WO2021231505A1 - Vectors and methods for in vivo transduction - Google Patents

Vectors and methods for in vivo transduction Download PDF

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Publication number
WO2021231505A1
WO2021231505A1 PCT/US2021/031878 US2021031878W WO2021231505A1 WO 2021231505 A1 WO2021231505 A1 WO 2021231505A1 US 2021031878 W US2021031878 W US 2021031878W WO 2021231505 A1 WO2021231505 A1 WO 2021231505A1
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Prior art keywords
mir
cells
cell
domain
receptor
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PCT/US2021/031878
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English (en)
French (fr)
Inventor
Doughlas J. JOLLY
Derek G. Ostertag
Noriyuki Kasahara
Carlos Ibanez
Cornelia A. Bentley
Mohd Mushtaq HUSAIN
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Abintus Bio Inc
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Abintus Bio Inc
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Priority to CN202180048754.2A priority Critical patent/CN115968300A/zh
Priority to JP2022567469A priority patent/JP2023525720A/ja
Priority to EP21803598.8A priority patent/EP4149495A4/en
Priority to CA3176697A priority patent/CA3176697A1/en
Priority to IL297514A priority patent/IL297514A/en
Priority to AU2021273253A priority patent/AU2021273253A1/en
Priority to US17/924,080 priority patent/US20230304031A1/en
Priority to KR1020227043191A priority patent/KR20230010231A/ko
Publication of WO2021231505A1 publication Critical patent/WO2021231505A1/en
Anticipated expiration legal-status Critical
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Definitions

  • compositions and methods for cellular therapies of cancer, infection, allergic, degenerative and immune disorders are provided.
  • Sequence-Listing_ST25.txt created on May 11, 2021 and having 239,881 bytes of data, machine formatted on IBM-PC, MS-Windows operating system.
  • sequence listing is hereby incorporated herein by reference in its entirety for all purposes.
  • T cells can be engineered to express the genes of chimeric antigen receptors (CARs) that recognize tumor associated antigens.
  • CARs are engineered immune- receptors, which can redirect T cells to selectively kill tumor cells.
  • the general premise for their use in cancer immunotherapy is to rapidly generate tumor-targeted T cells.
  • the ideal gene delivery system would be easy to produce, easy to administer, and non-toxic to normal cells; it would deliver the genetic information efficiently and specifically via the bloodstream to the targeted tissues and integrate the genetic material into the host cell so that the transgene would be stably expressed.
  • the novel systems described here fit this prescription.
  • the disclosure provides a recombinant vector comprising (a) a polynucleotide encoding a chimeric antigen receptor (CAR); and (b) polynucleotide comprising at least one miRNA targeting sequence, wherein (a) and (b) are linked on the same polynucleotide.
  • CAR chimeric antigen receptor
  • the vector further comprising (c) a polynucleotide encoding a cytotoxic polypeptide that converts a prodrug to a cytotoxic drug.
  • (a) comprises a first polynucleotide domain encoding one or more antigen binding domain(s); an optional polynucleotide domain encoding a linker; and a second polynucleotide domain operably linked to the first polynucleotide domain, wherein the second polynucleotide domain encodes a transmembrane; and a third polynucleotide domain encoding an intracellular signaling domain.
  • the first polynucleotide domain encodes an antibody fragment, single domain antibody, single chain variable fragment, single domain antibody, camelid VHH domain, a non-immunoglobulin antigen binding scaffold, a receptor or receptor fragment, or a bispecific antibody.
  • the optional polynucleotide encoding a linker encodes a Gly3 sequence.
  • the transmembrane domain is from a member selected from the group consisting alpha, beta or zeta chain of a T-cell receptor, CD3y,
  • CRT AM Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMFl, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or NKG2C.
  • the third polynucleotide domain encodes an intracellular signaling domain selected from the group consisting of CD3 zeta, common FeR gamma (FCER1G), Fe gamma Rlla, FeR beta (Fe Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b,
  • the cytotoxic polypeptide that converts a prodrug to a cytotoxic drug is selected from the group consisting of a polypeptide having cytosine deaminase activity, a polypeptide having thymidine kinase activity and a combination thereof.
  • the vector is an integrating vector.
  • the vector is a retroviral vector.
  • the retroviral vector is a non-replicating gammaretroviral vector.
  • the at least one miRNA targeting sequence is bound by an miRNA selected from the group consisting of hsa-miR-223-3p, hsa- miR143-3p, hsa-mirl82-5p, hsa-miR-lObp, hsa-miR141-3p, has-miR486-5p and any combination of the foregoing.
  • the disclosure also provides a recombinant retroviral particle comprising a gag polypeptide; a pol polypeptide; an env polypeptide; and a retroviral polynucleotide contained within a capsid of the retroviral vector, wherein the retroviral polynucleotide comprises from 5' to 3': (R-U5 domain)-(optional signal peptide coding sequence domain)-(Binding domain coding sequence domain)-(optional hinge/linker coding sequence domain)- (transmembrane (TM) coding sequence domain)-(miRNA target domain(s))-(U3-R domain).
  • TM transmembrane
  • the R-U5 domain has a sequence that is at least 80% identical to SEQ ID NO:25 from nucleotide 1 to about nucleotide 145.
  • the binding domain coding sequence is preceded by a signal sequence.
  • the binding domain coding sequence is followed by an optional linker/spacer domain sequence.
  • the retroviral polynucleotide further comprises a kill switch domain coding sequence.
  • the kill switch coding domain comprises an IRES operably linked to a coding sequence for a polypeptide that converts a prodrug to a cytotoxic drug.
  • the polypeptide has thymidine kinase (TKO) activity or cytosine deaminase (CD) activity.
  • the retroviral polynucleotide comprises at least one miRNA targeting sequence.
  • the at least one miRNA targeting sequence comprises a plurality of miRNA targeting sequences.
  • the plurality of miRNA targeting sequences are the same.
  • at least two of the plurality of miRNA targeting sequences are different.
  • the U3-R domain comprises a sequence that is at least 80% identical to SEQ ID NO:25 from about nucleotide 5537 to about 6051.
  • FIG. 1 is an illustration of RNV engineered to deliver
  • CAR or GFP transgenes with or without varying 3' UTR microRNA target sequences.
  • Examples include miR233 (causes transcript degradation in monocytes); or miRaBCT and miRaNKT (cause transcript degradation in B and NK cells, respectively). These target sequences are added in multiples of 1-4 and in varying combinations to direct transgene degradation in monocytes, B, and NK cells.
  • FIG. 2 is an illustration of RNV engineered to deliver
  • CAR or GFP transgenes with or without varying 3' UTR microRNA target sequences.
  • further examples include miR122a and miR199a which specifies transgene transcript degradation in hepatocytes. These target sequences are added in multiples of 1- 4 and in varying combinations with miRNA target sequence describe in figure XI to direct transgene
  • FIG. 3 provides an expected CD19 CAR expression profile in peripheral T-cells from mixed lymphocyte (PBMC) transduction.
  • PBMC mixed lymphocyte
  • FIG. 3 provides an expected CD19 CAR expression profile in peripheral T-cells from mixed lymphocyte (PBMC) transduction.
  • PBMC mixed lymphocyte
  • CD3, CD4 and CD8 positive cells show CD19CAR positive staining (APC) compared to RNV-GFP infected PBMCs.
  • FIG. 4A-C provide schematic presentations of the three safety-modified MLV-based retroviral components amphotropic env (4070A-derived), gag/pol (MoMLV-derived), and vector (MoMLV- derived).
  • Amphotropic env 4070A-derived
  • gag/pol MoMLV-derived
  • vector MoMLV- derived
  • FIG. 5 depict constructs comprising microRNA target sequences used to down regulate expression of GFP sequences in myeloid, B and NK cells by using multiple miR sequences as described above.
  • FIG. 6 provide flow charts outlining the process for generating a packaging cell line (PCL) and high-titer vector- producing line (VPCL) starting with a parent cell line.
  • PCL packaging cell line
  • VPCL high-titer vector- producing line
  • FIG. 7A-E show the results of an experiment in the A20 B- cell lymphoma animal model where (B) tumor-bearing mice injected IV with the vector of (A) mCD19 (1D3)-IRES YCD(V), at a dose of 1E7 per day for four consecutive days, starting at day 3 after A20 implantation.
  • Tumor burden was assessed at days 12, 18 and 25 after A20 B cell lymphoma implantation by imaging (C) and luminescent signal (D, radiance) in vehicle control and RNV-lD3-treated animals. Imaging of luminescent signal show a visual decrease in tumor burden
  • FIG. 8 shows a Western blot could confirm expression of yCD2 in cells transduced with construct 8 at high (MOI 10) and low MOI (0.1) compared to construct 14 which does not have the yCD2 transgene.
  • FIG. 9 shows kill-curves for CD and thymidine kinase (TK) encoding vectors: for CD encoding vectors +/- 5-FC (flucytosine, D.Ostertag et al. Neuro-Oncology 2012), and for TK vectors +/- ganciclovir (GCV).
  • TK thymidine kinase
  • Test cells are plated and after 24-48h drug is added at the various concentrations shown; viability was determined after 5-7 days and to generate a kill curve and IC50 determination using the MTS assay (Abeam abl97010). Results show IC501 to 3 logs greater IC50's for cells carrying vectors without the kill-switches genes compared to those without.
  • FIG. 10A-B shows the effect of including a miRNA target sequence in an RNV vector.
  • the target sequence for the miR223-3p was inserted into a GFP vector to give the sequence pBA-9B-GFPmiR223- 3pB-4TX (construct 7) and used to make infectious vector.
  • miR223-3p is a microRNAs which is produced at significant concentrations only in monocytic or myeloid cells. (A) Conceptual picture of the desired result.
  • FIG. B shows the expression of the original GFP vector (left column), GFP miR 223-3p vector (center column)and GFP vector with an irrelevant miRNA target for miRaBC, in the U937 monocytic cell line, showing a 190-100foldredictio in GFP expression in the GFPmiR223 infected cell line, compared to the two other vectors. All three vectors produced equivalent amounts of GFP in HT1080 fibrosarcoma cells or other non-monocytic cells.
  • FIG. 11 provides a representation of an anti-BCMA-CAR RNV structure.
  • FIG. 12 shows a box-plot example of miRNA expression identification.
  • FIG. 13 shows miRNA expression de-targeting example.
  • the figure shows the expression of the original GFP vector (left column), GFP miR 223-3p vector (center column) and GFP vector with an irrelevant miRNA target for miRaBC, in the U937 monocytic cell line, showing a 100 fold reduction in GFP expression in the GFPmiR223 infected cell line, compared to the two other vectors.
  • FIG. 14 shows the 4070A amphotropic envelope modified to contain anti-CD8 scFV sequences in both orientations inserted into the proline rich region of the env sequence.
  • Glycoprotein (G) and Hemagglutinin (H) for attachment to proteinaceous receptors.
  • FIG. 15A-B shows the proviral integrated forms of (A) the pBA-9b vector with the mouse CD19CAR construct based on the anti mouse CD19-1D3 scFV monoclonal antibody linked to hinge-TM and signaling domains or (B) the pBA-9b vector with the human CD19CAR construct based on the anti-human CD19-FMC63 scFV monoclonal antibody linked to hinge-TM and signaling domains.
  • FIG. 16 provides microRNA target sequences used to down regulate expression of CAR sequences in myeloid, B and NK cells.
  • FIG. 17 provides examples of a SIN vector design that drives expression using the constitutive CMV promoter with the EFla enhancer ensuring expression of the human anti-CD19 CAR sequence.
  • the vector includes an insertional ribosome entry site (IRES) sequence for expression of a human codon optimized thymidine kinase (TKO)gene or a yeast cytosine deaminase gene (yCD2) as a vector kill switch when exposed to its corresponding prodrug, followed by a woodchuck hepatitis post-translational (WPRE) element for enhanced expression of the gene of interest.
  • IRS insertional ribosome entry site
  • TKO thymidine kinase
  • yCD2 yeast cytosine deaminase gene
  • WPRE woodchuck hepatitis post-translational
  • FIG. 18A-D shows retroviral constructs comprising
  • CRISPR/CAS9 system(s) to (A) CCR5, (B) CCR2, (C) CCR5 and CCR2, and
  • FIG. 19A-D shows retroviral constructs (A-D) to treat HIV infection or multiple sclerosis by blocking activity of the HIV co receptors CCR5 and CCR2.
  • FIG. 20A-D shows retroviral constructs (A-D) to treat HIV infection or multiple sclerosis using siRNA of the HIV co-receptors CCR5 and CCR2.
  • FIG. 21 shows retroviral construct to treat HIV infection or multiple sclerosis using CRISPR/CAS systems to CCR5 and CCR2.
  • FIG. 22 shows time line used in the protocol for mobilization and transduction in vivo of hematopoietic stem/progenitor cells (HSPCs).
  • HSPCs hematopoietic stem/progenitor cells
  • FIG. 23 shows RNV-GFP in vivo transduction for 2 hours in mobilized Balb/c mice. Splenocytes harvested and examined after 3 days in culture for GFP transduction of HSPC by FACS analysis and MethoCultassay. Photomicrographs taken after FACS analysis (upper panels show GFP+cells under UV light, lower panels show phase contrast).
  • the term "about,” as used herein can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which can depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. Alternatively, “about” can mean a range of plus or minus 20%, plus or minus 10%, plus or minus 5%, or plus or minus 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about” meaning within an acceptable error range for the particular value can be assumed.
  • the ranges and/or subranges can include the endpoints of the ranges and/or subranges. In some cases, variations can include an amount or concentration of 20%, 10%, 5%, 1 %, 0.5%, or even 0.1 % of the specified amount.
  • Retroviridae family of viruses may be used to create vectors that integrate into their host genome and provide long-term gene expression to the transduced cell and its descendants.
  • gammaretroviral vectors, lentiviral vectors and foamy viral vectors are usable and useful to transduce cells including mobilized stem cells.
  • the disclosure focuses on gammaretroviral vectors, but one skilled in the art will quickly realize that the invention can use all types of integrating vectors including viral and non-viral vectors such as adenoviral-retroviral hybrids, piggy-bac and sleeping beauty transposons etc.
  • compositions and methods to transduce cells in vivo by direct administration of the vector to achieve therapeutic effects in many typed of disease including genetic diseases, cancer, infectious disease and autoimmune disease.
  • cells can be mobilized prior to in vivo infection.
  • the vector constructs of the disclosure can be considered modular with domains (sometimes referred to as cassettes) described below.
  • the vector comprises Long Terminal Repeats, a binding domain, a hinge or linker domain, a transmembrane domain, an intracellular domain, one or more miRNA target domains, an optional kill switch domain and an optional cell-activity-regulating domain.
  • the binding domain, hinge/linker, transmembrane domain and intracellular domain generally comprise chimeric antigen receptors (CAR) including 1 st generation, 2 nd generation, 3 rd generation and related constructs.
  • CAR chimeric antigen receptors
  • antibody refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen.
  • Antibodies can be monoclonal, or polyclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources.
  • Antibodies can be tetramers of immunoglobulin molecules.
  • the antibody may be 'humanized', 'chimeric' or non-human.
  • antibody fragment refers to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen.
  • antibody fragments include, but are not limited to, Fab, Fab 1 , Fv fragments, scFv antibody fragments, disulfide- linked Fvs, a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies (sdAb) such as either vL or vH, camelid vHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody.
  • An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005).
  • Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see U.S.
  • Patent No.: 6,703,199 which describes fibronectin polypeptide mini bodies).
  • antibody heavy chain refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.
