1BB, or ICOS are referred to as activation elements because of their ability to activate resting T cells.
  • nucleic acids encoding such an activating element are found in the genome of a replication incompetent recombinant retroviral particle that contains the activating element on its surface.
  • nucleic acids encoding an activating element are not found in the replication incompetent recombinant retroviral particle genome. In still other embodiments, the nucleic acids encoding an activating element are found in the genome of the virus packaging cell.
  • the activation element is a polypeptide capable of binding to CD3.
  • the polypeptide capable of binding to CD3 is an anti-CD3 antibody, or a fragment thereof that retains the ability to bind to CD3.
  • the anti-CD3 antibody or fragment thereof is a single chain anti-CD3 antibody, such as but not limited to, an anti-CD3 scFv.
  • the polypeptide capable of binding to CD3 is anti-CD3scFvFc.
  • a number of anti-human CD3 monoclonal antibodies and antibody fragments thereof are available, and can be used in the present invention, including but not limited to UCHT1, OKT-3, HIT3A, TRX4, X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, Fl 11409, CLB-T3.4.2, TR- 66, WT31, WT32, SPv-T3b, 11D8, XIII-141, XIII46, XIII-87, 12F6, T3/RW2-8C8, T3/RW24B6, OKT3D, M-T301, SMC2 and F101.01.
  • the activation element is a polypeptide capable of binding to CD28.
  • the polypeptide capable of binding to CD28 is an anti-CD28 antibody, or a fragment thereof that retains the ability to bind to CD28.
  • the polypeptide capable of binding to CD28 is CD80, CD86, or a functional fragment thereof that is capable of binding CD28 and inducing CD28-mediated activation of Akt, such as an external fragment of CD80.
  • an external fragment of CD80 means a fragment that is typically present on the outside of a cell in the normal cellular location of CD80, that retains the ability to bind to CD28.
  • the anti-CD28 antibody or fragment thereof is a single chain anti-CD28 antibody, such as, but not limited to, an anti-CD28 scFv.
  • the polypeptide capable of binding to CD28 is CD 80, or a fragment of CD 80 such as an external fragment of CD80.
  • Anti-CD28 antibodies are known in the art and can include, as non-limiting examples, monoclonal antibody 9.3, an IgG2a antibody (Dr. Jeffery Ledbetter, Bristol Myers Squibb Corporation, Seattle, Wash.), monoclonal antibody KOLT-2, an IgGl antibody, 15E8, an IgGl antibody, 248.23.2, an IgM antibody and EX5.3D10, an IgG2a antibody.
  • an activation element includes two polypeptides, a polypeptide capable of binding to CD3 and a polypeptide capable of binding to CD28.
  • the polypeptide capable of binding to CD3 or CD28 is an antibody, a single chain monoclonal antibody or an antibody fragment, for example a single chain antibody fragment.
  • the antibody fragment can be, for example, a single chain fragment variable region (scFv), an antibody binding (Fab) fragment of an antibody, a single chain antigen-binding fragment (scFab), a single chain antigen-binding fragment without cysteines (scFabAC), a fragment variable region (Fv), a construct specific to adjacent epitopes of an antigen (CRAb), or a single domain antibody (VH or VL).
  • an activation element can include a dimerizing or higher order multimerizing motif. Dimerizing and multimerizing motifs are well-known in the art and a skilled artisan will understand how to incorporate them into the polypeptides for effective dimerization or multimerization.
  • the activation element that includes a dimerizing motif can be one or more polypeptides capable of binding to CD3 and/or CD28.
  • the polypeptide capable of binding to CD3 is an anti-CD3 antibody, or a fragment thereof that retains the ability to bind to CD3.
  • the anti-CD3 antibody or fragment thereof is a single chain anti-CD3 antibody, such as but not limited to, an anti-CD3 scFv.
  • the polypeptide capable of binding to CD3 is anti- CD3scFvFc, which in some embodiments is considered an anti-CD3 with a dimerizing motif without any additional dimerizing motif, since anti-CD3scFvFc constructs are known to be capable of dimerizing without the need for a separate dimerizing motif.
  • the dimerizing or multimerizing motif, or a nucleic acid sequence encoding the same can be an amino acid sequence from transmembrane polypeptides that naturally exist as homodimers or multimers.
  • the dimerizing or multimerizing motif, or a nucleic acid sequence encoding the same can be an amino acid sequence from a fragment of a natural protein or an engineered protein.
  • the homodimeric polypeptide is a leucine zipper motif- containing polypeptide (leucine zipper polypeptide).
  • leucine zipper polypeptide derived from c-JUN, non-limiting examples of which are disclosed related to chimeric lymphoproliferative elements (CLEs) herein.
  • these transmembrane homodimeric polypeptides can include early activation antigen CD69 (CD69), Transferrin receptor protein 1 (CD71), B-cell differentiation antigen (CD72), T-cell surface protein tactile (CD96), Endoglin (Cdl05), Killer cell lectin-like receptor subfamily B member 1 (Cdl61), P-selectin glycoprotein ligand 1 (Cdl62), Glutamyl aminopeptidase (Cd249), Tumor necrosis factor receptor superfamily member 16 (CD271), Cadherin-1 (E-Cadherin) (Cd324), or active fragments thereof.
  • CD69 early activation antigen CD69
  • CD71 Transferrin receptor protein 1
  • CD72 B-cell differentiation antigen
  • T-cell surface protein tactile CD96
  • Endoglin Cdl05
  • Killer cell lectin-like receptor subfamily B member 1 Cdl61
  • P-selectin glycoprotein ligand 1 Cdl62
  • the dimerizing motif, and nucleic acid encoding the same can include an amino acid sequence from transmembrane proteins that dimerize upon ligand (also referred to herein as a dimerizer or dimerizing agent) binding.
  • the dimerizing motif and dimerizer can include (where the dimerizer is in parentheses following the dimerizer-binding pair): FKBP and FKBP (rapamycin); GyrB and GyrB (coumermycin); DHFR and DHFR (methotrexate); or DmrB and DmrB (AP20187).
  • rapamycin can serve as a dimerizer.
  • rapamycin derivative or analog can be used (see, e.g., W096/41865; WO 99/36553; WO 01/14387; and Ye et al (1999) Science 283:88-91).
  • analogs, homologs, derivatives, and other compounds related structurally to rapamycin include, among others, variants of rapamycin having one or more of the following modifications relative to rapamycin: demethylation, elimination or replacement of the methoxy at C7, C42 and/or C29; elimination, derivatization or replacement of the hydroxy at C13, C43 and/or C28; reduction, elimination or derivatization of the ketone at C14, C24 and/or C30;
  • dimerizing agents suitable for use as an alternative to rapamycin include those described in U.S. Patent Publication No. 2012/0130076.