  • antibody light chain refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (K) and lambda (l) light chains refer to the two major antibody light chain isotypes.
  • Anticancer agent refers to agents that inhibit aberrant cellular division and growth, inhibit migration of neoplastic cells, inhibit invasiveness or prevent cancer growth and metastasis. The term includes chemotherapeutic agents, biological agent (e.g., siRNA, viral vectors such as engineered MLV, adenoviruses, herpes virus that deliver cytotoxic genes), antibodies and the like.
  • anticancer effect refers to a biological effect which can be manifested by various means including, but not limited to, a decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, decrease in cancer cell proliferation, decrease in cancer cell survival, or amelioration of various physiological symptoms associated with the cancerous condition.
  • An “anticancer effect” can also be manifested by the ability of a CAR in prevention of the occurrence of cancer in the first place.
  • antigen refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically- competent cells, or both.
  • antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein.
  • an antigen need not be encoded solely by a full length nucleotide sequence of a gene.
  • the disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response.
  • an antigen need not be encoded by a "gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide.
  • a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.
  • Non-limiting examples of antigens that can be targeted include: CD5; CD19; CD20; CD22; CD24; CD30; CD33, CD34; CD38, CD72; CD97; CD123; CD171; CS1 (also referred to as CD2 subset 1, CRACC, MPL, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2- 3)bDGalp(l-4 )bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase like orphan receptor 1 (ROR1);
  • CD43 epitope Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha (IL-llRa); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage- specific embryonic antigen-4 (SSEA-4); Folate receptor alpha (FRa or FR1); Folate receptor beta (FRb); Receptor tyrosine-protein kinase ERBB2 (Her2/neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; pro
  • HAVCR1 Hepatitis A virus cellular receptor 1
  • ADRB3 adrenoceptor beta 3
  • PANX3 pannexin 3
  • GPR20 G protein-coupled receptor 20
  • LY6K lymphocyte antigen 6 complex, locus K 9
  • OR51E2 Olfactory receptor 51E2
  • TCR Gamma Alternate Reading Frame Protein TARP
  • WT1 Wilms tumor protein
  • NY-ESO-1 Cancer/testis antigen 1
  • Cancer/testis antigen 2 (LAGE-la); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member
  • XAGE1 angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumor antigen-1 (PCT A-l or Galectin 8); melanoma antigen recognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N- Acetyl glucosaminyl-transferase V (NA17); paired box protein Pax-3 (
  • Cytochrome P450 IB 1 (CYP1B 1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation End products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD
  • GPC3 Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1); MPL; Biotin; c-MYC epitope Tag; LAMP1 TROP2; GFRalpha4; CDH17; CDH6; NYBR1; CDH19; CD200R; Slea (CA19.9; Sialyl Lewis Antigen); Fucosyl-GMl; PTK7; gpNMB; CDH1-CD324; DLL3;
  • an "antigen binding domain” refers to a polypeptide or peptide that due to its primary, secondary or tertiary sequence, post-translational modifications and/or charge binds to an antigen with a high degree of specificity.
  • the antigen binding domain may be derived from different sources, for example, an antibody (full length heavy chain, Fab fragments, single chain Fv (scFv) fragments, divalent single chain antibodies or diabodies), a non-immunoglobulin binding protein, a ligand or a receptor.
  • the antigen binding domain comprises T cell receptors (TCRs) or portions thereof.
  • anti-infection effect refers to a biological effect that can be manifested by various means, including but not limited to, e.g., decrease in the titer of the infectious agent, a decrease in colony counts of the infectious agent, amelioration of various physiological symptoms associated with the infectious condition.
  • anti-cancer effect refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, inhibition of metastasis, or a decrease in tumor cell survival.
  • “beneficial results” may include, but are not limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient or subject developing the disease condition and prolonging a patient's or subject's life or life expectancy.
  • “beneficial results” may be alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of cancer progression, delay or slowing of metastasis or invasiveness, and amelioration or palliation of symptoms associated with the cancer.
  • biological equivalent thereof is intended to be synonymous with “equivalent thereof” when referring to a reference protein, antibody or fragment thereof, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure and/or functionality.
  • an equivalent intends at least about 70% homology or identity, or at least 80% homology or identity and alternatively, or at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively at least 98% percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide, antibody or fragment thereof or nucleic acid.
  • an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement and which has the same biological function (e.g., binds to a specific miRNA or encodes a protein or polypeptide having the same or similar biological effect to the polynucleotide to which it is being compared).
  • an equivalent thereof is an expressed polypeptide or protein from a polynucleotide that hybridizes under stringent conditions to the polynucleotide or its complement that encodes the reference polypeptide or protein.
  • Cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • Examples of cancer include, but are not limited to, B-cell lymphomas (Hodgkin's lymphomas and/or non- Hodgkin's lymphomas), T cell lymphomas, myeloma, myelodysplastic syndrome, myeloproliferative disorders (e.g., polycythemia vera, myelofibrosis, essential thrombocythemia etc.), skin cancer, brain tumor, breast cancer, colon cancer, rectal cancer, esophageal cancer, anal cancer, cancer of unknown primary site, endocrine cancer, testicular cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, cancer of reproductive organs thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, brain cancer (e.g., B-cell lymph
  • tumor and cancer are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors.
  • cancer or tumor includes premalignant, as well as malignant cancers and tumors.
  • cancer is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • cell-activity-regulating domain refers to any one or more of PDL1, PDL2, CD80, CD86, crmA, p35, NEMO-K277A (or derivative thereof), K13-opt, IKK2-SS/EE, IKK1-SS/EE, 41BBL, CD40L, VFLIP-K13, MC159, and the like and combination thereof that is expressed in an immune cell (e.g., T cell, e.g., CAR-T cell etc.) to decrease, regulate or modify the activity of the immune cell.
  • an accessory module is co-expressed with an immune receptor such as a CAR to increase, decrease, regulate or modify the expression or activity of a CAR or a CAR-expressing cell.
  • the accessory module can be co-expressed with a CAR using a single vector or using two or more different vectors.
  • chemotherapeutic agents are compounds that are known to be of use in chemotherapy for cancer.
  • Non-limiting examples of chemotherapeutic agents can include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; a camptothecin (including the synthetic analogue topotecan); bryostatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin),
  • CARs Chimeric antigen receptors
  • T cell immune cell
  • chimeric T-cell receptors or chimeric immunoreceptors.
  • CARs are constructed specifically to stimulate T cell activation and proliferation in response to a specific antigen to which the CAR binds.
  • a CAR refers to a set of polypeptides, typically two in the simplest embodiments, which when expressed in an immune effector cell, provides the cell with specificity for a target antigen or cell, typically a cancer cell, and with intracellular signal generation.
  • a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "an intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and/or costimulatory molecule.
  • the set of polypeptides are contiguous with each other.
  • the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane.
  • CARs are recombinant polypeptides comprising an antigen binding domain, a hinge region (HR), a transmembrane domain (TMD), an optional co-stimulatory domain (CSD) and an intracellular signaling domain (ISD).
  • the optional costimulatory domain is generally absent in 1 st generation CAR constructs.
  • Second (2 nd ) generation CARs comprising antigen binding domains (e.g., vL and vH fragments, vHH, ligands and receptors etc.) typically incorporate a costimulatory domain (e.g., 41BB).
  • CAR CAR
  • CARs also encompasses newer approaches to conferring antigen specificity onto cells, such as Antibody-TCR chimeric molecules or Ab-TCR (W02017/070608A1 incorporated herein by reference), TCR receptor fusion proteins or TFP (WO2016/187349A1 incorporated herein by reference), Tri functional T cell antigen coupler (Tri-TAC or TAC) (see, WO2015/117229A1, incorporated herein by reference).
  • Tri-TAC or TAC Tri functional T cell antigen coupler
  • the term “CAR-T cell” is used, to refer to T-cells that have been engineered to express a chimeric antigen receptor.
  • CAR-T lymphocytes T lymphocytes bearing such CARs are generally referred to as CAR-T lymphocytes.
  • CARs can be also expressed in cells other than T cells, such as hematopoietic stem cells, induced pluripotent stem cells (iPSC), NK cells and macrophage.
  • iPSC induced pluripotent stem cells
  • Codon optimization or "controlling for species codon bias” refers to the preferred codon usage of a particular host cell.
  • the genetic code is redundant with 64 possible codons, but most organisms typically use a subset of these codons.
  • the codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons.
  • the coding sequences of a vector of the disclosure can be modified to limit ApoBec mediated mutations.
  • the vectors of the disclosure can be engineered to modify their stability and/or expression.
  • changes in expression can occur due to the frequency with which inactivating or attenuating mutations accumulate in the vector as it replicates in a cell. Investigation shows that one of the most frequent events is G to A mutations (corresponds to the C to T mutation) characteristic ApoBec mediated mutations in the negative strand of single stranded DNA from the first replicative step. This can cause changes in amino acid composition of vector-encoded proteins and a devastating change from TGG (Tryptophan) to stop codons (TAG or TGA). In one embodiment this inactivating change is avoided by substitution codons of other amino acids with similar chemical or structural properties such as phenylalanine or tyrosine at position of ApoBec modifications.
  • Such mutations can include modifications of one or more codons in the coding sequences of vector domains that change a tryptophan codon to a permissible codon that maintains the biological activity of the encoded protein.
  • the codon for tryptophan is UGG (TGG in DNA).
  • the "stop codon” is UAA, UAG or UGA (TAA, TAG or TGA in DNA).
  • a single point mutation in the tryptophan codon can cause an unnatural stop codon (e.g., UGG -> UAG or UGG -> UGA).
  • human APOBEC3GF hA3G/F
  • inhibits retroviral replication through G -> A hypermutations Neogi et al., J. Int.
  • the disclosure contemplates modifications to the coding sequences of vectors of the disclosure to reduce ApoBec hypermutations by modifying tryptophan codons to permissible non-tryptophan codons.
  • a “conservative substitution” or “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics or function of the encoded protein.
  • “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics or function of a CAR construct of the disclosure (e.g., a conservative change in the constant chain, antibody, antibody fragment, or non- immunoglobulin binding domains). Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g.
  • Co-stimulatory domain refers to a biological agent that enhances the proliferation, survival and/or development of T cells.
  • a co-stimulatory domain can comprises the costimulatory domain of any one or more of, for example, members of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (0X40), DaplO,
  • CD30 Fas, CD30, CD40 or combinations thereof.
  • Other co-stimulatory domains e.g., from other proteins
  • Cytokine Release Syndrome is a complication of cell therapies (e.g., CAR-T, bispecific T cell engaging antibodies etc.) that manifests itself with signs and symptoms such as fever, hypotension, shortness of breath, renal dysfunction, pulmonary dysfunction and/or capillary leak syndrome.
  • CRS is usually due to excessive production of cytokines, such as IL6 and IL1.
  • “Derived from” indicates a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and a second molecule and does not include a process or source limitation on a first molecule that is derived from a second molecule. For example, in the case of an antigen binding domain that is derived from an antibody molecule, the antigen binding domain retains sufficient antibody structure such that is has the required function, namely, the ability to bind to an antigen. It does not include any limitation to a particular process of producing the antibody.
  • Domain or “module” refers to a discrete section or part of a larger construct that can be replaced with a similar domain without affecting the function of other domains or module of the construct.
  • a chimeric antigen receptor polypeptide or coding nucleic acid sequence can be described as having a binding domain, a transmembrane domain and an intracellular domain.
  • Each "domain” of the CAR can be modified or changed without affecting the other domains of the CAR.
  • the binding domain can be one of a number of different binding domains as described herein.
  • the binding domain can be a polypeptide sequence that binds to a CD19 antigen.
  • This CD19 binding domain can be replaced with a binding domain that binds to CD20 without affecting of having to change the transmembrane domain.
  • a retroviral vector of the disclosure contained in a viral capsid comprises a polynucleotide sense RNA strand having a number of domains including (from 5' to 3'): 5'Repeat(5'R)—U5—packaging sequence—CAR sequence— (optional kill switch comprising an IRES domain linked to, e.g., thymidine kinase coding sequence)—miRNA targeting sequence(s)-U3- 3'Repeat(3'R).
  • Each domain/module of the viral polynucleotide can be changed such that different CAR sequences can be provided, different kill switches (e.g., TKO or CD), different miRNA targeting sequences etc.
  • the construct of the disclosure are modular in design.
  • Each domain/module of the construct, whether a polynucleotide construct or an encoded polypeptide construct can comprise minor variations in sequence so long as the variations do not destroy the biological activity of the domain.
  • a transmembrane domain can have 80-100% identity to a specific transmembrane sequence.
  • genetically engineered cells or “modified cells” as used herein refer to cells that express, for example, a CAR.
  • the genetically modified cells comprise vectors that encode a CAR.
  • Hinge region refers to the hydrophilic region which is between the antigen binding domain and the transmembrane domain of a CAR.
  • the hinge region includes, but is not limited to, Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions of antibodies, artificial spacer sequences or combinations thereof.
  • Examples of hinge regions include, but are not limited to, CD8a hinge, and artificial spacers made of polypeptides which may be as small as, for example, Gly3 or CHI and CH3 domains of IgGs (such as human IgG4).
  • the hinge region is any one or more of (i) a hinge, CH2 and CH3 regions of IgG4, (ii) a hinge region of IgG4, (iii) a hinge and CH2 of IgG4, (iv) a hinge region of CD8a, (v) a hinge, CH2 and CH3 regions of IgGl, (vi) a hinge region of IgGl or (vi) a hinge and CH2 region of IgGl.
  • Other hinge regions will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the disclosure.
  • Immuno cell refers to the cells of the mammalian immune system including, but not limited to, antigen presenting cells, B-cells, basophils, cytotoxic T-cells, dendritic cells, eosinophils, granulocytes, helper T-cells, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monocytes, natural killer cells, neutrophils, phagocytes, plasma cells and T- cells.
  • In vivo Immune cells refer to immune cells present in the body of a subject that have not been isolated or removed from the subject.
  • Immuno effector cell refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response.
  • immune effector cells include T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
  • T cells e.g., alpha/beta T cells and gamma/delta T cells
  • B cells e.g., natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
  • NK natural killer
  • NKT natural killer T
  • cytoplasmic domain refers to an intracellular signaling portion of a molecule.
  • the intracellular signaling domain generates a signal that promotes an immune effector function of the cell.
  • immune effector function include cytolytic activity and helper activity, including the secretion of cytokines.
  • domains that transduce the effector function signal include, but are not limited to, the z chain of the T-cell receptor complex or any of its homologs (e.g., h chain, FceRlg and b chains, MB1 (Iga) chain, B29 (Igb) chain, etc.), human CD3 zeta chain, CD3 polypeptides (D, d and e), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5 and CD28.
  • Other intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the disclosure.
  • the intracellular signaling domain can comprise a primary intracellular signaling domain.
  • Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation.
  • the intracellular signaling domain can comprise a costimulatory intracellular domain.
  • Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.
  • a primary intracellular signaling domain can comprise a cytoplasmic sequence of CD3z or CD3zlxx (Feucht et al. Nt.
  • a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from co-receptor or costimulatory molecule, such as CD28 or 41BB.
  • a primary intracellular signaling domain can comprise a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or ITAM.
  • ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, common FeR gamma (FCER1G), Fe gamma Rlla, FeR beta (Fe Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.
  • isolated refers to molecules or biologies or cellular materials being substantially free from other materials.
  • the term “isolated” refers to nucleic acid, such as DNA or RNA; protein or polypeptide; cell or cellular organelle(s); or tissue, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, which are present in the natural source.
  • isolated also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • an "isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
  • isolated is also used herein to refer to cells or tissues that are isolated from other cells or tissues and is meant to encompass both, cultured and engineered cells or tissues.
  • linker refers to an oligo or a peptide that joins together two or more domains or regions of a CAR polynucleotide or polypeptide, respectively.
  • the linker can be anywhere from 1 to 500 amino acids in length or 3 to 1500 nucleotide in length.
  • the "linker” is cleavable or non-cleavable.
  • linker used herein means a non- cleavable linker. Said non-cleavable linkers may be composed of flexible amino acids residues which allow freedom of motion of adjacent protein domains relative to one another.
  • linkers include non-flexible amino acid residues.
  • examples of cleavable linkers include 2A linkers (for example T2A), 2A-like linkers or functional equivalents thereof and combinations thereof.
  • the linkers include the picornaviral 2A-like linker, CHYSEL sequences of porcine teschovirus (P2A),
  • the linker sequences may comprise a motif that results in cleavage between the 2A glycine and the 2B proline.
  • Other cleavable linkers that may be used herein are readily appreciated by those of skill in the art.
  • flexible polypeptide linker refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link polypeptide chains together (e.g., variable heavy and variable light chain regions together).
  • the flexible polypeptide linker is a Gly/Ser linker and comprises the amino acid sequence (Gly3-Ser) n , where n is a positive integer equal to or greater than 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 etc.).
  • Mammalia including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.
  • CAR constructs and related sequences are optimized for use in a particular mammalian species (e.g., the encoded protein/polypeptide is derived from the mammalian species being treated).
  • off-target transduced cells refers to cells that are infected by a viral vector of the disclosure, but where expression of the vector's genes are unwanted or undesirable. It will be recognized in the art that viral vectors can be "targeted” through incorporation of targeting proteins on the viral envelop. In addition, or alternatively, the expression of a viral gene can be controlled through the use of tissue specific promoters. In still other or further embodiments, the expression of the viral gene/construct can be controlled through the use of cellular machinery that exists to control innate gene expression control. In this instance RNAi target sequence can be used, whereby binding of innate miRNA to the target sequences can be used to control expression in an off-target cell type.
  • operably linked refers to functional linkage or association between a first component and a second component such that each component can be functional.
  • operably linked includes the association between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a first polypeptide functions in the manner it would independent of any linkage and the second polypeptide functions as it would absent a linkage between the two.
  • Percent identity in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences that are related by percent sequence identity. Two sequences are "substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same ⁇ e.g., 60% identity, optionally 70%, 71%. 72%.
  • the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more typically over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
  • sequence comparison For sequence comparison, generally one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • Methods of alignment of sequences for comparison are well known in the art and are publicly available. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math.
  • NCBI National Center for Biotechnology Information
  • the percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (1970) J. Mol. Bioi.
  • RNAi target sequence or "miR target sequence” or “miR target cassette” as used herein, relates to a nucleic acid sequence specifically hybridizing to a dsRNA inducing RNA interference (interfering RNA).
  • the RNAi target sequence is a sequence essentially complementary to at least one RNAi inducing molecule (interfering RNA).
  • the RNAi target sequence is a miRNA target sequence or a siRNA target sequence.
  • the RNAi target sequence is a miRNA target sequence.
  • the disclosure provides polynucleotide constructs containing a coding sequence for a CAR, one or more RNAi targeting sequences and an optional kill switch coding sequence.
  • single chain variable region refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a synthetic linker e.g., a short flexible polypeptide linker
  • an scFv may have the vL and vH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise vL-linker-vH or may comprise vH-linker-vL. Alternatively, a scFv is also described as (vL+vH) or (vH+vL).
  • signaling domain refers to the functional region of a protein which transmits information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.
  • the term "subject” is intended to include living organisms in which an immune response can be elicited (e.g., any domesticated mammals or a human).
  • the terms "subject” or “individual” or “animal” or “patient” are used interchangeably herein to refer to any subject, particularly a mammalian subject, for whom administration of a composition or pharmaceutical composition of the disclosure is desired.
  • Mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like, with humans being preferred.
  • T-cell and “T-lymphocyte” are interchangeable and used synonymously herein. Examples include but are not limited to naive T cells ("lymphocyte progenitors”), central memory T cells, effector memory T cells, stem memory T cells (T SCm ), iPSC-derived T cells, synthetic T cells or combinations thereof.
  • the term "therapeutic effect” refers to a biological effect which can be manifested by various means, including but not limited to, e.g., decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, decrease in cancer cell proliferation, decrease in cancer cell survival, decrease in the titer of the infectious agent, a decrease in colony counts of the infectious agent, amelioration of various physiological symptoms associated with a disease condition.
  • a “therapeutic effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies in prevention of the occurrence of disease in the first place or in the prevention of relapse of the disease.
  • terapéuticaally effective amount refers to the amount of a pharmaceutical composition comprising vector or in vivo genetically engineered cells, to decrease at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect.
  • therapeutically effective amount means a sufficient amount of the composition to treat a disorder, at a reasonable benefit/risk ratio applicable to any medical treatment.
  • a therapeutically or prophylactically significant reduction in a symptom is, e.g. at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150% or more in a measured parameter as compared to a control or non-treated subject or the state of the subject prior to administering the a vector as described herein.
  • Measured or measurable parameters include clinically detectable markers of disease, for example, elevated or depressed levels of a biological marker, as well as parameters related to a clinically accepted scale of symptoms or markers for cancer.
  • Transmembrane domain refers to the region of the CAR which crosses the plasma membrane.
  • the transmembrane domain of the CAR of the disclosure is the transmembrane region of a transmembrane protein (for example Type I transmembrane proteins), an artificial hydrophobic sequence or a combination thereof.
  • Other transmembrane domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the disclosure.
  • the TMD is selected from the transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD3y, CD3s, CD36, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134,
  • NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM
  • Vector refers to the vehicle by which a polynucleotide sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.
  • Vectors include plasmids, phages, viruses, etc.
  • viral vector refers to a vector obtained or derived from a virus.
  • the virus is a retrovirus including, but not limited to, lentiviruses and gamma retroviruses.
  • the viral vector of the disclosure may be a retroviral vector, such as a gamma-retroviral vector.
  • the viral vector may be based on human immunodeficiency virus.
  • the viral vector of the disclosure may be a lentiviral vector.
  • the vector may be based on a non-primate lentivirus such as equine infectious anemia virus (EIAV).
  • EIAV equine infectious anemia virus
  • the viral vector of the disclosure comprises a mitogenic T-cell activating transmembrane protein and/or a cytokine-based T-cell activating transmembrane protein in the viral envelope.
  • the mitogenic T-cell activating transmembrane protein and/or cytokine-based T-cell activating transmembrane protein is/are derived from the host cell membrane, as explained above.
  • virus like particle or "VLP” refers to a viral particle lacking a viral genome. In some cases the VLP lacks an env protein. As with complete viral particles they contain an outer viral envelope made of the host cell lipid-bi-layer (membrane), and hence contain host cell transmembrane proteins.
  • a VLP can be used in the methods and compositions of the disclosure.
  • the disclosure provides a recombinant viral vector comprising a plurality of copies of one or more miRNA target sequences inserted into the vector to control expression of coding sequence contained in the vector (e.g., CAR coding sequences) in off-target transduced cells.
  • a recombinant viral vector may comprise miRNA target sequence(s) inserted into encapsidated viral polynucleotide.
  • miRNAs expressed in off-target cells can bind to such miRNA target sequence(s) and suppress expression of the viral polynucleotide containing the miRNA target sequence, thereby limiting viral replication and/or expression of vector-containing coding sequences (e.g., CARs) in the off-target transduced cells.
  • vector-containing coding sequences e.g., CARs
  • Such recombinant viral vectors can be referred to herein as "miR-attenuated”, “expression-restricted vectors” or “replication-restricted vectors” as they demonstrate reduced or attenuated replication and/or expression of vector-containing coding sequences in cells that express one or more miRNAs capable of binding to the incorporated miR target sequence(s) compared to cells that do not express, or have reduced expression of, the miR.
  • the one or more miRNA target sequence(s) is incorporated into the 3' untranslated region (UTR) and/or 3' UTR downstream of a CAR coding sequence.
  • the mRNA transcripts of a CAR coding sequence comprises an miR- target sequence (TS) comprising one or more miRNA target sequences (e.g., a miRNA target sequence cassette).
  • TS miR- target sequence
  • the miR-TS cassettes described herein comprise at least one miRNA target sequence.
  • the miR-TS cassettes described herein comprise a plurality of miRNA target sequences.
  • the miR-TS cassettes described herein comprise 2, 3, 4,
  • the miR-TS cassettes comprise two or more miRNA target sequences
  • the two or more target sequences can be the same of different.
  • the miR-TS cassettes comprise a plurality miRNA target sequences, wherein each miRNA target sequence of the plurality is a target sequence for the same miRNA.
  • the miR-TS cassettes may comprise 2, 3, 4, 5, 6, 7, 8, 9,
  • the miR-TS cassettes comprise between 2 to 6 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 3 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 4 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 5 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 6 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 7 copies of the same miR target sequence.
  • the miR-TS cassettes comprise 8 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 9 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 10 copies of the same miR target sequence. [0093] In some embodiments, the miR-TS cassettes described herein comprise a plurality of miRNA target sequences, wherein the plurality comprises at least two different miRNA target sequences.
  • the miR-TS cassettes described herein comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 different miRNA target sequences.
  • the miR-TS cassette may comprise one or more copies of a first miRNA target sequence and one or more copies of a second miRNA target sequence.
  • the miR-TS cassette comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a first miR target sequence and at least 2, 3, 4, 5,
  • the miR-TS cassette comprises 3 or 4 copies of a first miR target sequence and 3 or 4 copies of a second miR target sequence.
  • the plurality of miRNA target sequences comprises at least 3 different miRNA target sequences.
  • the miR-TS cassette comprises one or more copies of a first miR target sequence, one or more copies of a second miR target sequence, and one or more copies of a third miR target sequence.
  • the miR-TS cassette comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a first miR target sequence, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a second miR target sequence, and at least 2, 3, 4, 5, 6, 7, 8,
  • the miR-TS cassette comprises 3 or 4 copies of a first miR target sequence, 3 or 4 copies of a second miR target sequence, and 3 or 4 copies of a third miR target sequence.
  • the plurality of miRNA target sequences comprises at least 4 different miRNA target sequences.
  • the miR-TS cassette comprises one or more copies of a first miR target sequence, one or more copies of a second miR target sequence, one or more copies of a third miR target sequence, and one or more copies of a fourth miR target sequence.
  • the miR-TS cassette comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a first miR target sequence, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a second miR target sequence, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a third miR target sequence, and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a fourth miR target sequence.
  • the miR-TS cassette comprises 3 or 4 copies of a first miR target sequence, 3 or 4 copies of a second miR target sequence, 3 or 4 copies of a third miR target sequence, and 3 or 4 copies of a fourth miR target sequence.
  • the miR-TS cassettes comprise a plurality of miRNA target sequences
  • the plurality of miRNA target sequences may be arranged in tandem, without any intervening nucleic acid sequences.
  • the plurality of miRNA target sequences may be separated by a linker sequence.
  • the linker sequence comprises 2, 3, 4, 5, 6, 7, 8,
  • a miR-TS cassette may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the following subunits: a first miRNA target sequence-linker-a second miRNA target sequence, wherein adjacent subunits are separated by an additional linker sequence.
  • the first and the second miRNA target sequence are targets of the same miRNA. In some embodiments, the first and the second miRNA target sequence are targets of different miRNAs.
  • the miR target sequence is a target sequence for miR-1251-5p, miR-219a-5p, miR-219a-2-3p, miR-124-3p, miR-448, miR-138-2-3p, miR-490-5p, miR-129-l-3p, miR-1264, miR-3943, miR-490-3p, miR-383-5p, miR-133b, miR-129-2-3p, miR-128-2-5p, miR- 133a-3p, miR-129-5p, miR-l-3p, miR-885-3p, miR-124-5p, miR-759, miR- 7158-3p, miR-770-5p, miR-135a-5p, miR-885-5p, let-7g-5p, miR-100, miR-101, miR-106a, miR-124, miR-124a, miR-125a, miR-125a-5p, miR- 125b, miR-
  • the miR target sequence is a target sequence for miR-10b-5p, miR-126-3p, miR-145-3p, miR-451a, miR-199b- 5p, miR-5683, miR-3195, miR-3182, miR-1271-5p, miR-204-5p, miR-409- 5p, miR-136-5p, miR-514a-5p, miR-559, miR-483-3p, miR-l-3p, miR- 6080, miR-144-3p, miR-10b-3p, miR-6130, miR-6089, miR-203b-5p, miR- 4266, miR-4327, miR-5694, miR-193b, let-7a, let-7a-l, let-7a-2, let- 7a-3, let-7b, let-7c, let-7d, let-7e, let-7f-l, let-7f-2, let-7g, let-7i, miR-100, miR-107,
  • the miR target sequence is a target sequence for miR-143, miR-145, miR-17-5p, miR-203, miR-214, miR-218, miR-335, miR-342-3p, miR-372, miR-424, miR-491-5p, miR-497, miR-7, miR-99a, miR-99b, miR-100, miR-101, miR-15a, miR-16, miR-34a, miR- 886-5p, miR-106a, miR-124, miR-148a, miR-29a, and/or miR-375.
  • the vector comprises a CAR coding sequence used to treat cervical cancer.
  • the miR target sequence is a target sequence for miR-133a-5p, miR-490-5p, miR-124-3p, miR-137, miR-655- 3p, miR-376c-3p, miR-369-5p, miR-490-3p, miR-432-5p, miR-487b-3p, miR-342-3p, miR-223-3p, miR-136-3p, miR-136-3p, miR-143-5p, miR-1- 3p, miR-214-3p, miR-143-3p, miR-199a-3p, miR-199b-3p, miR-451a, miR- 127-3p, miR-133a-3p, miR-145-5p, miR-145-3p, miR-199a-5p, let-7a-l, let-7a-2, let-7a-3, let-7b, let-7c, let-7d, let-7e, let-7f-l, let- 7f-2, let-7g, let-7
  • the miR target sequence is a target sequence for miR-101, miR-130a, miR-130b, miR-134, miR-143, miR-145, miR-152, miR-205, miR-223, miR-301a, miR-301b, miR-30c, miR-34a, miR-34c, miR-424, miR-449a, miR-543, and/or miR-34b.
  • the vector comprises a CAR coding sequence used to treat endometrial cancer.
  • the miR target sequence is a target sequence for miR-125b, miR-138, miR-15a, miR-15b, miR-16, miR-16-1, miR-16-l-3p, miR-16-2, miR-181a, miR-181b, miR-195, miR-223, miR- 29b, miR-34b, miR-34c, miR-424, miR-lOa, miR-146a, miR-150, miR-151, miR-155, miR-2278, miR-26a, miR-30e, miR-31, miR-326, miR-564, miR- 27a, let-7b, miR-124a, miR-142-3p, let-7c, miR-17, miR-20a, miR-29a, miR-30c, miR-720, miR-107, miR-342, miR-34a, miR-202, miR-142-5p, miR-29c, miR-
  • the miR target sequence is a target sequence for miR-1, miR-145, miR-1826, miR-199a, miR-199a-3p, miR- 203, miR-205, miR-497, miR-508-3p, miR-509-3p, let-7a, let-7d, miR- 106a*, miR-126, miR-1285, miR-129-3p, miR-1291, miR-133a, miR-135a, miR-138, miR-141, miR-143, miR-182-5p, miR-200a, miR-218, miR-28-5p, miR-30a, miR-30c, miR-30d, miR-34a, miR-378, miR-429, miR-509-5p, miR-646, miR-133b, let-7b, let-7c, miR-200c, miR-204, miR-335, miR- 377, and/or miR-506.