  • coumermycin can serve as a dimerizing agent.
  • a coumermycin analog can be used (see, e.g., Farrar et al. (1996) Nature 383: 178-181; and U.S. Pat. No. 6,916,846).
  • the dimerizing agent is methotrexate, e.g., a non-cytotoxic, homo-bifunctional methotrexate dimer (see, e.g., U.S. Pat. No.
  • an activation element including a dimerizing motif when present on the surface of replication incompetent recombinant retroviral particles, can be active in the absence of a dimerizing agent.
  • activation elements including a dimerizing motif from transmembrane homodimeric polypeptides including CD69, CD71, CD72, CD96, Cdl05, Cdl61, Cdl62, Cd249, CD271, Cd324, active mutants thereof, and/or active fragments thereof can be active in the absence a dimerizing agent.
  • the activation element can be an anti-CD3 single chain fragment and include a dimerizing motif selected from the group consisting of CD69, CD71, CD72, CD96, Cdl05, Cdl61, Cdl62, Cd249, CD271, Cd324, active mutants thereof, and/or active fragments thereof.
  • an activation element including a dimerizing motif when present on the surface of replication incompetent recombinant retroviral particles, can be active in the presence of a dimerizing agent.
  • activation elements including a dimerizing motif from FKBP, GyrB, DHFR, or DmrB can be active in the presence of the respective dimerizing agents or analogs thereof, e.g. rapamycin, coumermycin, methotrexate, and AP20187, respectively.
  • the activation element can be a single chain antibody fragment against anti-CD3 or anti-CD28, or another molecule that binds CD3 or CD28
  • the dimerizing motif and dimerizing agent can be selected from the group consisting of FKBP and rapamycin or analogs thereof, GyrB and coumermycin or analogs thereof, DHFR and methotrexate or analogs thereof, or DmrB and AP20187 or analogs thereof.
  • an activation element is fused to a heterologous signal sequence and/or a heterologous membrane attachment sequence, both of which help direct the activation element to the membrane.
  • the heterologous signal sequence targets the activation element to the endoplasmic reticulum, where the heterologous membrane attachment sequence covalently attaches to one or several fatty acids (also known as posttranslational lipid modification) such that the activation elements that are fused to the heterologous membrane attachment sequence are anchored in the lipid rafts of the plasma membrane.
  • posttranslational lipid modification can occur via myristoylation, palmitoylation, or GPI anchorage.
  • Myristoylation is a post-translational protein modification which corresponds to the covalent linkage of a 14-carbon saturated fatty acid, the myristic acid, to the N-terminal glycine of a eukaryotic or viral protein. Palmitoylation is a post-translational protein modification which corresponds to the covalent linkage of a C 16 acyl chain to cysteines, and less frequently to serine and threonine residues, of proteins. GPI anchorage refers to the attachment of glycosylphosphatidylinositol, or GPI, to the C-terminus of a protein during posttranslational modification.
  • the heterologous membrane attachment sequence is a GPI anchor attachment sequence.
  • the heterologous GPI anchor attachment sequence can be derived from any known GPI-anchored protein (reviewed in Ferguson MAJ, Kinoshita T, Hart GW. Glycosylphosphatidylinositol Anchors. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 11).
  • GPI-anchored protein Reviewed in Ferguson MAJ, Kinoshita T, Hart GW. Glycosylphosphatidylinositol Anchors. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 11).
  • the heterologous GPI anchor attachment sequence is the GPI anchor attachment sequence from CD14, CD16, CD48, CD55 (DAF), CD59, CD80, and CD87.
  • the heterologous GPI anchor attachment sequence is derived from CD 16.
  • the heterologous GPI anchor attachment sequence is derived from Fc receptor FcyRIIIb (CD 16b) or decay accelerating factor (DAF), otherwise known as complement decay-accelerating factor or CD55.
  • one or both of the activation elements include a heterologous signal sequence to help direct expression of the activation element to the cell membrane.
  • Any signal sequence that is active in the packaging cell line can be used.
  • the signal sequence is a DAF signal sequence.
  • an activation element is fused to a DAF signal sequence at its N terminus and a GPI anchor attachment sequence at its C terminus.
  • the activation element includes anti-CD3 scFvFc fused to a GPI anchor attachment sequence derived from CD 14 and CD80 fused to a GPI anchor attachment sequence derived from CD 16b; and both are expressed on the surface of a replication incompetent recombinant retroviral particle provided herein.
  • the anti-CD3 scFvFc is fused to a DAF signal sequence at its N terminus and a GPI anchor attachment sequence derived from CD 14 at its C terminus and the CD80 is fused to a DAF signal sequence at its N terminus and a GPI anchor attachment sequence derived from CD 16b at its C terminus; and both are expressed on the surface of a replication incompetent recombinant retroviral particle provided herein.
  • the DAF signal sequence includes amino acid residues 1-30 of the DAF protein.
  • Some embodiments of the method and composition aspects provided herein include a membrane- bound cytokine, or polynucleotides encoding a membrane-bound cytokine.
  • Cytokines are typically, but not always, secreted proteins. Cytokines that are naturally secreted can be engineered as fusion proteins to be membrane-bound.
  • Membrane -bound cytokine fusion polypeptides are included in methods and compositions disclosed herein, and are also an aspect of the invention.
  • replication incompetent recombinant retroviral particles have a membrane-bound cytokine fusion polypeptide on their surface that is capable of binding a T cell and/or NK cell and promoting proliferation and/or survival thereof.
  • membrane -bound polypeptides are incorporated into the membranes of replication incompetent recombinant retroviral particles, and when a cell is transduced by the replication incompetent recombinant retroviral particles, the fusion of the retroviral and host cell membranes results in the polypeptide being bound to the membrane of the transduced cell.
  • the cytokine fusion polypeptide includes IL-2, IL-7, IL-15, or an active fragment thereof.
  • the membrane -bound cytokine fusion polypeptides are typically a cytokine fused to heterologous signal sequence and/or a heterologous membrane attachment sequence.
  • the heterologous membrane attachment sequence is a GPI anchor attachment sequence.
  • the heterologous GPI anchor attachment sequence can be derived from any known GPI-anchored protein (reviewed in Ferguson MAJ, Kinoshita T, Hart GW. Glycosylphosphatidylinositol Anchors. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 11).
  • the heterologous GPI anchor attachment sequence is the GPI anchor attachment sequence from CD14, CD16, CD48, CD55 (DAF), CD59, CD80, and CD87.
  • the heterologous GPI anchor attachment sequence is derived from CD 16. In an illustrative embodiment, the heterologous GPI anchor attachment sequence is derived from Fc receptor FcyRIIIb (CD 16b). In some embodiments, the GPI anchor is the GPI anchor of DAF.