  • miR-106a* miR
  • the miR target sequence is a target sequence for let-7a-l, let-7a-2, let-7a-3, let-7b, let-7c, let-7d, let-7e, let-7f, let-7f-l, let-7f-2, let-7g, let-7i, miR-1, miR-100, miR-101, miR-105, miR-122, miR-122a, miR-1236, miR-124, miR-125b, miR-126, miR-127, miR-1271, miR-128-3p, miR-129-5p, miR-130a, miR- 130b, miR-133a, miR-134, miR-137, miR-138, miR-139, miR-139-5p, miR- 140-5p, miR-141, miR-142-3p, miR-143, miR-144, miR-145, miR-146a, miR-148a, miR-148b, miR-150-5p, miR-141, miR-142
  • the miR target sequence is a target sequence for miR-143-3p, miR-126-3p, miR-126-5p, miR-1266-3p, miR- 6130, miR-6080, miR-511-5p, miR-143-5p, miR-223-5p, miR-199b-5p, miR-199a-3p, miR-199b-3p, miR-451a, miR-142-5p, miR-144, miR-150-5p, miR-142-3p, miR-214-3p, miR-214-5p, miR-199a-5p, miR-145-3p, miR- 145-5p, miR-1297, miR-141, miR-145, miR-16, miR-200a, miR-200b, miR- 200c, miR-29b, miR-381, miR-409-3p, miR-429, miR-451, miR-511, miR- 99a, let-7a-l, let-7a-2
  • the miR target sequence is a target sequence for let-7b, miR-101, miR-125b, miR-1280, miR-143, miR-146a, miR-146b, miR-155, miR-17, miR-184, miR-185, miR-18b, miR-193b, miR- 200c, miR-203, miR-204, miR-205, miR-206, miR-20a, miR-211, miR-218, miR-26a, miR-31, miR-33a, miR-34a, miR-34c, miR-376a, miR-376c, miR- 573, miR-7-5p, miR-9, and/or miR-98.
  • the vector comprises a CAR coding sequence used to treat melanoma.
  • the miR target sequence is a target sequence for let-7d, miR-218, miR-34a, miR-375, miR-494, miR-100, miR-124, miR-1250, miR-125b, miR-126, miR-1271, miR-136, miR-138, miR-145, miR-147, miR-148a, miR-181a, miR-206, miR-220a, miR-26a, miR-26b, miR-29a, miR-32, miR-323-5p, miR-329, miR-338, miR-370, miR-410, miR-429, miR-433, miR-499a-5p, miR-503, miR-506, miR-632, miR-646, miR-668, miR-877, and/or miR-9.
  • the vector comprises a CAR coding sequence used to treat oral cancer.
  • the miR target sequence is a target sequence for let-7i, miR-100, miR-124, miR-125b, miR-129-5p, miR- 130b, miR-133a, miR-137, miR-138, miR-141, miR-145, miR-148a, miR- 152, miR-153, miR-155, miR-199a, miR-200a, miR-200b, miR-200c, miR- 212, miR-335, miR-34a, miR-34b, miR-34c, miR-409-3p, miR-411, miR- 429, miR-432, miR-449a, miR-494, miR-497, miR-498, miR-519d, miR- 655, miR-9, miR-98, miR-101, miR-532-5p, miR-124a, miR-192, miR- 193a, and/or miR-7
  • the miR target sequence is a target sequence for miR-216a-5p, miR-802, miR-217, miR-145-3p, miR-143-3p, miR-451a, miR-375, miR-214-3p, miR-216b-3p, miR-432-5p, miR-216a-3p, miR-199b-5p, miR-199a-5p, miR-136-3p, miR-216b-5p, miR-136-5p, miR- 145-5p, miR-127-3p, miR-199a-3p, miR-199b-3p, miR-559, miR-129-2-3p, miR-4507, miR-l-3p, miR-148a-3p, miR-101, miR-1181, miR-124, miR- 1247, miR-133a, miR-141, miR-145, miR-146a, miR-148a, miR-148b, miR- 150*
  • the miR target sequence is a target sequence for let-7a-3p, let-7c, miR-100, miR-101, miR-105, miR-124, miR-128, miR-1296, miR-130b, miR-133a-l, miR-133a-2, miR-133b, miR- 135a, miR-143, miR-145, miR-146a, miR-154, miR-15a, miR-187, miR- 188-5p, miR-199b, miR-200b, miR-203, miR-205, miR-212, miR-218, miR- 221, miR-224, miR-23a, miR-23b, miR-25, miR-26a, miR-26b, miR-29b, miR-302a, miR-30a, miR-30b, miR-30c-l, miR-30c-2, miR-30d, miR-30e, miR-31, miR-330
  • the miR target sequence is a target sequence for miR-101, miR-183, miR-204, miR-34a, miR-365b-3p, miR- 486-3p, and/or miR-532-5p.
  • the vector comprises a CAR coding sequence used to treat retinoblastoma.
  • the miR target sequence is a target sequence for miR-143-3p, miR-133b, miR-1264, miR-448, miR-1298-5p, miR-490-5p, miR-138-2-3p, miR-144-3p, miR-144-5p, miR-150-5p, miR- 129-l-3p, miR-559, miR-l-3-p, miR-143-5p, miR-223-3p, miR-3943, miR- 338-3p, miR-124-3p, miR-219a-5p, miR-219a-2-3p, miR-451a, miR-142- 5p, miR-133a-3p, miR-145-5p, and/or miR-145-3p.
  • the vector comprises a CAR coding sequence used to treat glioblastoma.
  • the miR target sequence is a target sequence for miR-143-3p, miR-223-3p, miR-6080, miR-208b-3p, miR-206, miR-133a-5p, miR-133b, miR-199a-5p, miR-199b-5p, miR-145-3p, miR- 145-5p, miR-150-5p, miR-142-3p, miR-144-3p, miR-144-5p, miR-338-3p, miR-214-3p, miR-559, miR-133a-3p, miR-l-3p, miR-126-3p, miR-142-5p, miR-451a, miR-199a-3p, and/or miR-199b-3p.
  • the vector comprises a CAR coding sequence used to treat head and neck cancer.
  • the methods and compositions can be used to treat a number of disease and disorders.
  • binding domains targeting any number of the "Targets" listing in Table 1 can be used to treat diseases associated with the target: [00113] Table 1:
  • the disclosure provides methods to provide adoptive cell immunity to a subject in need thereof comprising administering a vector of the disclosure encoding a chimeric antigen receptor (CAR) to the subject such that the CAR is selectively expressed in a desired immune cell type or immune cell stem cell (e.g., hematopoietic stem cell).
  • the method includes administering a viral construct comprising a viral capsid and envelope containing a polynucleotide derived from a viral genome.
  • the polynucleotide comprises RNA.
  • the polynucleotide is derived from a gamma retrovirus.
  • the polynucleotide comprises long terminal repeats at the 5' and 3' end.
  • the polynucleotide comprises a coding sequence for a CAR. In yet another embodiment, the polynucleotide comprises one or more miRNA target sequence. In still another embodiment, the miRNA target sequence are targets for miRNA present in off-target cells. In still another embodiment, the polynucleotide comprises a sequence encoding a polypeptide that converts a prodrug to a toxic drug.
  • the disclosure provides a plasmid comprising a sequence that produces a polynucleotide that is encapsulated into a viral capsid.
  • the polynucleotide comprises RNA.
  • the polynucleotide is derived from a gamma retrovirus.
  • the polynucleotide comprises long terminal repeats at the 5' and 3' end.
  • the polynucleotide comprises a coding sequence for a CAR.
  • the polynucleotide comprises one or more miRNA target sequence.
  • the miRNA target sequence are targets for miRNA present in off-target cells, but not in target cells, nor in cells used to make infectious vectors.
  • the polynucleotide comprises a sequence encoding a polypeptide that converts a prodrug to a toxic drug.
  • the disclosure provide a viral polynucleotide construct comprising from 5' to 3', an "R-U5" domain from a gammaretrovirus operably linked to a coding sequence for a binding domain operably linked to a hinge/linker coding sequence operably linked to a transmembrane domain coding sequence operably linked to a signaling domain coding sequence followed by one or more miRNA target sequences (miR-TS; miR target cassette) followed by a "U3-R" domain from a gammaretrovirus.
  • the viral RNA can include a coding sequence for a kill switch operably linked to an IRES.
  • the IRES-Kill switch can be upstream or downstream (5' or 3') to the miRNA cassette.
  • the polynucleotide sequence can be schematically presented as (see also Fig. 1):
  • the R-U5 domain can comprise a sequence that is at least 80-100% identical to the sequence:
  • the R-U5-packaging domains can comprise a sequence that is at least 80-100% identical to the sequence:
  • the U3-R domain can comprise a sequence that is at least 80-100% identical to the sequence:
  • the binding domain of the "CAR” can be any sequence that encodes a polypeptide that binds to a desired target antigen.
  • the binding domain can be an antibody fragment such as an scFv directed to a desired target antigen (see, e.g., Table 1). Sequences encoding various binding domains to the targets set forth in Table 1 are known in the art and published in numerous applications.
  • the CARs of the disclosure are modular in nature and thus different "binding domains" can be attached depending upon the desired target.
  • a 'hinge' or linker coding sequence can be operably linked to the binding domain of the CAR.
  • the 'hinge' is optional and the binding domain can be directly linked to the transmembrane domain coding sequence.
  • the binding domain and transmembrane domain are separated by a minimal peptide coding sequence or spacer.
  • Various hinge domains and spacers are known in the art and described herein.
  • the miR targeting sequence or cassette will typically comprise a target for an miRNA molecules that would inhibiting expression of a polynucleotide of the viral construct.
  • the miR target sequence will be typically bind an miRNA that is expressed in a tissue or cell where expression of, e.g., a CAR is undesirable or unwanted, but the miRNA is not expressed in target cells nor in vector producer cells where expression from the viral construct is desired.
  • sequences or miRNA are not already known, they can be readily identified and characterized by making total RNA and performing deep bulk sequencing on such samples, from several examples of target tissues for which expression is not wanted (e.g., a tumor) and from several examples of cells where expression is desirable or needed (e.g., T cells) then using bioinformatic techniques known to those skilled in the art, candidate miRNAs and corresponding targets for further testing are identified.
  • binding domain for treating a particular cancer or disease, as well as an miRNA targeting sequence that would prevent expression of the CAR in undesirable tissues and/or cells and suitable 'hinge', 'transmembrane domain' and intracellular domains.
  • a vector construct of the disclosure will include a kill switch as a further safety mechanism, such that expression of the vector construct will result in expression of, e.g., a suicide gene (e.g., a polypeptide having thymidine kinase (TK) or cytosine deaminase (CD) activity).
  • a suicide gene e.g., a polypeptide having thymidine kinase (TK) or cytosine deaminase (CD) activity.
  • the subject, tissue or cell is contacted with a pro-drug (e.g., 5- flurocytosine) such that the cells expressing, e.g., a polypeptide having cytosine deaminase activity are contacted by the 5-FC wherein the 5-FC is converted to cytotoxic 5-FU at the site of the kill- switch's expression thereby killing the vector-infected cell.
  • a pro-drug e.g., 5- flurocytosine
  • the disclosure provides a retroviral vector comprising a gag polypeptide, a pol polypeptide and an env polypeptide and a retroviral polynucleotide contained within the capsid of the retroviral vector.
  • the retroviral polynucleotide comprising from 5' to 3': R-U5-Binding domain-hinge/linker-TM domain-signaling domain-miRNAtarget-U3-R.
  • the retroviral polynucleotide comprises an R-U5 nucleic acid sequence having at least 80%, 85%, 87%, 90%, 92%, 95%, 98%, 99% or 100% identity to SEQ ID NO:25 from nucleotide 1 to about nucleotide 145 (e.g., about nucleotide 140, 141, 142, 143, 144, 145, 126, 147, 148, 149 or 150).
  • the retroviral polynucleotide comprises a chimeric antigen receptor coding sequence 3' to the R-U5 domain.
  • the CAR coding sequence comprises a coding sequence for an antigen binding domain (e.g., an scFv to CD19).
  • the binding domain coding sequence can be preceded by a signal sequence.
  • the binding domain coding sequence is followed by an optional linker/spacer domain sequence.
  • the binding domain coding sequence an optional spacer/linker coding sequence is followed by a nucleic acid sequence encoding a transmembrane domain.
  • the transmembrane coding sequence is followed by a nucleic acid sequence encoding a cytoplasmic signaling domain.
  • the retroviral polynucleotide can comprise an optional kill switch domain coding sequence.
  • the optional kill switch coding domain comprises an IRES operably linked to a coding sequence for a polypeptide that converts a prodrug to a cytotoxic drug.
  • the polypeptide is thymidine kinase (TKO) or cytosine deaminase (CD).
  • the retroviral polynucleotide comprises at least one, typically a plurality of the same of different miRNA targeting sequences. The miRNA targeting sequences are 3' to the CAR domain.
  • the retroviral polynucleotide further includes a U3-R domain at the 3' end of the polynucleotide.
  • the U3-R domain comprises a sequence that is at least 80%, 85%, 87%, 90%, 92%, 95%, 98%, 99% or 100% identity to SEQ ID NO:25 from about nucleotide 5537 to about 6051.
  • the domains can be separated by small spacer sequences of about 2-20 nucleotides that can be intentional or artifacts of cloning.
  • the disclosure also provides a plasmid sequence that when expressed in a suitable host cell produces the retroviral vectors of the disclosure.
  • the disclosure provides retroviral vectors comprising a recombinant viral genome having a R-U5 domain, a packaging domain, a CAR domain and a miRNA detargeting domain (e.g., a miRNA targeting domain).
  • the recombinant viral genome can further comprise a kill switch comprising a coding sequence for a suicide gene (e.g., a gene that produced a polypeptide that has TKO or CD activity).
  • the CAR domain can comprise a 1 st , 2 nd or 3 rd generation CAR construct (as are known in the art) which when expressed in a desired immune cells is capable of binding to a target antigen.
  • the retroviral vector of the disclosure comprises retroviral capsid comprising an envelope that can infect mammalian cells and deliver the recombinant viral genome into the mammalian cell.
  • the retroviral vector can be used to transform cell in vivo thus eliminating the need for ex vivo isolation of cells as is performed in current adoptive cell therapy.
  • the miRNA detargeting domain comprises targeting sequences that can be bound by miRNA produced in cells where expression of the viral genome is undesirable. The binding of the miRNA in these cells binds the miRNA targeting sequence and prevents expression of the viral genome in the cell.
  • the disclosure also provides methods of treating a subject with cancer.
  • the method comprises inducing, in vivo, expression of a CAR, without ex vivo immune cell manipulation.
  • the method can include identifying a target antigen specific to a disease or disorder to be treated. Constructing a chimeric antigen receptor having a binding domain that targets the antigen specific for the disease or disorder. Cloning the CAR coding sequence into a vector of the disclosure. Generating a viral construct containing a polynucleotide coding for the CAR construct and administering the viral construct such that the subject's immune cells are transduced in vivo to express the CAR.
  • a vector of the disclosure can be generated as described herein, purified and prepared in a pharmaceutical formulation for administration to a subject.
  • the disclosure provides a method of mobilizing stem cells (e.g., hematopoietic stem cells) in a subject that is planning to, undergoing or has undergone therapy with a vector of the disclosure.
  • stem cells e.g., hematopoietic stem cells
  • Hematopoietic stem and progenitor cells reside in distinct niches within the bone marrow environment, populated by several cell types such as osteoblasts, reticular/mesenchymal cells, endothelial cells, macrophages, and megakaryocytes. These niche cells serve a regulatory function, limiting the entry of HSPCs into the cell cycle, and ensuring life long repopulation of the hematopoietic system by a limited number of HSPCs that can maintain and regenerate the HSPC pool.
  • HSPCs Hematopoietic stem and progenitor cells
  • HSPCs can be mobilized from these niches into the blood, and this mobilization can be induced by growth factors, drugs, antibodies, etc. Once mobilized, HSPCs travel through the bloodstream and can home back to sites of hematopoiesis.
  • the disclosure provides several different ways that effectively do this to allow transduction of hematopoietic stem cells (HSC) in the blood before returning to the relatively inaccessible marrow niche.
  • HSC hematopoietic stem cells
  • the disclosure also provides methods of targeting retroviral vectors to HSC using envelopes on a retroviral capsid having moieties designed to bind to epitopes displayed on the surface of these cells.
  • Osteoblasts regulate the quiescence or proliferation of HSPCs through their expression of soluble and membrane-localized factors.
  • osteoblasts produce hematopoietic growth factors like granulocyte colony stimulating factor (G-CSF) and hepatocyte growth factor (HGF) upon contact with CD34+ HSPC or stimulation with either parathyroid hormone (PTH) or the locally produced PTH-related protein (PTHrP) through the PTH/PTHrP receptor (PPR).
  • G-CSF granulocyte colony stimulating factor
  • HGF hepatocyte growth factor
  • PTHrP hepatocyte growth factor
  • PPR PTH/PTHrP receptor
  • bone marrow stromal cells cultured in the presence of PTH gained capacity to maintain long-term bone marrow-initiating cells (LTC-IC), and application of PTH increases HSPCs with bone marrow-repopulating activity.
  • LTC-IC long-term bone marrow-initiating cells
  • PTH
  • Osteoblasts supporting HSPCs further have a distinct phenotype of N-cadherin+ CD45-, and are regulated by bone morphogenetic protein (BMP). These osteoblasts express chemokines such as C-X-C motif chemokine-12 (CXCL12; also known as Stromal- derived factor-1 (SDF1)), as well as stem cell factor (SCF), interleukin-6 (IL-6) and the Notch ligand, Jagged 1 (Jagl).
  • CXCL12 also known as Stromal- derived factor-1 (SDF1)
  • SCF stem cell factor
  • IL-6 interleukin-6
  • Jagged 1 Jagged 1
  • Notch signaling in HSPCs for example, through PTH/PTHrP activation of PRR of Jagl in osteoblasts, increases numbers of HSPCs without affecting mature hematopoietic cells, whereas blocking Notch signaling by gamma-secretase inhibition of Notch activation decreases numbers of long-term repopulating HSPCs (Calvi et al., 2003; Stier et al., 2002).
  • Osteoblasts further express Angiopoietin-1, which binds to the Tie2 receptor on HSPCs to support HSPCs quiescence, adhesion of HSPCs to bone areas, and maintenance of HSPCs (Arai et al., 2004).
  • Osteoblasts also express thrombopoietin (TPO), which activates the MPL receptor that is expressed on quiescent HSPCs in the bone marrow; TPO/MPL interaction upregulates betal-integrin and cyclin- dependent kinase inhibitors in HSPCs and thereby induce quiescence of HSPC, whereas inhibition of the TPO/MPL pathway with anti-MPL- neutralizing antibody reduces the number of quiescent HSPCs (Yoshihara et al., 2007; Qian et al., 2007).
  • TPO thrombopoietin
  • Osteopontin Another factor in the regulation of primitive HSPC proliferation in the osteoblastic niche is Osteopontin, which restricts primitive cell expansion in the bone marrow niche. Osteopontin is produced by osteoblasts, and primitive HSPC demonstrate specific adhesion to Osteopontin in vitro via betal integrin (Nilsson et al., 2005). Deficiency or inhibition of Osteopontin results in significantly increased stromal Jagl and Notchl receptor expression on human CD34+ HSPCs, resulting in increased numbers of long-term repopulating HSPC (Stier et al.,
  • the bone marrow stroma further contains fibroblast-like cells which are part of the adherent fraction of bone marrow cells and which forms a hematopoiesis-supporting adherent layer when bone marrow is placed into long-term culture conditions.
  • fibroblastic mesenchymal cells can differentiate into various lineages such as osteoblasts, chondrocytes, or adipocytes, and have been variously termed as marrow stromal cells, mesenchymal stem cells (MSC), adventitial reticular cells (ARCs), and STRO-1 cells.
  • Cell surface markers such as STRO-1, SH2, SH3, SH4, Nestin, platelet-derived growth factor receptor-a (PDGFRa), CD51, CD146 and are negative for CD45, CD31, and Terll9 (Simmons et al., 1994; Sacchetti et al., 2007; Mendez-Ferrer et al., 2010; Pnho et al., 2013).
  • Arteriolar perivascular cells which express the mesenchymal cell marker NG-2 (Cspg4) also maintain HSPC quiescence (Kunisaki et al., 2013).
  • Nestin is a marker for a small subpopulation of non-hematopoietic MSCs, which are spatially associated with HSPCs and adrenergic nerve fibers (Mendez-Ferrer et al., 2010). Most HSPCs are in close contact with stromal cells expressing high amounts of the chemokine CXCL12, termed 'CXCL12-abundant reticular' cells, which either surround sinusoidal endothelial cells or are located near the endosteum (Sugiyama et al., 2006). CXCL12 expression is >50-fold higher in Nestin+ than in Nestin- stromal cells, and 10- fold higher than in primary osteoblasts.
  • Nestin+ MSCs highly express HSPC maintenance genes SCF/c-kit Ligand, IL-7, and vascular cell adhesion molecule-1 (VCAM-1) and, as a counter-regulator of HSPC maintenance, osteopontin.
  • CXCL12-abundant reticular cells are innervated by the sympathetic nervous system, as confirmed by high expression of connexins 43 and 45 in Nestin+ MSCs, indicating their electromechanical coupling with ?-adrenergic nerve terminals, and administration of a ?3 adrenergic receptor agonist enhances mobilization of HSPCs (Katayama et al., 2006).
  • Endothelial cells also contribute to HSPC niche signals. Imaging of bone marrow vascular structures showed that 85% of long term repopulating HSPCs were within 10 pm of a sinusoidal blood vessel and in contact with leptin receptor ⁇ and CXCL12-high stromal niche cells (Acar et al., 2015). Recent work also demonstrated that more than 94% of HSPCs in the bone marrow marked by expression of HoxB5 are found in an abluminal position of the vessel and are directly attached to VE-cadherin (Cdh5)-expressing endothelial cells (Chen et al., 2016).
  • Notch signaling is not essential for homeostasis of adult HSPCs, Notch-ligand adhesive interaction maintains HSC quiescence and niche retention, and it has recently been reported that Notch2 blockade (but not Notchl blockade) sensitizes HSPCs to mobilization stimuli and leads to enhanced egress from marrow to the peripheral blood (Wang et al., 2017).
  • VEGFR2 vascular-endothelial growth factor receptor 2
  • SCF Stem Cell Factor
  • leptin receptor-expressing perivascular cells Other factors regulating HSPC proliferation include Stem Cell Factor (SCF) in endothelial cells and leptin receptor- expressing perivascular cells (Ding et al., 2012).
  • SCF Stem Cell Factor
  • the heparin binding growth factor pleiotrophin which is expressed and secreted by bone marrow sinusoidal endothelial cells, has also been shown to regulate hematopoietic stem cell self-renewal and retention (Himburg et al., 2012).
  • E-selectin induces increased HSPC quiescence, suggesting that endothelial cells can also regulate HSPC proliferation (Winkler et al., 2012).
  • HSPCs are also found in contact with bone marrow macrophages, which support osteoblast survival and retention of HSPCs in their niche (Chow et al. 2011; Christopher et al. 2011).
  • Depletion of CD169+ (Siglecl) macrophages leads to decreased retention of HSPCs in the mesenchymal (ARC) niche in the bone marrow, and consequently HSPCs are mobilized in the bloodstream (Chow et al. 2011).
  • ARC mesenchymal
  • G-CSF induces depletion of endosteal macrophages, which leads to suppression of osteoblast function and HSPC mobilization (Winkler et al. 2010).
  • HSPCs are also found in direct contact with megakaryocytes, which also contribute to the HSPC niche.
  • Megakaryocytes normally secrete cell cycle regulators such as thrombopoietin (TPO), transforming growth factor ?1 (TGF-?1), and chemokine C-X-C motif ligand-4 (CXCL4), which keep HSPCs in the GO phase of the cell cycle (Nakamura-Ishizu et al. 2014; Bruns et al. 2014; Zhao et al. 2014).
  • TPO thrombopoietin
  • TGF-?1 transforming growth factor ?1
  • CXCL4 chemokine C-X-C motif ligand-4
  • TGF-bI is reported to be expressed more highly in megakaryocytes than any other stromal cell type in the bone marrow, including osteoblasts, endothelial cells, Nestin+ perivascular cells, and CXCL12-abundant reticular cells; however, under the chemotoxic stress of chemotherapeutic drugs such as 5-fluoruracil (5-FU), megakaryocytes secrete fibroblast growth factor 1 (FGF-1) and down-regulate TGF-bI, stimulating the expansion of HSPCs (Zhao et al. 2014).
  • chemotherapeutic drugs such as 5-fluoruracil (5-FU)
  • FGF-1 fibroblast growth factor 1
  • HSPCs The mobilization of HSPCs is a multifactorial process and is regulated at the level of the bone marrow microenvironment through modulation of the interaction between HSPC and bone marrow stroma.
  • Adhesion molecules, paracrine cytokines, and chemokines have been implicated in this interaction.
  • the main factors that anchor HSPCs in the niche, and/or induce their quiescence are vascular cell adhesion molecule (VCAM)-l, CD44, hematopoietic growth factors, e.g. stem cell factor (SCF) and FLT3 Ligand, chemokines including CXCL12, growth-regulated protein beta and IL-8, proteases, peptides, and other chemical transmitters such as nucleotides.
  • Mobilization is initiated by disengagement from stromal adhesion, followed by directed migration toward bone marrow sinuses, and subsequent egress through the basement membrane and the endothelial layer.
  • HSPC exhibit a wide range of cell adhesion molecules (CAM), with ligands found on bone marrow stromal cells.
  • CAM cell adhesion molecules
  • the expression of several leukocyte adhesion molecules, including the integrins LFA-1 (Lymphocyte function-associated antigen 1) and VLA-4 (Very Late Antigen-4 or integrin a4b1) is reduced on circulating progenitors when compared with progenitor cells resident in the bone marrow.
  • the respective ligands Intercellular Adhesion Molecule-1 or ICAM-1, and Vascular Cell Adhesion Molecule-1 or VCAM-1) for these adhesion molecules are found on bone marrow endothelial and stromal cells.
  • the VCAM-1 protein is an endothelial ligand for VLA-4 (a4b1), and binds weakly to LPAM (Lymphocyte Peyer patch adhesion molecule or integrin a4b7). More than 90 percent of all purified peripheral blood CD34+ cells express VLA-4 (a4b1) integrins, whereas only 10 to 15 percent express VLA-5 (integrin a5b1 or fibronectin receptor).
  • VLA-4 (a4b1) integrin alone influences adhesion whereas VLA-4 (a4b1) and VLA-5 (a5b1) both mediate chemotaxis of clonogenic CD34+ progenitor cells on recombinant fibronectin (Carstanjen et al. 2005).
  • HSPCs The release of HSPCs from stromal cells within the bone marrow is effected by proteolytic degradation of VCAM-1 by elastase and cathepsin G, and of CXCL12 by neutrophil proteases (Levesque et al. 2002). Also, shedding of membrane-bound SCF by matrix metalloproteinase 9 (MMP-9) has been found to contribute to HSPC mobilization (Heissig et al. 2002). These discoveries pointed to a common 'end pathway' in HSPC mobilization, after stimulation with G- SCF, but also after stimulation with other stimuli such as chemokines or chemotherapy.
  • MMP-9 matrix metalloproteinase 9
  • VLA-4 and VCAM were shown to mobilize progenitor cells and/or inhibit their homing to the bone marrow in non-human primates (Papayannopoulou et al. 1995; Papayannopoulou and Nakamoto 1993).
  • VLA-4 VCAM-1
  • VLA-5 ?5?1; fibronectin receptor
  • CD44 / hyaluronan / osteopontin interactions between HSPCs and stromal cells for retention of HSPCs in the bone marrow niche
  • function-blocking anti-VLA-5 and anti-CD44 antibodies all resulting in liberation of HSPCs and their mobilization into the blood.
  • antibodies against VLA-4 (a4b1) and VLA-5 (a5b1) independently reduce the repopulation of bone marrow after transplantation of human peripheral blood CD34+ cells (Carstanjen et al. 2005).
  • Natalizumab is a humanized monoclonal anti-VLA-4 antibody which is FDA approved for the treatment of patients with multiple sclerosis and Crohn's disease. Serial measurements in patients receiving their natalizumab infusion therapy for multiple sclerosis showed a significant increase in circulating CD34+ cells of approximately 3-fold (Zohren et al. 2008). In the context of hematopoietic stem cell transplantation and stem cell diseases, the use of natalizumab alone or in combination with either cytotoxic drugs or other antibodies might be a new modality for stem cell mobilization (Neumann, Zohren, and Haas 2009).
  • Low expression or reduced avidity of the adhesion molecules on HSPC may also facilitate their mobilization from the bone marrow.
  • specific cytokines such as interleukin (IL)-3, granulocyte-macrophage CSF (GM-CSF), and KIT ligand (KL) are capable of modifying the function of VLA-4 and VLA-5 expressed on CD34+ cells and thereby modulating adhesion to fibronectin (Levesque et al. 1995).
  • SCF Stem Cell Factor
  • HSPC has been reported to increase by approximately 2-fold and 4- to 10-fold, respectively, resulting in increased adhesion to fibronectin after stimulation with SCF (Hart et al. 2004).
  • compositions of the disclosure may comprise a viral vector, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • compositions of the disclosure can be formulated for intravenous administration.
  • the composition may further comprise a secondary active agent (e.g., an anticancer, antiviral or antibiotic agent).
  • a secondary active agent e.g., an anticancer, antiviral or antibiotic agent.
  • Pharmaceutical compositions of the disclosure may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease.
  • a pharmaceutical composition of the disclosure comprises vector at 10 3 to 10 11 transforming units/dose (e.g., 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 ,
  • the dose may be administered one time to several times per day and may be administered for consecutive days, weeks or months as necessary to induce immune effector cells in vivo.
  • the pharmaceutical composition comprising vectors of the disclosure can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • pBA9-9b-hCD19 - miRT223T4X means the plasmid (e.g., construct 7) carrying the viral backbone from pBA-9b (SEQ ID NO:l wherein "U” can be "T") containing a coding sequence for an anti-human CD19CAR and 4 copies of a target sequence for miR223; BA-9b-hCD19-miRT223T4X or vBA-9b-hCDl9- miRT223T4X or BA-9b-hCD19-miRT223T4X(V) represents the corresponding infectious vector.
  • Cells that express the cognate microRNA will degrade RNA that include the miRNA target sequence thereby limiting expression of the transgene in that cell type.
  • Transgenes may include 1 or more copies of each microRNA target sequence.