  • the membrane-bound cytokine is a fusion polypeptide of a cytokine fused to DAF.
  • DAF is known to accumulate in lipid rafts that are incorporated into the membranes of replication incompetent recombinant retroviral particles budding from packaging cells. Accordingly, not to be limited by theory, it is believed that DAF fusion proteins are preferentially targeted to portions of membranes of packaging cells that will become part of a recombinant retroviral membrane.
  • the cytokine fusion polypeptide is an IL-7, or an active fragment thereof, fused to DAF.
  • the fusion cytokine polypeptide includes in order: the DAF signal sequence (residues 1-31 of DAF), IL-7 without its signal sequence, and residues 36-525 of DAF.
  • the present disclosure provides mammalian packaging cells and packaging cell lines that produce replication incompetent recombinant retroviral particles.
  • the cell lines that produce replication incompetent recombinant retroviral particles are also referred to herein as packaging cell lines.
  • a non- limiting example of such method is provided in Example 1 herein.
  • the cells of the packaging cell line can be adherent or suspension cells.
  • the packaging cell line can be a suspension cell line, i.e. a cell line that does not adhere to a surface during growth.
  • the cells can be grown in a chemically-defined media and/or a serum-free media.
  • the packaging cell line can be a suspension cell line derived from an adherent cell line, for example, the HEK293 cell line can be grown in conditions to generate a suspension-adapted HEK293 cell line according to methods known in the art.
  • the packaging cell line is typically grown in a chemically defined media.
  • the packaging cell line media can include serum.
  • the packaging cell line media can include a serum replacement, as known in the art.
  • the packaging cell line media can be serum-free media.
  • Such media can be a chemically defined, serum-free formulation manufactured in compliance with Current Good Manufacturing Practice (CGMP) regulations of the US Food and Drug Administration (FDA).
  • CGMP Current Good Manufacturing Practice
  • FDA US Food and Drug Administration
  • the packaging cell line media can be xeno-free and complete.
  • the packaging cell line media has been cleared by regulatory agencies for use in ex vivo cell processing, such as an FDA 510(k) cleared device.
  • the media includes the composition of the basal media with a media supplement of a catalog number such as A1048501 or A1048503 that includes a media supplement, that the recited composition is intended to mean the composition of the media with the added supplement.
  • the manufacture of the media and supplement provides instructions for volumes of supplement to be added.
  • a method of making a replication incompetent recombinant retroviral particle including: A. culturing a packaging cell in suspension in serum-free media, wherein the packaging cell comprises nucleic acid sequences encoding a packageable RNA genome of the replication incompetent retroviral particle, a REV protein, a gag polypeptide, a pol polypeptide, and a pseudotyping element; and B. harvesting the replication incompetent recombinant retroviral particle from the serum-free media.
  • a method of transducing a lymphocyte with a replication incompetent recombinant retroviral particle comprising: A.
  • the packaging cell comprises nucleic acid sequences encoding a packageable RNA genome of the replication incompetent retroviral particle, a REV protein, a gag polypeptide, a pol polypeptide, and a pseudotyping element; B. harvesting the replication incompetent recombinant retroviral particle from the serum-free media; and C.
  • the lymphocyte contacting the lymphocyte with the replication incompetent recombinant retroviral particle, wherein the contacting is performed for less than 24 hours, 20 hours, 18 hours, 12 hours, 8 hours, 4 hours or 2 hours (or between 1, 2, 3, or 4 hours on the low end of the range and 4, 6, 8, 12, 18, 20, or 24 hours on the high end of the range), thereby transducing the lymphocyte.
  • a retroviral packaging system including: a mammalian cell including: a) a first transactivator expressed from a constitutive promoter and capable of binding a first ligand and a first inducible promoter for affecting expression of a nucleic acid sequence operably linked thereto in the presence versus absence of the first ligand; b) a second transactivator capable of binding a second ligand and a second inducible promoter, and affecting expression of a nucleic acid sequence operably linked thereto in the presence versus absence of a second ligand; and c) a packageable RNA genome for a retroviral particle, wherein the first transactivator regulates expression of the second transactivator, and wherein the second transactivator regulates expression of retroviral polypeptides involved in viral packaging, such as, for example, a gag polypeptide, a pol polypeptide, and/or a pseudotyping element, and optionally other polypeptides that
  • the second transactivator itself is cytotoxic to packaging cell lines.
  • Pseudotyping elements are typically capable of binding to a cell membrane of a target cell and facilitating fusion thereto, as discussed in detail herein.
  • the system provides the ability to accumulate certain polypeptides/proteins that do not inhibit, or do not substantially inhibit, or are not believed to inhibit proliferation or survival of the mammalian cells, for example, non-toxic proteins, while culturing a population of the mammalian cells for days or indefinitely, and controlling induction of polypeptides that are desired for retroviral product but that are inhibitory or can be inhibitory or have been reported to be inhibitory to the survival and/or proliferation of the mammalian cell, for example toxic polypeptides, until a later time closer to the time of when replication incompetent recombinant retroviral particles will be produced and harvested.
  • the packageable RNA genome is typically encoded by a polynucleotide operably linked to a promoter, sometimes referred to herein as a third promoter for convenience, wherein said third promoter is typically inducible by either the first transactivator or the second transactivator.
  • the packageable RNA genome is encoded by a polynucleotide operably linked to a third promoter, wherein said third promoter is inducible by the second transactivator.
  • the packageable RNA genome can be produced at the later time point, closer to when the replication incompetent recombinant retroviral particles will be harvested.
  • inducible promoters can be used in the retroviral packaging system.
  • Such inducible promoters can be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of inducible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art.
  • Such inducible promoters, and systems based on such inducible promoters but also including additional control proteins include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters,
  • a mifepristone-regulated system can be used.
  • a mifepristone-inducible system with an autoregulatory feedback loop can be used.
  • a GAL4 regulatory fusion protein is expressed from one construct that also contains the transposon terminal repeats and lox and FRT sites.
  • the GAL4 regulatory fusion protein controls expression of a reverse tet transactivator (rtTA) and BiTRE.
  • another construct with lox and FRT sites contains a GAL4 upstream activating sequences (UAS) and an Elb TATA box promoter driving a reporter like mCherry.
  • a GAL4 regulatory fusion protein binds to GAL4 upstream activating sequences (UAS) in both the promoter controlling expression of the GAL4 regulatory fusion protein and the promoter controlling expression of a target polynucleotide.
  • UAS GAL4 upstream activating sequences
  • mifepristone, doxycycline, and puromycin will be used for induction and selection of packaging cell line.
  • either or both transactivators can be split into two or more polypeptides.
  • the two or more polypeptides can include a DNA binding domain and an activation domain capable of stimulating transcription on separate polypeptides.
  • This "activation domain” is not to be confused with an "activation element,” such as a polypeptide that binds CD3, which is capable of activating a T cell and/or NK cell, and typically does activate such T cell and/or NK cell when contacted with it, as discussed in detail herein.
  • the separate polypeptides can further include fusions with polypeptides capable of dimerization through the addition of a ligand.
  • the activation domain can be the p65 activation domain or a functional fragment thereof.
  • the DNA binding domain can be the DNA binding domain from ZFHD 1 or a functional fragment thereof.
  • one polypeptide can be a fusion with FKBP, or functional mutants and/or fragments thereof, or multiple FKBPs and another polypeptide can be a fusion with the FRB domain of mTOR, or functional mutants and/or fragments thereof, and the ligand can be rapamycin or a functional rapalog.
  • the FRB contains the mutations K2095P, T2098L, and/or W210 IF.
  • the separate polypeptides can be FKBP, or functional fragments thereof, and CalcineurinA, or functional fragments thereof, and the dimerizing agent can be FK506.
  • the separate polypeptides can be FKBP, or functional fragments thereof, and CyP-Fas, or functional fragments thereof, and the dimerizing agent can be FKCsA.
  • the separate polypeptides can be GAI, or functional fragments thereof, and GID 1, or functional fragments thereof, and the dimerizing agent can be gibberellin.
  • the separate polypeptides can be Snap-tag and HaloTag, or functional fragments thereof, and the dimerizing agent can be HaXS.
  • the separate polypeptides can include the same polypeptide.
  • the DNA binding domain and activation domain can be expressed as fusion proteins with FKBP or GyrB and the dimerizing agent can be FK1012 or coumermycin, respectively.
  • the inducible promoter can be the DNA sequence where the DNA binding domain typically binds. In some embodiments, the inducible promoter can vary from the DNA sequence where the DNA binding domain typically binds. In some embodiments, either transactivator can be an rtTA, the ligand can be tetracycline or doxycycline, and the inducible promoter can be a TRE.
  • the first transactivator is the p65 activation domain fused to FRB and the ZFHD 1 DNA binding domain fused to three FKBP polypeptides and the first ligand is rapamycin.
  • the second transactivator can be an rtTA, the second ligand can be tetracycline or doxycycline, and the inducible promoter can be a TRE.
  • the first transactivator can regulate expression of an element to control the nuclear export of transcripts containing a consensus sequence, such as an HIV Rev and the consensus sequence can be the Rev response element.
  • a consensus sequence such as an HIV Rev
  • the consensus sequence can be the Rev response element.
  • the target cell is a T cell.
  • the pseudotyping element is a retroviral envelope polypeptide.
  • the pseudotyping element typically includes a binding polypeptide and a fusogenic polypeptide for binding to and facilitating membrane fusion of the target cell and viral membranes, as discussed in more detail herein.
  • the pseudotyping element is the feline endogenous virus (RDl 14) envelope protein, the oncoretroviral amphotropic envelope protein, the oncoretroviral ecotropic envelope protein, and/or vesicular stomatitis virus envelope protein (VSV-G) .
  • VSV-G vesicular stomatitis virus envelope protein
  • pseudotyping element includes a binding polypeptide and a fusogenic polypeptide derived from different proteins, as discussed in further detail herein.
  • the binding polypeptide is a hemagglutinin (H) polypeptide of a Measles Virus (such as the Edmonston strain of the Measles Virus), or a cytoplasmic domain deletion variant thereof
  • the fusogenic polypeptide other is a fusion (F) polypeptide of a Measles Virus (such as the Edmonston strain of the Measles Virus), or a cytoplasmic domain deletion variant thereof.
  • the fusogenic polypeptide can include multiple elements expressed as one polypeptide.
  • the binding polypeptide and the fusogenic polypeptide can be translated from the same transcript but from separate ribosome binding sites, or the polypeptide is cleaved after translation using a peptide cleavage signal or a ribosomal skip sequence, as disclosed elsewhere herein, to generate the binding polypeptide and the fusogenic polypeptide.
  • the binding polypeptide is a Measles Virus H polypeptide, or a cytoplasmic domain deletion thereof
  • the fusogenic polypeptide is a Measles Virus F polypeptide, or a cytoplasmic domain deletion thereof
  • translation of the F and H polypeptides from separate ribosome binding sites results in a higher amount of the F polypeptide as compared to the H polypeptide.
  • polypeptides (or cytoplasmic domain deletions thereof) to H polypeptides (or cytoplasmic domain deletions thereof) is at least 2: 1, at least 3: 1, at least 4: 1, at least 5: 1, at least 6: 1, at least 7: 1, or at least 8: 1.
  • the first transactivator can regulate the expression of an activation element capable of binding to and activating a target cell, such as a T cell or NK cell.
  • the activation element can include: a.) a membrane-bound polypeptide capable of binding to and activating CD3; and/or b.) a membrane-bound polypeptide capable of binding to and activating CD28.
  • the membrane-bound polypeptide capable of binding to and activating CD3 can be an anti-CD3 antibody.
  • the anti-CD3 antibody can be anti-CD3 scFvFc.
  • the membrane-bound polypeptide capable of binding to and activating CD28 is CD80, CD86, or functional fragments thereof, such as an extracellular domain of CD80.
  • the second transactivator can regulate the expression of a packageable RNA genome that can include an R A that encodes one or more target polypeptides, including as a non- limiting example, any of the engineered signaling polypeptides disclosed herein and/or one or more (e.g. two or more) inhibitory RNA molecules.
  • a packageable RNA genome can include an R A that encodes one or more target polypeptides, including as a non- limiting example, any of the engineered signaling polypeptides disclosed herein and/or one or more (e.g. two or more) inhibitory RNA molecules.
  • the retroviral packaging system aspect, and the method of making a replication incompetent recombinant retroviral particle aspect are not limited to making replication incompetent recombinant retroviral particles for transduction of T cell and/or NK cells, but rather for any cell type that can be transduced by replication incompetent recombinant retroviral particles.
  • the packageable RNA genome in certain illustrative embodiments, is designed to express one or more target polypeptides, including as a non-limiting example, any of the engineered signaling polypeptides disclosed herein and/or one or more (e.g. two or more) inhibitory RNA molecules in opposite orientation (e.g., encoding on the opposite strand and in the opposite orientation), from retroviral components such as gag and pol.
  • target polypeptides including as a non-limiting example, any of the engineered signaling polypeptides disclosed herein and/or one or more (e.g. two or more) inhibitory RNA molecules in opposite orientation (e.g., encoding on the opposite strand and in the opposite orientation), from retroviral components such as gag and pol.