  • the transgene encodes for a CD19 chimeric antigen receptor without and with 4 copies of each miRNA target sequence at the 3' end of the transcript.
  • the microRNA target sequence codes for 4 repeats of the miR223 target ("miR223T(4X) ") to reduce transgene expression in transduced monocytes (FIG. 1; construct 7; SEQ ID NO:2).
  • the microRNA target sequence codes for a B cell (lymphoma) specific miRaBCT(4X) to reduce transgene expression in transduced B cells (FIG. 1).
  • microRNA target sequence codes for miRaNKT(4X) to reduce transgene expression in transduced NK cells ( Figure 1). In one embodiment the microRNA target sequence codes for miRaBCT(4X) and miR223T(4X) to reduce transgene expression in transduced B cells and monocytes ( Figure 1; construct 5). In one embodiment the microRNA target sequence codes for miRaNKT(4X) and miR223T(4X) to reduce transgene expression in transduced B cells and monocytes ( Figure 1).
  • the microRNA target sequence codes for miRaNKT(4X), miRaNKT(4X) and miR223T(4X) to reduce transgene expression in transduced B cells and monocytes (Figure 1).
  • RNV may also contain miRNA target sequences in the transgene UTR that result in transcript degradation in hepatocytes. Addition of miRNA122aT(4X) and miR199aT(4X) sequences as single targets or combined to the UTR reduces transgene expression in the liver ( Figure 2; construct 36, 37, 38). These liver detargeting miRNAs may be combined with components from Figure 1 including a combination where all 5 miRNA target sequences are encoded in the RNV transgene UTR ( Figure 2).
  • RNA viral polynucleotide (vRNA genome) of the disclosure is provided in SEQ ID NO:l; Bold/underlined portion identifies multiple cloning site:
  • a CAR construct and (without or without additional domains, e.g., miRNA targeting domain, kill switch domain etc.) can be cloned into the multiple cloning site.
  • RNV containing emerald GFP is used in place of CD19CAR to look at transgene expression across PMBC cell types and varying cell lines.
  • Vector BA9B-emdGFP-containing miR variants described in Figure 1 was used to transduce cell lines: Jurkat (T cell); TALL-104 (T cell); Raji (B); NALM-6 (B); THP-1 (monocyte); U937 (monocyte) and NK-92 (NK).
  • HT1080 cells are transduced with a 3 rd generation SIN-lentiviral vector expressing a single or varying combinations of precursor miRNAs along with puromycin.
  • Post-transduction HT1080s are selected with puromycin (6ug/mL) for two weeks, before RNV transduction that encodes the desired transgene with miRNA target sequences in the UTR. This ensures at least one cell line that is transduced will express the desired miRNA(s) to target the RNV transgene UTR for degradation.
  • BA9B-CD19CAR-miRaBCT4X-miRaNKT4X-miR223T4X vector is used at 10 MOI to transduce PBMCs in a mix lymphocyte reaction in the presence of polybrene (4-8 pg/ml).
  • PBMCs are seeded at le6/mL density in RPMI1640 medium supplemented with 10%FBS in a single well of a 24 well tissue culture plate. 24-48 hours post-transduction, cells are sampled and run on a cytometer to evaluate cell type specific expression of CD19CAR (Figure 3).
  • T cells which expressed CD4 or CD8 canonical markers show CD19CAR expression by flow cytometry while other cell types, including B cells, NK cells and monocytes, do not show CD19CAR expression. Also, reduced CD19+ B cells frequencies can be seen in cultures of PBMCs due to CD19CAR activity from T cells. Further, CD19CAR expressing T cells may be enriched during the performance of some in vitro assays described below.
  • Tumor line Nalm6-CD19WT is purchased from ATCC and maintained in medium RPMI-1640 supplemented with 10% fetal bovine serum for the co-culture with reprogrammed T cells to test CD19 CAR activity.
  • a CD19 antigen deficient variant of Nalm6 (Nalm6-CD19KO) is generated using CRISPR technology from the original Nalm6-CD19WT parental tumor line.
  • Supernatants from the co-cultures of reprogrammed CD19 CAR-T cells with Nalm6 at different effectors to target (E:T) ratios of 16:1, 8:1, 4:1, 2:1, 1:1, 1:2, 1:4 and 1:8 are collected to measure secreted cytokines levels using Enzyme-linked Immunosorbent assay (ELISA) and flow cytometry using Biolegend's Legendplex assay.
  • E:T effectors to target
  • Reprogrammed CAR-T cells secrete cytokines such as IL2, IFNy and TNF only in the supernatants from culture with Nalm6-CD19WT not with Nalm6-CD19KO in a CD19 CAR- specific manner.
  • cytokines such as IL2, IFNy and TNF only in the supernatants from culture with Nalm6-CD19WT not with Nalm6-CD19KO in a CD19 CAR- specific manner.
  • Flow cytometry-based methods are also used to detect degranulation (CD107 mobilization) and intracellular cytokines on reprogrammed-CAR-expressing T cells after 4 hrs of short-term co-culture with NALM6-CD19WT and Nalm6-CD19KO cell lines separately.
  • Reprogrammed CAR-T cells degranulate and retain cytokines such as IL2, IFNy and TNF intracellularly in the co cultures with Nalm6-CD19WT and not with Nalm6-CD19KO in a CD19 CAR- specific manner.
  • cytokines such as IL2, IFNy and TNF intracellularly in the co cultures with Nalm6-CD19WT and not with Nalm6-CD19KO in a CD19 CAR- specific manner.
  • CTV cell trace violet
  • Reprogrammed T cells also show CD19 CAR-specific tumor cytotoxicity when co-cultured with Nalm6-CD19WT compared to co culture with Nalm6-CD19KO as measured in short and long-term co culture assays.
  • Luminescence based method are used to measure short term immediate CAR-specific cytotoxicity against Luciferase expressing-Naml6-CD19WT.
  • Agilent's xCELLigence assays are also used to measure short and long-term CAR-specific tumor cytotoxicity against Nalm6-CD19WT.
  • xCELLigence assay performs continuous real time cell analysis (RTCA) on any changes in numbers of Nalm6 tumor line using biosensors embedded in the culture plates.
  • HAL2 Packaging Cell Line Encoding Plasmid Constructions Expressing Engineered Versions of MLV derived Gag/Pol and Env in a parental HT-1080 Cell
  • the HAL2 packaging cell line was developed in similar fashion to the HA-LB packaging cell as described in Sheridan et al., MOLECULAR THERAPY Vol. 2, No. 3, September 2000.
  • the HAII packaging cell line also described in Sheridan et al. Mol. Ther. 2000, can be used.
  • the HAL2 packaging cell plasmid constructs as well as the constructs for the HAII packaging cell line are described herein.
  • the plasmids constructs were stably transfected into the parental HT-1080 cell line (ATCC-CCL-121).
  • the gag/pol and env MLV derived sequences can also be inserted and expressed using a lentiviral vector pseudotyped with VSG-g to stably transduce the MLV sequences in sequential transduction events to obtain a similar HAL2 packaging cell lines.
  • the gag/pol expressing HT-1080 intermediate is dilution cloned, with individual clones screened for high gag/pol p30 expression by Western blot analysis.
  • the clones can also be screened for functional titer production by transducing the gag/pol clonal intermediate with a MLV vector that contains both 5' and 3' LTRs flanking a packaging signal, a selectable marker (e.g., neomycin resistance) or a marker gene (e.g., Emerald GFP) which also expresses a functional MLV env sequence, to confirm that the gag/pol packaging cell line intermediate is capable of producing high titer MLV viral particles.
  • the MLV env sequence is inserted into the selected gag/pol intermediate by stable transfection or stable transduction using a lenti-viral vector encoding the MLV env sequence.
  • the packaging cell line expressing both gag/pol and env is dilution cloned and screened to identify the highest env expression as well as for confirmation of functional titer production using a similar MLV test vector capable of expressing a selectable marker or marker gene except without a viral env sequence to evaluate individual clone packaging cell line performance.
  • the specific MLV gag/pol and env constructs used to create the HAL2 packaging cell line are further described below.
  • the HAL2 packaging cell line uses the original MoMLV-derived gag/pol plasmid pSCVIO (see, e.g., WIPO patent publications WO 91/06852 and WO 92/05266, the disclosures of each of which are incorporated herein by reference in their entirety) as used for the HA-LB MLV packaging cell line designed to reduce replication competent virus by homologous recombination events.
  • the gag/pol construct in the HAII packaging cell line can also be used, which has reduced sequence homology to the retroviral vector and env expression constructs.
  • the gag/pol construct expression cassette pCI-WGPM contains degenerate code in approximately the first 400 nt of the coding region for gag, as well as deletions of all 5' and 3' untranslated sequences. In addition, the sequence coding for the last 28 amino acids of the pol gene is deleted, resulting in a truncated integrase gene. Plasmids pCI-GPM and pSCVl0/5',3'tr contain the same gag/pol cDNA as pCI-WGPM except that the 5' area of gag contains the native sequence.
  • VPCL Vector Producing cell line
  • Plasmid construct pBA-9b-emdGFPmir233-3p4TXv2 (construct 7) has been modified for cell-specific detargeting.
  • the basic pBA- 9B-emdGFP sequence can be modified to also encode microRNA (miR) target sequences as an effective method of down regulating vector expression in cell type specific manner to increase the safety profile of the vector and prevent expression in non-intended cell types.
  • Fig. 5 shows examples of miR target sequences used to impede expression in myeloid, B and NK cell types.
  • Retroviral non-clonal vector producing cell lines as well as subsequent production clones are established from the selected HAL2 clonal packaging cell line using a high multiplicity of transduction ("m.o.t.") "high m.o.t.” approach with m.o.t.'s of >20 using a single or multiple back-to-back rounds of transductions using a VSV-G pseudotyped MLV vector particles.
  • the multiplicity of transduction is defined as the number of infectious viral particles used per PCL cell for the production of VPCL non-clonal cell line.
  • a PCL culture is seeded at 1 X 10 5 cells/well in a 6-well plate one day prior to transduction.
  • the appropriate volumes of vector supernatants are then added to PCLs (in the presence of 4, 5, 6, 7 or 8 pg/ml polybrene; corresponding to m.o.t.'s of 0.1, 0.5, 5,
  • the vector supernatant is replaced with 2 ml of fresh media.
  • the transduction procedure can be repeated for a second day using the same volume of vector supernatant.
  • Producer pools are grown to confluence and supernatants collected daily at 24, 48, and 72 h post-confluence to determine PCR transduction titers and show transfer of gene expression. Selected non-clonal pools are cloned using limited dilution seeding into 96 well plates that result in a single cell per well that are analyzed in several rounds of titer determination and transfer of expression assays as individual groups of clones expand.
  • VPCL clones that have sustainable high titer production are cryopreserved to prepare frozen down a working stock that is tested for safety (sterility, mycoplasma, replication competent retrovirus as well as other viral adventitious agents as described in FDA points-to-consider and Guidance publications).
  • Genomic viral sequencing of the viral particles that derive from individual clones is also performed to ensure accuracy of the genomic MLV sequences, as part of qualified cell bank stock characterization. Once qualified, a vial from the qualified cell bank stock can be further expanded and further tested under GMP to produce a GMP Master and Working cell bank stocks.
  • the series of events for creating either Packaging Cell Line or Vector Producing Cell Line is summarized in Fig. 6.
  • the following example reviews cell culture viral production methods for growing adherent vector producer cell line (VPCL) cells to produce murine leukemia virus (MuLV) for small scale R&D Studies.
  • VPCL adherent vector producer cell line
  • MuLV murine leukemia virus
  • Production MuLV virus based on the pBA-9b- emdGFPmir233-3p4TXv2 construct is used as an example, however this process can also be used for all MLV viral vectors described in this disclosure.
  • VPCL produced from the parental HT1080 cells (ATCC CCL-121) is described, however the method can also be used for VPCLs produced from HEK 293T, D17 or CF2 derived cells (CRL-1573, CCL-183, or CRL-1430, respectively), under the conditions of 37°C under 5% CO conditions.
  • VPCLs are cryopreserved under liquid nitrogen conditions, stored in cryoprotectant plastic vials containing up to 1.0 x 10 7 cells frozen in a cryoprotectant cell culture media solution containing 10% DMSO, and 50-90% fetal bovine serum in cell culture growth media solution.
  • cells are expanded by initially seeding into a T-75 flask with subsequent expansions into two T-175's and then subsequently cultured into multiple T-175 flasks with the following growth medium:
  • TrpZean® Sigma
  • TrpZean® Sigma
  • the cells are seeded into three 10- layer CellSTACKs ® (Corning) at a seeding density of about 3.1 x 10 4 viable cells/cm 2 in complete DMEM medium, to produce the virus.
  • Each Cell Stack contained 1.1L of the growth medium.
  • the CellSTACKs ® are incubated at 37°C and 5% CO .
  • the CellSTACKs ® cultures will approach or reach confluence. After reaching confluence, the medium in each culture is replaced on a daily bases with fresh medium with spent cell culture media, containing produced virus, harvested after a fresh media exchange. Alternatively, fresh media feedings and vector harvesting can occur every 10-12 hours with same volume (1.1 L) of fresh growth medium. After a desired volume is collected, the harvest collections are then pooled for purification. Viral titers for the 3 harvests and the pool are listed in the following table.
  • large-scale retroviral vector production using adherent cell culture expansion trains can be performed using multiple single use systems such as fibrous disks, micro-carrier beads or fixed bed like systems like the iCellis® system (Pall Corporation, NY).
  • iCellis® system Pall Corporation, NY.
  • expansion of the VPCLs are performed in 225-cm 2 tissue culture flasks using DMEM formulated with 10% g-irradiated defined fetal bovine serum. Cells are allowed to expand for about 3-4 days until sub-confluence.
  • the cells are then progressively passaged while increasing the surface area to 4 X 10-layer Cell Factories (Nalge Nunc International, IL) to reach the required number of cells to inoculate the CellCube® system.
  • Fresh media perfusion feeding is controlled based on glucose consumption with up to a maximum media exchange of four system volumes per day. Production volume depending on the number of Cell Cube modules used from 200 to 1000 liters over a period of up to about 13-16 days. Representative bioreactor samples are taken for metabolic profile and PCR transduction titer analysis.
  • a serum free adaptation process is performed after screening and identification of a suitable dilution clone of HT-1080 vector producing cell line.
  • the adaptation process is initiated by seeding approximately 2xl0 7 cells into a 125 mL shaker flask containing 10 mL of 5% serum containing conditioned media and 10 mL of a selected serum free media of choice, resulting into a reduced serum concentration of 2.5%.
  • the serum free media is Freestyle 293 Expression Media distributed through Invitrogen Corp, Carlsbad, CA, however for those skilled in the art, a comparable or custom serum-free media can also be used.
  • the culture is placed on a shaking platform located in a tissue culture incubator with both temperature and CO2 gas control.
  • the shaking platform is set to about 80 RPM and the incubator is set to a 37°C and a preferred 5% CO2 conditions.
  • the culture is re-fed by collecting cells that are in suspension and reseeded into a new shaker flask containing 10 mL of the same initial conditioned media and 10 mL of fresh serum free media maintaining a level of serum of approximately 2.5%.
  • the culture is examined at each re feeding event with viable cell counts performed as needed to check for cell propagation. When the cells show evidence of growth based on cell doubling or glucose consumption, a serum concentration of 1.67% is then targeted by adjusting the volume amount of condition media and fresh serum free media.
  • the culture again is examined and refed every 3- 7 days.
  • a serum concentration of 1.25% is targeted by again adjusting the volume of conditioned media and fresh serum free media. This process is continued targeting subsequent serum conditions of 1.0%, 0.9%, 0.83% serum conditions until the cells are in 100% serum free conditions.
  • the cell culture is expanded to approximately 200 mL volume in a 1,000 mL shaking flask targeting a minimal viable culture of approximately 0.5 to 1.0 xlO 6 cells/mL. Once the cells reach 100% serum free conditions, the cells are continuously passaged under serum free conditions isolating single suspended cells by allowing heavier clumping cells to settle for short periods of time without agitation. Once the culture consists of approximately 95% population of the single cell suspension consistently, the culture can be frozen in cryopreservation media consisting of 10% DMSO and 90% serum free media using standard mammalian cell freezing conditions.
  • Example 5 Adaptation of producer cell line to suspension culture and producing virus in a rocking bag bioreactor system for pilot/pre-clinical scale production.
  • cell expansion is initiated using a series of shaker flasks containing the indicated amount of cell culture media per flask: 125-mL (20 mL culture); 250-mL (40 mL); 500-mL (100 mL); and 1-L (200 mL) all from Corning.
  • Cell expansion is performed using the fully defined serum-free medium (Freestyle 293 Expression Media, Gibco Cat# 12338), supplemented with 0.1% human serum albumin (HSA, from Octapharma USA, NJ).
  • the cultures are incubated at 37°C and 5% CO2 with shaker speed of about 80 rpm.
  • Five 1-L shake flask cultures are used to inoculate a WAVE bioreactor (WAVE 20/50 EHT, Cytiva Healthcare Life Sciences/GE Healthcare, MA) containing a 20-L Cellbag with 10L working volume.
  • the initial cell density in the bioreactor is about 4 x 10 5 viable cells/mL (viability 91%) at 37°C.
  • the initial operating conditions are: 5% CO2, rocker speed 15 rpm, angle 6°, air flow rate 0.2 L/min.
  • the pH control is set at about 7.2, and DO control at about 40%. Both pH and DO controls are implemented by a WAVE POD console system.
  • the feed (and permeate) rate is initially set at -0.25 volume/day, and progressively increased with cell density for up to an -3.8 volumes/day.
  • a total of 180 L of permeate containing the virus is harvested within a 15 day period.
  • Approximately 300L of harvested material can be collected using the 50L WAVE bags using a working volume of 25L.
  • the viral titer in the 180L - 300L harvests can be 5-8 x 10 6 TU/mL.
  • the cell culture medium is Gibco Freestyle 293 Expression Medium (Thermo Fisher Scientific) supplemented prior to use with 0.1% human serum albumin (HSA) with the addition of a low level concentration of an antifoam agent based on polydimethylsiloxane such as Anti-Foam B (Sigma-Aldrich).
  • HSA human serum albumin
  • suspension cells are retained in cell culture circulation, while the cell culture supernatant containing the vector product is harvested following tangential flow microfiltration through a hollow-fiber cartridge with a 0.45 pm nominal pore.
  • the "permeate”, or clarified vector harvest is collected into a single-use harvest bag within a jacketed single-use mixer (SUM), which is controlled to 2-8°C.
  • SUM jacketed single-use mixer
  • Harvest collection starts between days 5-9, when cell density reaches 3-5 x 10 6 viable cells/mL.
  • the collection process lasts for 11 to 17 days.
  • the production run is ended when approximately 900-1000 L of clarified vector harvest is obtained, which takes approximately 14 to 21 days from the time of seeding the bioreactor.
  • the temperature of the bioreactor is controlled at 37°C. pH is controlled at 7.2010.15 by the controller-automated addition of sodium bicarbonate, and dissolved oxygen is controlled at 40% saturation set point by sparging of O2 into the bioreactor.
  • the CO2 gas flow rate is set at 5% of that of the air flow rate.
  • Glucose level is on-line monitored as an indicator for feed rate increases.
  • the density of cells in the bioreactor reaches approximately 2.5 x 10 6 viable cells/mL, or the level of glucose in bioreactor decreases to 0.5 g/L (from an initial 5 g/L), perfusion of fresh medium is initiated.
  • the feed rate increases.
  • Feed rate increase is automated via an Applikon controller utilizing a user specific and developed automation code based on BioXpert W7 software (Applikon Biotechnology).
  • Fresh medium is fed to the bioreactor to supplement glucose and nutrient depletions due to increased consumption of nutrients in the bioreactor as cell density increases.
  • the rate of clarified vector harvest withdrawn from the tangential-flow hollow- fiber cartridge is increased as the medium feed rate increases.
  • Both the feed and permeate rate increases are automated based on on line glucose measurement from the bioreactor.
  • the weight (volume) of the bioreactor is also controlled using the system by adjusting the permeate rate (volume withdrawn from the bioreactor), after its concurrent increase with the feed rate, by the same user specific automation code.
  • Virus of the disclosure are manufactured under various modalities by either transient transfection either 293T, a 293 cell line expressing MLV gag-pol (e.g., see Burns et al. PNAS 90:8033-80371993) or HT1080 cells, or from a vector producing non-clonal cell line pool, or from a cloned vector producer cell line.
  • the medium can be with serum or serum free, and the cells can be grown as adherent cells or in suspension, with cell culture supernatant collected in batch harvests or collected under perfusion mode and stored under refrigerated 2-8°C conditions.
  • the culture supernatant is harvested, and stored for up to 2 weeks at 4°C. This bulk harvest is filtered through a dead end 0.45 micron filter cartridge or through using tangential flow microfiltration using hollow fiber cartridges or plate and frame filtration systems used to remove large cellular debris.
  • the vector is treated with 2-5 Units/mL Benzonase® (EMD Millipore, Damstadt Germany) in the presence of 2mM MgCl2 incubated overnight in the range of 15-30 hours at a minimum temperature of 4°C to digest cellular DNA (Shastry et al., Hum Gene Ther., 15:221, 2004) and then subjected to a concentration step.
  • EMD Millipore EMD Millipore, Damstadt Germany
  • the concentration step can be performed using tangential flow ultra-filtration or by anion ex-change chromatography.
  • concentration of viral vector material is achieved using a 500 MW cut-off membrane designed to retain the large viral particles within the recirculating loop.
  • post treated vector material is concentrated in a re-circulation loop that starts in the concentrating vessel, through a peristaltic pump, through a 500MW hollow-fiber cartridge, and then returned to the concentrating vessel.
  • the permeate outlet is open to begin the concentration process.
  • the flow of permeate is set to approximately 1/10 the flow rate of the recirculation rate.
  • the recirculation rate is dependent on the size of the hollow fiber cartridge but typically it is set to approximately 75% of the maximum speed at which the pump speed and shear rate is not damaging to the virus.
  • additional vector material is added to the loop until the material reaches a desired concentrated range normally a 10 to 50x fold increase in titer and a corresponding reduction in volume.
  • formulation buffer is used to diafilter and buffer exchange the vector into the desired pH neutral, isotonic Tris-buffered sucrose solution.
  • This material can then be filter sterilized and used as is or can be subject to further chromatography purification.
  • Polishing chromatography can be used by either using (1) a multimodal resin such as Capto Core 400 (Cytiva/GE Healthcare) which can further remove small molecular weight charged proteins or (2) the use of standard size exclusion chromatography, such as S-500 resin (Cytiva/GE Healthcare), which serves as a buffer exchange as well as removes small molecular weight protein.
  • the vector can be formulated with required excipients to provide stability, 0.2 m filter sterilized, vialed and frozen at minus 65°C or below.
  • vector concentration can also be achieved using AEX chromatography (see, e.g., US Pat. No. 5,792,643; Rodriguez et al., J Gene Med., 9:233, 2007; Sheridan et al., Mol. Ther., 2:262-275, 2000).
  • the benzonase treated viral preparation is loaded on an anion exchange column and the virus is eluted in a stepwise NaCl gradient.
  • the fraction containing the virus can be identified by PCR assay, or by A215,
  • A280 as well as A400 Positive fractions are collected and pooled.
  • the pooled preparation is subsequently loaded on a size exclusion column (SEC) to remove salt as well as other remaining small molecular weight contaminants as well as condition the virus into SEC formulation buffer (20 mM Tris based isotonic solution consisting of 90 mM NaCl; 1% sucrose, 1% mannitol adjusted to pH 7.2 with HC1).
  • SEC size exclusion column
  • the SEC is run under isocratic conditions with formulation buffer and the viral fraction from the SEC column is collected from the void volume.
  • Positively identified fractions are pooled and supplemented to a final 1 mg/mL human serum albumin, filtered through a formulation pre-wetted sterile 0.2 pm filter, aliquoted and frozen at minus 65°C or below. All components used for the process are USP compendial grade materials with the manufacturing process, which can be performed under GMP requirements, for clinical use.
  • the viral preparation is released based on standard testing such as sterility, mycoplasma and endotoxins, with purity and consistency evaluated by SDS PAGE gel analysis. Titer is determined as Transducing Units (TU) by PCR quantitation of integrated viral DNA in target cells.
  • the final product is targeted to have a titer range of lxlO 8 to lxlO 9 TU/ml.
  • MLV-measles hybrid vectors are provided to target CD8 positive T-cells
  • Measles hemagglutin "H" constructs are modified to encode a chimeric sequence of a single chain antibody specific for CD8 expressing cells.
  • the H protein cytoplasmic tail is used to anchor the chimeric protein to the virus with the receptor binding sequences, are either mutated or deleted to destroy H receptor binding.
  • receptor binding is rather achieved using the fused CD8 targeting scFv sequences through a linker sequence.
  • HstalkscFvlh improvements or alternative targeting is achieved by identifying the orientation of alternative heavy and light sequences of this and other receptor specific scFv binding sequences.
  • the Edmonston B strain measles sequence is used but other strains of measles virus can be used to donate F and H proteins for equivalent modification.
  • a separate chimeric MLV env construct, without the Pit2 binding recognition sequences is fused to the measles "F" fusion protein as described for construct EdB fusion P DNA-4070A tail.
  • This chimeric construct is bound to the viral particle using the cytoplasmic tail of the amphotropic envelope.
  • a tail-less F protein can also be used to achieve viral particle fusion.
  • the measles H-CD8scFv and truncated Measles F protein constructs described in Example 7 are used in place of either the pCMVenv am DraLBGH or pCMV-/env am envelope constructs described in Example 2 to create a packaging cell line.
  • the plasmids constructs encoding H-CD8scFv (construct 52, pCMVenv MFhlCD8 DraLBGH) and the truncated Measles F protein expression vectors (construct 53, pCMVenv MtF DraLBGH) , are transfected sequentially into the characterized gag/pol packaging cell line intermediate by stable plasmid transfection.
  • construct 52 pCMVenv MFhlCD8 DraLBGH
  • truncated Measles F protein expression vectors (construct 53, pCMVenv MtF DraLBGH)
  • the measles F protein vector is initially delivered to create a gag/pol intermediate cell line that also expresses the measles F protein.
  • This allows for an alternative gag/pol-mF intermediate cell line that only requires the addition of viral hybrid envelope targeting sequences to create a packaging cell line with alternative viral targeting potential.
  • This new gag/pol-F protein intermediate is serially diluted to produce dilution clones, with clones screened for the highest co-expression of both the gag/pol and mF protein sequences by Western blot analysis.
  • naive test cells to test transfer of expression will need to be CD8 positive cells such as TALL-104 (ATCC CRL-11386); Molt4 (ATCC CRL-1582) or on adherent cells modified to stably express the CD8 receptor such as with PC-3 (ATCC CRL-1435) or HT0180 (ATCC CCL-121).
  • the cells can then be stably transfected or transduced with the targeting H-CD8scFV expression vector.
  • the targeting H-CD8scFV packaging cell line is subsequently serially diluted, with individual clones screened for all relevant viral protein sequences: gag/pol, mF, and H-CD8scFV. Clones with the highest expression are again tested for functional performance by introducing a MLV test vector expressing a fluorescent marker and/ or drug selectable resistance marker to evaluate stable titer production by transfer of expression of viral particles onto naive CD8 positive titering cells.
  • VPCL Vector Producing cell line
  • the identical MLV vector pBA-9b-emdGFPmir233-3p4TXv2 as demonstrated in Example 2, or an alternative MLV vector construct is used to stably transfect the CD8 targeting PCL.
  • the MLV vector construct is used to produce transiently produced VSV-g or ampho-derived vector particles to stably transduce the CD8 targeting MLV packaging cell line.
  • retroviral non-clonal vector producing cell lines, as well as subsequent production clones are established according to the outline referenced in Fig. 6.
  • the CD8 targeting PCL is used in lieu of the HAL2 clonal packaging cell line using the same high multiplicity of transduction "high m.o.t.” approach with m.o.t.'s of >20 used in a single or multiple back-to-back rounds of transductions using VSV-G pseudotyped MLV produced from a gag/pol intermediate cell line or using amphotropic vector generated particles by transiently transfecting into HAL2 cells with the MLV vector.
  • a PCL culture is seeded at 1 X 10 5 cells/well in a 6-well plate one day prior to transient transduction.
  • the appropriate volumes of vector supernatants are then added to PCLs (in the presence of 4-8 pg/ml polybrene) corresponding to m.o.t.'s of 0.1, 0.5, 5, 25, and 125. After 20-24 h the vector supernatant is replaced with 2 ml of fresh media. To increase the m.o.t.'s, the transduction procedure can be repeated for a second day using the same volume of vector supernatant.
  • Producer pools are grown to confluence and supernatants collected daily using a daily media refeed schedule at 24, 48, and 72 h post-confluence to determine PCR transduction titers and/or assess transfer of gene expression as described above using a CD8 positive cell line as the titering cell line.
  • Selected non-clonal pools are cloned using limited dilution seeding into 96 well plates that result in a single cell per well that are analyzed using several rounds of titer determination and transfer of expression assays as individual clones expand.
  • VPCL clones that have sustainable high titer production are cryopreserved to prepare frozen down working stocks that is tested for safety (sterility, mycoplasma, replication competent retrovirus as well as other viral adventitious agents as described in points to consider FDA publications).
  • Genomic viral sequencing of the viral particles should also be performed that derive from individual clones to ensure accuracy of the genomic MLV sequences, as part of a qualified cell bank characterization. Once qualified, a vial from the qualified cell bank stock can be further expanded and further tested under GMP to produce a GMP Master and Working cell bank stocks for eventual clinical production.
  • the plasmids constructs H-CD4scFv and the truncated Measles F protein expression vectors are transfected sequentially into the characterized gag/pol packaging cell line intermediate by stable plasmid transfection.
  • those skilled in the art can also use engineered lentiviral vectors to stably transduce the gag/pol intermediate cell line with the targeting H-CD4scFV and truncated measles F (mF) sequences.
  • the measles F protein vector is initially delivered to create an alternative gag/pol intermediate that also expresses the measles F protein.
  • gag/pol-mF intermediate cell line that only requires the addition of viral targeting sequences to create a packaging cell line with alternative viral targeting potential.
  • This gag/pol-F protein cell line intermediate is serially diluted to produce dilution clones, with clones screened for the highest co-expression of both the gag/pol and mF protein sequences by Western blot analysis. Once several clones have been identified for stable expression, the clones can be functionally tested by introducing into the cell line a similar MLV test vector capable of expressing a selectable marker as well as a viral targeting H-CD4scFV sequence to evaluate viral packaging cell line performance.
  • naive test cells to test transfer of expression will need to be CD4 positive cells such as CCRF-CEM (ATCC CCL-119); A301 or on adherent cells modified to stably express the CD4 receptor such as with PC-3 (ATCC CRL-1435) or HT0180 (ATCC CCL-121).