  • the packageable RNA genome can include from 5' to 3': a 5' long terminal repeat, or active truncated fragment thereof; a nucleic acid sequence encoding a retroviral cis-acting RNA packaging element; a nucleic acid sequence encoding a first and optionally second target polypeptide, such as, but not limited to, an engineered signaling polypeptide (s) in opposite orientation, which can be driven off a promoter in this opposite orientation with respect to the 5' long terminal repeat and the cis-acting RNA packaging element, which in some embodiments is called a "fourth" promoter for convenience only (and sometimes referred to herein as the promoter active in T cells and/or NK cells), which is active in a target cell such as a T cell and/or an NK cell but in illustrative examples is not active in the packaging cell or is only inducibly or minimally active in the packaging cell; and a 3' long terminal repeat, or active truncated fragment thereof.
  • the retroviral cis-acting RNA packaging element can be HIV Psi. In some embodiments, the retroviral cis-acting RNA packaging element can be the Rev Response Element.
  • the engineered signaling polypeptide driven by the promoter in the opposite orientation from the 5 ' long terminal repeat is one or more of the engineered signaling polypeptides disclosed herein and can optionally express one or more inhibitory RNA molecules as disclosed in more detail herein and in WO2017/165245 A2, WO2018/009923A1, and WO2018/161064A1.
  • promoter number such as a first, second, third, fourth, etc. promoter is for convenience only.
  • a promoter that is called a "fourth" promoter should not be taken to imply that there are any additional promoters, such as first, second or third promoters, unless such other promoters are explicitly recited.
  • each of the promoters are capable of driving expression of a transcript in an appropriate cell type and such transcript forms a transcription unit.
  • the engineered signaling polypeptide can include a first
  • the lymphoproliferative element can be expressed as a fusion with a recognition domain, such as an eTag, as disclosed herein.
  • the packageable RNA genome can further include a nucleic acid sequence encoding a second engineered polypeptide including a chimeric antigen receptor, encoding any CAR embodiment provided herein.
  • the second engineered polypeptide can include a first antigen-specific targeting region, a first transmembrane domain, and a first intracellular activating domain. Examples of antigen-specific targeting regions, transmembrane domains, and intracellular activating domains are disclosed elsewhere herein.
  • the promoter that is active in a target cell is active in a T cell, as disclosed elsewhere herein.
  • the engineered signaling polypeptide can include a CAR, and the nucleic acid sequence can encode any CAR embodiment provided herein.
  • the engineered polypeptide can include a first antigen-specific targeting region, a first transmembrane domain, and a first intracellular activating domain. Examples of antigen-specific targeting regions, transmembrane domains, and intracellular activating domains are disclosed elsewhere herein.
  • the packageable RNA genome can further include a nucleic acid sequence encoding a second engineered polypeptide.
  • the second engineered polypeptide can be a lymphoproliferative element.
  • the promoter that is active in a target cell is active in a T cell or NK cell, as disclosed elsewhere herein.
  • the packageable RNA genome can further include a riboswitch, as discussed in WO2017/165245 A2, WO2018/009923 A 1 , and WO2018/161064A 1.
  • the nucleic acid sequence encoding the engineered signaling polypeptide can be in a reverse orientation with respect to the 5' to 3' orientation established by the 5' LTR and the 3' LTR.
  • the packageable RNA genome can further include a riboswitch and, optionally, the riboswitch can be in reverse orientation.
  • a polynucleotide including any of the elements can include a primer binding site.
  • transcription blockers or polyA sequences can be placed near genes to prevent or reduce unregulated transcription.
  • a nucleic acid sequence encoding Vpx can be on the second or an optional third transcriptional unit, or on an additional transcriptional unit that is operably linked to the first inducible promoter.
  • RNA genome for a replication incompetent retroviral particle, wherein said packageable RNA genome comprises:
  • RNA packaging element b. a nucleic acid sequence encoding a retroviral cis-acting RNA packaging element
  • 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 one or more (e.g. 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 a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain; and
  • CAR chimeric antigen receptor
  • the inhibitory RNA molecules in the above aspect can include any of the inhibitory RNA molecules, as non-limiting examples, shRNA or miRNA, provided herein in other sections of this disclosure.
  • the polynucleotide of (c) can be in reverse orientation to the nucleic acid sequence encoding the retroviral cis-acting RNA packaging element (b), the 5' long terminal repeat (a), and/or the 3' long terminal repeat (d), for example with respect to the orientation established by the 5' LTR and the 3' LTR.
  • expression of the packageable RNA genome is driven by an inducible promoter active in the mammalian packaging cell line.
  • the promoter active in T cells and/or NK cells that drives expression of the inducible RNA and the CAR in these aspects provided immediately above, in illustrative embodiments is not active or is only minimally or inducibly active in the packaging cell line.
  • This promoter active in T cells and/or NK cells in illustrative embodiments is located on the packageable RNA genome between the nucleic acids encoding the one (e.g. two) or more inducible RNAs and the CAR and the 3' LTR.
  • At least one and in some embodiments all inhibitory RNA molecules can include a 5' strand and a 3' strand that are partially or fully complementary to one another, wherein said 5' strand and said 3' strand are capable of forming an 18-25 nucleotide RNA duplex.
  • the 5' strand can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length
  • the 3' strand can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
  • the 5' strand and the 3' strand can be the same or different lengths.
  • the RNA duplex can include one or more mismatches. In alternate embodiments, the RNA duplex has no mismatches.
  • the inhibitory RNA molecule can be a miRNA or an shRNA.
  • the inhibitory molecule can be a precursor of a miRNA, such as for example, a Pri-miRNA or a Pre-miRNA, or a precursor of an shRNA.
  • the one or more inhibitory RNA molecules can be an artificially derived miRNA or shRNA.
  • the inhibitory RNA molecule can be a dsRNA (either transcribed or artificially introduced) that is processed into an siRNA or the siRNA itself.
  • the inhibitory RNA molecule can be a miRNA or shRNA that has a sequence that is not found in nature, or has at least one functional segment that is not found in nature, or has a combination of functional segments that are not found in nature. In illustrative embodiments, at least one or all of the inhibitory RNA molecules are miR-155.
  • the one or more inhibitory RNA molecule(s) in some embodiments, can comprises from 5' to 3' orientation: a 5' arm, a 5' stem, a loop, a 3' stem that is partially or fully complementary to said 5' stem, and a 3' arm.
  • at least one of the two or more inhibitory RNA molecules has this arrangement.
  • all of the two or more inhibitory RNA molecules have this arrangement.
  • the 5' stem can be 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
  • the 3' stem can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
  • the loop can be 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,2 5, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.
  • the 5' arm, 3' arm, or both are derived from a naturally occurring miRNA.
  • the 5' arm, 3 ' arm, or both are derived from a naturally occurring miRNA is selected from the group consisting of: miR-155, miR-30, miR- 17-92, miR-122, and miR-21.
  • the 5' arm, 3' arm, or both are derived from miR-155. In some embodiments, the 5' arm, 3' arm, or both are derived from Mus musculus miR-155 or Homo sapiens miR-155.
  • the 5' arm has the sequence set forth in SEQ ID NO: 256 or is a functional variant thereof, such as, for example, a sequence that is the same length as SEQ ID NO:256, or 95%, 90%, 85%, 80%,75%, or 50% as long as SEQ ID NO: 256 or is 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less; and is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 9
  • the 3' arm has the sequence set forth in SEQ ID NO:260 or is a functional variant thereof, such as, for example, the same length as SEQ ID NO:260, or 95%, 90%, 85%, 80%,75%, or 50% as long as SEQ ID NO: 260 or is a sequence that is 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less; and is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%
  • a method for making replication incompetent recombinant retroviral particles including: culturing a population of packaging cells to accumulate a first
  • the packaging cells include the first transactivator expressed from a constitutive promoter, wherein the first transactivator is capable of binding a first ligand and a first inducible promoter for affecting expression of a nucleic acid sequence operably linked thereto in the presence versus absence of the first ligand, and wherein expression of a second transactivator is regulated by the first
  • transactivator incubating the population of packaging cells including accumulated first transactivator in the presence of the first ligand to accumulate the second transactivator, wherein the second transactivator is capable of binding a second ligand and a second inducible promoter for affecting expression of a nucleic acid sequence operably linked thereto in the presence versus absence of the second ligand; and incubating the population of packaging cells including accumulated second transactivator in the presence of the second ligand thereby inducing expression of retroviral polypeptides involved in viral packaging, such as, for example, a gag polypeptide, a pol polypeptide, and/or a pseudotyping element, and optionally other polypeptides that are believed to inhibit mammalian cell proliferation or survival that will become incorporated in or on the replication incompetent recombinant retroviral particle, thereby making the replication incompetent recombinant retroviral particle.
  • retroviral polypeptides involved in viral packaging such as, for example, a gag polypeptid
  • a packageable RNA genome is encoded by a polynucleotide operably linked to a promoter, sometimes referred to for convenience as a "third" promoter wherein said third promoter is either constitutively active or inducible by either the first transactivator or, in illustrative embodiments, the second transactivator, thereby making the replication incompetent recombinant retroviral particle.
  • the pseudotyping elements are typically capable of binding to a cell membrane of a target cell and facilitating fusion of the target cell membrane to the replication incompetent recombinant retroviral particle membrane.
  • the pseudotyping elements can be any envelope proteins known in the art.
  • the envelope protein can be vesicular stomatitis virus envelope protein (VSV-G), feline endogenous virus (RD114) envelope protein, oncoretroviral amphotropic envelope protein, and/or oncoretroviral ecotropic envelope protein.
  • VSV-G vesicular stomatitis virus envelope protein
  • RD114 feline endogenous virus envelope protein
  • the first transactivator can regulate expression of an element to control the nuclear export of transcripts containing a consensus sequence, such as an HIV Rev and the consensus sequence can be the Rev Response Element (RRE).
  • the target cell is typically a T cell.
  • the HIV RREs and the polynucleotide region encoding HIV Rev can be replaced with HIV-2 RREs and a polynucleotide region encoding the HIV-2 Rev, respectively.
  • the HIV RREs and the polynucleotide region encoding HIV Rev can be replaced with SIV RREs and a polynucleotide region encoding the SIV Rev, respectively.
  • the HIV RREs and the polynucleotide region encoding HIV Rev can be replaced with RemREs and a
  • the HIV RREs and the polynucleotide region encoding HIV Rev can be replaced with a deltaretrovirus RexRRE and a polynucleotide region encoding a deltaretrovirus Rex, respectively.
  • a Rev-like protein is not required and the RREs can be replaced with cis-acting RNA elements, such as the constitutive transport element (CTE).
  • the pseudotyping element is a viral envelope protein.
  • the pseudotyping element typically includes a binding polypeptide and a fusogenic polypeptide for binding to and facilitating membrane fusion of viral and target cell membranes.
  • the pseudotyping element can be the feline endogenous virus (RD 114) envelope protein, the oncoretroviral amphotropic envelope protein, the oncoretroviral ecotropic envelope protein, and/or vesicular stomatitis virus envelope protein (VSV-G).
  • the pseudotyping element includes a binding polypeptide and a fusogenic polypeptide derived from different proteins, as discussed in further detail herein.
  • the binding polypeptide can be a cytoplasmic domain deletion variant of a Measles Virus H polypeptide and the fusogenic polypeptide can be the cytoplasmic domain deletion variant of a Measles Virus F polypeptide.
  • the fusogenic polypeptide can include multiple elements expressed as one polypeptide.
  • the binding polypeptide and fusogenic polypeptide can be translated from the same transcript and translated from separate ribosome binding sites, or the polypeptide can be cleaved after translation using a peptide cleavage signal or a ribosomal skip sequence, as disclosed elsewhere herein, to generate the binding polypeptide and the fusogenic polypeptide.
  • the translation of the binding polypeptide and fusogenic polypeptide from separate ribosome binding sites results in a higher amount of the fusogenic polypeptide as compared to the binding polypeptide.
  • the ratio of the fusogenic polypeptide to the binding polypeptide is at least 2: 1, at least 3: 1, at least 4: 1, at least 5: 1, at least 6: 1, at least 7: 1, or at least 8: 1.
  • the first transactivator can regulate the expression of an activation element capable of binding to and activating a target cell, such as a T cell.
  • the activation element can include: a.) aa membrane-bound polypeptide capable of binding to and activating CD3: and/or b.) a membrane -bound polypeptide capable of binding to and activating CD28.
  • the membrane -bound polypeptide capable of binding to and activating CD28 is CD80, CD86, or functional fragments thereof.
  • the replication incompetent recombinant retroviral particle can include the activation element on a retroviral membrane and the retroviral RNA within a nucleocapsid, thereby making a replication incompetent recombinant retroviral particle.
  • the second transactivator can regulate the expression of an RNA including from 5' to 3': a 5' long terminal repeat, or active truncated fragment thereof; a nucleic acid sequence encoding a retroviral cis-acting RNA packaging element; a nucleic acid sequence encoding a first target polypeptide and optional second target polypeptide, as non-limiting example, one or two engineered signaling polypeptides; a promoter that is active in a target cell; and a 3' long terminal repeat, or active truncated fragment thereof.
  • the RNA can include a cPPT/CTS element.
  • the RNA can include a primer binding site.
  • the retroviral cis-acting RNA packaging element can be HIV Psi. In some embodiments, the retroviral cis-acting RNA packaging element can be the Rev Response Element. In any of the embodiments disclosed herein, retroviral components on the RNA, including RRE and Psi, can be located in any position, as a skilled artisan will understand.
  • the engineered signaling polypeptide in illustrative embodiments, is one or more of the engineered signaling polypeptides disclosed herein.
  • the engineered signaling polypeptide can include a first
  • the lymphoproliferative element is an IL-7 receptor mutant fused to a recognition domain, such as an eTag.
  • the packageable RNA genome can further include a nucleic acid sequence encoding a second engineered polypeptide including a chimeric antigen receptor, encoding any CAR embodiment provided herein.
  • the second engineered polypeptide can include a first antigen-specific targeting region, a first transmembrane domain, and a first intracellular activating domain. Examples of antigen-specific targeting regions, transmembrane domains, and intracellular activating domains are disclosed elsewhere herein.
  • the promoter that is active in a target cell is active in a T cell, as disclosed elsewhere herein.
  • the packageable RNA genome can further include a riboswitch, as discussed in WO2017/165245 A2, WO2018/009923 A 1 , and WO2018/161064A 1.
  • the nucleic acid sequence encoding the engineered signaling polypeptide can be in reverse orientation.
  • the packageable RNA genome can further include a riboswitch and, optionally, the riboswitch can be in reverse orientation.
  • a polynucleotide including any of the elements can include a primer binding site.
  • transcription blockers or polyA sequences can be placed near genes to prevent or reduce unregulated transcription.
  • a nucleic acid sequence encoding Vpx can be on the second or an optional third transcriptional unit, or on an additional transcriptional unit that is operably linked to the first inducible promoter.
  • the encoded RNA can include an intron, which can be transcribed, for example, from the same promoter for expressing the target polypeptide(s). Such intron can encode 1, 2, 3, or 4 miRNAs, in certain illustrative embodiments.
  • the packageable RNA genome is 11,000 KB or less and in some instances 10,000 KB or less in size.
  • the first transactivator can affect the expression of one or more polypeptides that are non-toxic.
  • the second transactivator can affect the expression of one or more polypeptides that are toxic.
  • the first transactivator can induce expression of the retroviral proteins Rev and Vpx in addition to polypeptides that will be transported to the cell membrane of the packaging cell and the second transactivator can induce expression of the retroviral proteins GAG, POL, MV(Ed)-FA30, and either MV(Ed)-HA18 or MV(Ed)-HA24 and expression of the lentiviral genome.
  • the first transactivator can affect the expression of one or more polypeptides that are toxic and/or the second transactivator can affect the expression of one or more polypeptides that are non-toxic.
  • a mammalian packaging cell including: a.) a first transcriptional unit in the genome of the mammalian packaging cell, including a nucleic acid sequence encoding a first transactivator, wherein said first transcriptional unit is operably linked to a constitutive promoter and wherein said transactivator is capable of binding a first inducible promoter and affecting expression of a nucleic acid sequence operably linked thereto in the presence versus absence of a first ligand, and wherein said first transactivator is capable of binding said first ligand; b.) a second and optional third transcriptional unit in the genome of the mammalian packaging cell, including a nucleic acid sequence encoding a retroviral REV protein and a nucleic acid sequence encoding a second transactivator capable of binding a second inducible promoter and affecting expression of a nucleic acid sequence operably linked thereto in the presence versus absence of a second ligand, wherein the
  • a method for making a replication incompetent recombinant retroviral particle including: 1.) culturing a population of packaging cells to accumulate a first transactivator, wherein the packaging cells include: a.) a first transcriptional unit in the genome of the mammalian packaging cell, including a nucleic acid sequence encoding a first transactivator, wherein said first transcriptional unit is operably linked to a constitutive promoter and wherein said transactivator is capable of binding a first inducible promoter and affecting expression of a nucleic acid sequence operably linked thereto in the presence versus absence of a first ligand, and wherein said first transactivator is capable of binding said first ligand; b.) a second and optional third transcriptional unit in the genome of the mammalian packaging cell, including a nucleic acid sequence encoding a retroviral REV protein and a nucleic acid sequence encoding a second transactivator capable of binding said first ligand;
  • the retroviral packaging system can include a mammalian cell including: 1.) a first transactivator expressed from a constitutive promoter and capable of binding a first ligand and a first inducible promoter for affecting expression of a nucleic acid sequence operably linked thereto in the presence versus absence of the first ligand; 2.) a second transactivator capable of binding a second ligand and a second inducible promoter and affecting expression of a nucleic acid sequence operably linked thereto in the presence versus absence of a second ligand; and 3.) a packageable RNA genome for a retroviral particle, wherein the first transactivator regulates expression of the second transactivator, HIV REV, an IL7 GPI DAF, and an activation element, and wherein the second transactivator regulates expression of a gag polypeptide, a pol polypeptide, a retroviral cis-acting RNA packaging element, and one or more envelope polypeptide
  • the first transactivator can be an FRB domain fused to a p65 activation domain and one or more FKBP domains fused to a ZFHD 1 DNA binding domain
  • the first ligand can be rapamycin
  • the first inducible promoter can be one or more ZFHDl binding sites.
  • the second transactivator can be an rtTA protein
  • the second ligand can be tetracycline or doxycycline
  • the second inducible promoter can be a TRE promoter or a bi-directional TRE promoter.
  • the retroviral cis-acting RNA packaging element can be HIV Psi.
  • the one or more envelope proteins include the cytoplasmic domain deletion variants of F and H polypeptides of a Measles Virus.
  • transcription blockers or polyA sequences can be placed near genes to prevent or reduce unregulated transcription.
  • a rapamycin-doxycycline inducible lentiviral genome with riboswitch can be used (SEQ ID NO:83).
  • a rapamycin-doxycycline inducible GAG POL ENV can be used (SEQ ID NO: 84).
  • a rapamycin-inducible TET activator can be used (SEQ ID NO: 85).
  • a rapamycin inducer inducible REV srcVpx can be used (SEQ ID NO: 86).
  • Some aspects of the present disclosure include or are cells, in illustrative examples, mammalian cells, that are used as packaging cells to make replication incompetent recombinant retroviral particles, such as lentiviruses, for transduction of T cells and/or NK cells.
  • recombinant retroviral particles such as lentiviruses
  • Any of a wide variety of cells can be selected for in vitro production of a virus or virus particle, such as a redirected recombinant retroviral particle, according to the invention.
  • Eukaryotic cells are typically used, particularly mammalian cells including human, simian, canine, feline, equine and rodent cells. In illustrative examples, the cells are human cells.
  • the cells reproduce indefinitely, and are therefore immortal.
  • examples of cells that can be advantageously used in the present invention include NIH 3T3 cells, COS cells, Madin-Darby canine kidney cells, human embryonic 293T cells and any cells derived from such cells, such as gpnlslacZ ⁇ cells, which are derived from 293T cells.
  • Highly transferable cells such as human embryonic kidney 293T cells, can be used.
  • “highly transferable” it is meant that at least about 50%, more preferably at least about 70% and most preferably at least about 80% of the cells can express the genes of the introduced DNA.
  • Suitable mammalian cells include primary cells and immortalized cell lines.
  • Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.
  • Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No.
  • CCLIO PC12 cells
  • COS cells COS-7 cells
  • RATI cells mouse L cells
  • HEK human embryonic kidney
  • HLHepG2 cells Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.
  • the methods of making a replication incompetent recombinant retroviral particle can include growing a mammalian packaging cells to 50%, 60%, 70%, 80%, 90% or 95% confluence or confluence or to 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% peak cell density or peak cell density and then splitting or diluting the cells.
  • a stirred tank reactor can be used to grow the cells.
  • the cells can be split at least about 1:2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 1 : 12, 1: 15, or 1 :20 using methods a skilled artisan will understand.
  • the cells can be diluted to 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% peak cell density.
  • after splitting or diluting the cells the cells can be grown for 1, 2, 3, 4, 5, 6, 7, 8, 10, or 16 hours or 1, 2, 3, 4, 5, 6, or 7 days before adding the first ligand.
  • the cells are grown in the presence of the first ligand for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, or 28 days in the presence of the first ligand, which in illustrative embodiments can be rapamycin or a rapalog.
  • the second ligand can be added and the cells can be grown for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, or 28 days which in illustrative embodiments can be tetracycline or doxycyline.
  • Conditions for culturing will depend on the cells and ligands used and the methods are known in the art. A specific example of conditions for culturing and inducing HEK293S cells is shown in Example 2.
  • replication incompetent recombinant retroviral particles are a common tool for gene delivery (Miller, Nature (1992) 357:455-460).
  • the ability of replication incompetent recombinant retroviral particles to deliver an unrearranged nucleic acid sequence into a broad range of rodent, primate and human somatic cells makes replication incompetent recombinant retroviral particles well suited for transferring genes to a cell.
  • the replication incompetent recombinant retroviral particles can be derived from the Alpharetrovirus genus, the Betaretrovirus genus, the Gammaretrovirus genus, the Deltaretrovirus genus, the Epsilonretrovirus genus, the Lentivirus genus, or the Spumavirus genus.
  • retroviruses suitable for use in the methods disclosed herein.
  • murine leukemia virus MMV
  • human immunodeficiency virus HIV
  • equine infectious anaemia virus EIAV
  • mouse mammary tumor virus MMTV
  • Rous sarcoma virus RSV
  • Fujinami sarcoma virus FuSV
  • Moloney murine leukemia virus Mo-MLV
  • FBR MSV FBR murine osteosarcoma virus
  • Mo-MSV Abelson murine leukemia virus
  • A-MLV Avian myelocytomatosis virus-29
  • AEV Avian erythroblastosis virus
  • retroviruses A detailed list of retroviruses may be found in Coffin et al ("Retroviruses” 1997 Cold Spring Harbor Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmus pp 758-763). Details on the genomic structure of some retroviruses may be found in the art. By way of example, details on HIV may be found from the NCBI Genbank (i.e. Genome Accession No. AF033819).
  • the replication incompetent recombinant retroviral particles can be derived from the Lentivirus genus. In some embodiments, the replication incompetent recombinant retroviral particles can be derived from HIV, SIV, or FIV. In further illustrative embodiments, the replication incompetent recombinant retroviral particles can be derived from the human
  • HIV immunodeficiency virus
  • Lentiviruses are complex retroviruses which, in addition to the common retroviral genes gag, pol and env, contain other genes with regulatory or structural function. The higher complexity enables the lentivirus to modulate the life cycle thereof, as in the course of latent infection.
  • a typical lentivirus is the human immunodeficiency virus (HIV), the etiologic agent of AIDS. In vivo, HIV can infect terminally differentiated cells that rarely divide, such as lymphocytes and macrophages.
  • replication incompetent recombinant retroviral particles contain Vpx polypeptide.
  • Vpx polypeptide can be expressed in a packaging cell line, after integration of a Vpx coding nucleic acid in its genome, for example as a cell membrane bound protein that gets incorporated into a retrovirus membrane (Durand et al., J. Virol. (2013) 87: 234-242).
  • a retroviral membrane bound Vpx can be constructed with a processing sequence for a viral protease such that free Vpx is released once incorporated in a viral particle.
  • Vpx fusion with this functionality is Src-Flag-Vpx, which includes a membrane-targeting domain (MGSSKSKPKDP) (SEQ ID NO:227) of the first 11 amino acids of c-Src followed by a viral protease cleavage domain
  • KARVLAEA (SEQ ID NO:228) followed by Flag-tagged Vpx.
  • Vpx polypeptide aids in transduction of resting cells by stimulating the efficiency of the process of reverse transcription by degrading the restriction factor SAMHD1. Accordingly, it is believed that in the methods provided herein where Vpx is present in a replication incompetent recombinant retroviral particle used to transduce T cells and/or NK cells, Vpx is released into the cytoplasm of a resting T cell or a resting NK cell upon transduction of the cell by a replication incompetent recombinant retroviral particle that contains Vpx. Vpx then degrades SAMHD1, which causes an increase in free dNTPs, which in turn, stimulates reverse transcription of the retroviral genome.
  • replication incompetent recombinant retroviral particles provided herein comprise and/or contain Vpu polypeptide.
  • Vpu polypeptide can be expressed from a plasmid or in a packaging cell line, after integration of a Vpu coding nucleic acid in its genome, for example, as a viral membrane protein that gets incorporated into the retrovirus membrane.
  • a recombinant form of Vpu, with its sequence codon optimized for expression in humans, can be constructed and expressed such that free Vpu can incorporate into the viral particles.
  • Vpu is an accessory protein found in HIV-1 and some HIV-1- related simian immunodeficiency virus (SIV) isolates, such as SIVcpz, SIVgsn, and SIVmon, but not in HIV-2 or the majority of SIV isolates.
  • SIV HIV-1-related simian immunodeficiency virus
  • the Vpu protein participates in the downregulation of CD4, and antagonism oftetherin for particle release. More importantly, Vpu shares structural similarities with viroporins, and particularly the viroporin protein M2 from Influenza A virus (IAV), As such, Vpu has been shown to form cation-selective ion channels and permeabilize membranes, in various models. It has been shown that IAV-M2 helps acidify the particles and thus favors an early release from the endosomal pathway used for entry, reducing the amount of dead-end transit products.
  • IAV-M2 Influenza A virus

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