  • CCRF-CEM ATCC CCL-119
  • adherent cells modified to stably express the CD4 receptor
  • PC-3 ATCC CRL-1435
  • HT0180 ATCC CCL-121
  • the targeting H-CD4scFV packaging cell line is subsequently serially diluted, with individual clones screened for all relevant viral protein sequences: gag/pol, mF, and H-CD4scFV. Clones with the highest expression of all proteins are again tested for functional performance by introducing an MLV test vector expressing a fluorescent marker and/or drug selectable sequence to evaluate stable titer production by transfer of expression of viral particles on naive CD4 positive test cells.
  • VPCL Vector Producing cell line
  • the identical MLV vector pBA-9b-emdGFPmir233-3p4TXv2 as demonstrated in Example 2 or an alternative MLV vector construct is used to directly stably transfect the CD4 targeting PCL.
  • the MLV vector construct is used to produce transiently produced VSV-g or ampho derived vector particles to stably transduce the CD4 targeting MLV packaging cell line.
  • retroviral non-clonal vector producing cell lines, as well as subsequent production clones are established according to the outline referenced in Fig. 6.
  • the CD8 targeting PCL is used in lieu of the HAL2 clonal packaging cell line using the same high multiplicity of transduction "high m.o.t.” approach with m.o.t.'s of >20 used in a single or multiple back-to- back rounds of transductions using VSV-G pseudotyped MLV produced from a gag/pol intermediate cell line or using amphotropic vector generated particles by transiently transfecting into HAL2 cells with the MLV vector.
  • a PCL culture is seeded at 1 X 10 5 cells/well in a 6-well plate one day prior to transient transduction.
  • the appropriate volumes of vector supernatants are then added to PCLs (in the presence of 4-8 pg/ml polybrene) corresponding to m.o.t.'s of 0.1, 0.5, 5, 25, and 125. After 20-24 h the vector supernatant is replaced with 2 ml of fresh media. To increase the m.o.t.'s, the transduction procedure can be repeated for a second day using the same volume of vector supernatant.
  • Producer pools are grown to confluence and supernatants are collected daily using a daily fresh media refeed schedule at 24, 48, and 72 h post-confluence to determine PCR transduction titers and/or assess transfer of gene expression as described above using a CD4 positive cell line.
  • Selected non-clonal pools are cloned using limited dilution seeding into 96 well plates that result in a single cell per well that are analyzed using several rounds of titer determination and transfer of expression assays as individual clones expand.
  • VPCL clones that have sustainable high titer production are cryopreserved to prepare frozen down working stocks that is tested for safety (sterility, mycoplasma, replication competent retrovirus as well as other viral adventitious agents as described in points to consider FDA publications).
  • Genomic viral sequencing of the viral particles that derive from individual clones is also performed to ensure accuracy of the genomic MLV sequences, as part of qualified cell bank stock characterization. Once qualified, a vial from the qualified cell bank stock can be further expanded and further tested under GMP to produce a GMP Master and Working cell bank stocks for eventual clinical production.
  • RNAs small noncoding RNA that act as posttranscriptional regulators of gene expression by degrading their target mRNAs.
  • Addition of a specific miRNA sequences to the transcriptional cargo of a non replicating retrovirus leads to cell type-specific degradation of the retrovirus such that the retrovirus can be effectively turned off in cells in which retroviral transduction and expression is undesirable.
  • HT1080 human fibrosarcoma cells were engineered to overexpress the miRNA R223-3p, which it does not normally express, using a lentviral vector.
  • the plasmid pBA-9b-mCD19(1D3)-IRESyCD (c40) encodes an anti-mouse CD19(ID3) CAR vector described in W02020/142780 with an IRES-CD cassette, and is used to make infectious vector as described in Examples 2-6.
  • the A20 lymphoma line (luciferized) in Balb/c mice was used a model to evaluate the efficacy of in vivo infection of the mouse antiCD19 CAR RNV construct, (mCDl9-RNVCAR, mCD19 (1D3)-IRES yCD(v)), a mouse anti-CD19 scFv 1D3, followed by the murine CDS transmembrane domain, followed by murine 4-1BB intracellular domain, followed by murine CD3i (construct 4) intracellular domain.
  • A20 lymphoma cells 6 to 8-week old BALB/c mice were administered 150 mg/kg cyclophosphamide into the peritoneum (IP). Cyclophosphamide pretreatment allows A20 robust tumor engraftment.
  • A20 B-cell lymphoma cells were injected IV (1E5 cells in 200 pL) on day 0.
  • the vector, mCD19 (1D3)-IRES yCD(v) was injected at a dose of 1E8 TU per day for four consecutive days, starting at day 3 after A20 implantation.
  • mCD19 (1D3)-IRES yCD(v) treatment led to lower A20 tumor burden compared to vehicle treated controls by day 25 as assessed by luminescent signal from A20 lymphoma cells (see Figure 7).
  • the significant decrease in numeric radiance is also visually apparent (see Figure 7C, and there was an apparent survival advantage for the treatment group of 6/10 with two animals with no detectable tumor survivors compared to 4/10, no animals with no detectable tumor for the control group).
  • the decrease in A20 tumor burden shows that IV administration of mCD19-RNVCAR vector inhibits A20 tumor growth.
  • the vector For tumor growth inhibition to occur after IV administration of mCD19 (1D3)-IRES yCD(v), the vector must enter the circulating T cells, the mCD19-CAR must get expressed on the surface of those T cells, those T cells must then home to sites of the disseminated tumor, and finally, the mCD19-CAR on the surface of the T cells must engage CD19 on the A20 tumor cells and activate killing of the A20 tumor cell.
  • CD yeast cytosine deaminase
  • CRS acute cytokine release syndrome
  • the in vitro and in vivo assays to measure Cytosine Deaminase activity and ability to kill cells is described in US Pat. Publ. No: US20140178340A1 and US9732326B2.
  • the CD is used as a kill switch to ablate RNV-transduced CD19CAR positive cells thereby ending therapeutic treatment of the CD19 CAR activity.
  • the acute, CRS, and long-term, B cell aplasia can both be mitigated by deploying the yeast CD kill switch built into a CD19 CAR vector e.g. constructs 8 and 11
  • Expression and activity of polypeptides having cytosine deaminase engineered to be expressed in CD19CAR RNV can be confirmed through in vitro assays described in US Pat. No. 9,732,326B2 and 5-FC kill curves compared for cells transduced with yeast CD+ construct 8) and CD-vectors (constructl4). These data show an almost 100 fold increased sensitivity to 5FC for CD+ cells compared to CD- cells.
  • the polypeptide having cytosine deaminase is replaced with optimized thymidine kinase (TKO) as the kill switch.
  • TKO thymidine kinase
  • TKO allows activation of common anti-herpetic drugs such as ganciclovir, acyclovir, valacyclovir (ValtrexTM) or other analogues by phosphorylation in situ leading to cell killing of RNV- transduced and neighboring cells.
  • ganciclovir acyclovir
  • valacyclovir valacyclovir
  • Acute CRS, and long-term B cell aplasia can therefore both be mitigated by deploying the TKO kill switch built into a CD19 CAR vector when combined with anti-herpetic drugs.
  • CD19CAR treatment in humans is associated with higher levels of certain cytokines produced by CAR-T function, cytokine release syndrome (CRS). This side effect can lead to extended hospitalization and can be life threatening.
  • CRS cytokine release syndrome
  • the ability to decrease the cytokine response mediated by CAR-T cell function by reduction in the number of CAR-T cells without eliminating the response entirely can increase the safety and reduce side effects associated with traditional CAR-T.
  • CD34+ transplanted mice are treated IV with CD19CAR(v) or CD19CAR-yCD(v) and two days later administered various concentrations of 5-flucytosine between 50 and 500 mg/kg.
  • mice Without flucytosine, approximately 30% of mice develop CRS-like symptoms and show elevated blood cytokine concentration of IL-6, IFN-g, GM-CSF, and TNFa. Those mice showing elevated cytokine levels also show strong CD19CAR persistence in the spleen and bone marrow by PCR and flow cytometry.
  • flucytosine 500 mg/kg
  • none of the mice treated with CD19CAR-yCD(v) show elevated peripheral blood cytokines or CD19CAR persistence in spleen and bone marrow, while the percent of mice experiencing CRS-like symptoms remains unchanged in mice treated with CD19CAR(v). This demonstrates that cytokine elevation and CRS-like symptoms are associated with the presence of the CD19CAR.
  • yeast cytosine deaminase is replaced with TKO in the CD19CAR-expressing RNV.
  • TKO yeast cytosine deaminase
  • B cell aplasia effects, and acute, CRS, effects associated with CAR-T activity can both be mitigated by deploying the TKO kill switch built into a CD19 CAR vector. Similar in vivo studies are run as described as above with similar results: activation of the TKO kill switch with a low concentration of prodrug (e.g.
  • ganciclovir IP 5-20 mg/kg ganciclovir IP, twice per day reduces peripheral cytokines associated with CRS while allowing for the persistence of CD19CAR- transduced T cells to kill tumor cells; and, activation of the TKO kill switch with a high dose of prodrug (e.g. 50 mg/kg ganciclovir IP, twice per day) effectively eliminates the CAR transduced cells and the activity of the CAR-transduced cells, namely B cell depletion.
  • prodrug e.g. 50 mg/kg ganciclovir IP, twice per day
  • human IL-12p70 as a single chain construct (construct 16; SEQ ID NO:3) to a human CAR-expressing construct supports sustained and potent T cell activity of the CAR and promotes anti-cancer immune activity of non-CAR transduced immune cells.
  • Primary peripheral T cells or T cell lines e.g.
  • TALL- 104 are transduced with RNV encoding human CD19-targeted CAR or encoding CD19-targeted CAR that co-expresses IL-12p70 transgene (pBA9b-CDl9CAR, construct 6 or 8, and pBAb-CD19CAR-IL- 12p70, construct 16) at varying MOIs (e.g., 0.1, 1, and 10).
  • cytokines levels using Enzyme-Linked Immunosorbent assay (ELISA) and flow cytometry-based Biolegend's LegendplexTM assay.
  • ELISA Enzyme-Linked Immunosorbent assay
  • flow cytometry-based Biolegend's LegendplexTM assay Reprogrammed CAR-T cells secrete cytokines such as IL2, IFNg and TNF only in the supernatants from culture with Nalm6-CD19WT not with Nalm6-CD19KO in a CAR-specific manner.
  • An ELISA confirms increased production of interferon gamma across MOIs when IL-12p70 is co expressed by the CD19CAR RNV. The greatest differences in interferon gamma production were seen at the lower MOIs.
  • IL12p70 is replaced with IL-2 (construct 14; SEQ ID NO:5) in the CD19CAR expressing RNV. Similar in vitro studies were run as described above with similar results. Western blot and ELISA confirms that IL-2 is expressed along with CD19 CAR in transduced cells. When IL-2 is co-expressed along with CD19 CAR in RNV-transduced T cells, those T cells show increased activity (proliferation), cytokine secretion upon activation by CD19 (interferon gamma), and increased killing of CD19-expressing cells by the transduced T cells in the assays.
  • IL12p70 is replaced with IL-242A (IL-2supmut, construct 13; SEQ ID NO:6) CD19CAR expressing RNV.
  • IL-2supmut construct 13; SEQ ID NO:6
  • CD19CAR expressing RNV.
  • Western blot and ELISA confirms that IL-2supmut is expressed along with CD19 CAR in transduced cells.
  • IL-2supmut is co-expressed along with CD19 CAR in RNV-transduced T cells, those T cells show increased activity (proliferation), cytokine secretion upon activation by CD19 (interferon gamma), and increased killing of CD19-epxressing cells by the transduced T cells.
  • in vitro studies are done comparing T cell lines 1:1 mixed with Treg cells (primary or MT-2 cell line) transduced with constructs 6, 8,
  • IL12p70 is replaced with IL-7R-CPT (construct 15; SEQ ID NO:4) in the CD19CAR expressing RNV.
  • Interleukin-7 receptor subunit alpha IL7Ra
  • CD antigen CD127 which belongs to the type I cytokine receptor family and type 4 subfamily.
  • IL7Ra /CD127 is expressed on various cell types, including naive and memory T cells and many others.
  • the IL7Ra forms a heterodimer with Iinterleukin-2 receptor subunit gamma (IL2RG) to transmit IL7 signal.
  • IL2RG Iinterleukin-2 receptor subunit gamma
  • IL7Ra can acquire cysteine and/or noncysteine mutations to form a homodimer, leading to ligand (IL7) independent signaling events.
  • This ligand independent IL7Ra homodimer supports continued proliferation and long term persistence of T cells. Similar in vitro studies were run as described above with similar results. Western blot and ELISA confirm that IL-7Ra-CPT is expressed along with CD19 CAR in transduced cells.
  • IL-7R-CPT When IL-7R-CPT is co-expressed along with CD19 CAR in RNV- transduced T cells, those T cells show increased activity (proliferation), cytokine secretion upon activation by CD19 (interferon gamma), and increased killing of CD19-expressing cells by the transduced T cells similar to the assays run above.
  • IL12p70 is replaced with IL-12 (construct 16) or IL-12 derivatives not related to IL-12p70 in the CD19CAR expressing RNV. Similar in vitro studies were run as described above with similar results. Western blot and ELISA confirms that IL-12 is expressed along with CD19 CAR in transduced cells. When IL-12 is co-expressed along with CD19 CAR in RNV- transduced T cells, those T cells show increased activity (proliferation), cytokine secretion upon activation by CD19 (interferon gamma), and increased killing of CD19-expressing cells by the transduced T cells similar to the assays above.
  • IL12p70 is replaced with cJun (construct 12; SEQ ID NO:7) in the CD19CAR expressing RNV.
  • c-Jun supports cellular proliferation while overexpression accelerates it.
  • T cells' dysfunction or exhaustion have been associated with decreased c-Jun levels and overexpression of it restored T effector functions while supporting continued cell cycle progression and long-term persistence.
  • Western blot confirms that cJun is expressed along with CD19 CAR in transduced cells.
  • T cells When cJun is co-expressed along with CD19 CAR in RNV-transduced T cells, those T cells show increased activity (proliferation) and survival, cytokine secretion upon activation by CD19 binding (interferon gamma), and increased killing of CD19-expressing cells by the transduced T cells similar to the assays run above. Increase survival is determined by extending the time course out to 10 days after co-culturing with CD19 positive cells and comparing to results without cJun expression using construct 6.
  • IL12p70 is replaced with IL-15 (construct 9 or 10) in the CD19CAR expressing RNV.
  • IL-15 is a cytokine that stimulates CD8 T cell and natural killer (NK) cell activation, proliferation, and cytolytic activity. Survival signals that maintain memory T cells in the absence of antigen are provided by IL-15 and may be helpful in generating durable CAR T responses. Similar in vitro studies were run as described above with similar results.
  • IL-15 When IL-15 is co-expressed along with CD19 CAR in RNV- transduced T cells, those T cells show increased activity (proliferation), cytokine secretion upon activation by CD19 (IL2, IFNg and TNF), and increased killing of CD19-expressing cells by the transduced T cells. Additional in vitro studies demonstrate IL-15 expression contribute towards long lasting memory T cell pool in culture.
  • TALL-104; Jurkat are transduced with RNV encoding CD19-targeted CAR (pBA9b-CDl9CAR) or encoding CD19-targeted CAR that co-expresses IL-15 transgene in either orientation (pBAb-CD19CAR-IL-15, construct 9 or 10) at varying MOIs (e.g., 0.1, 1, and 10).
  • MOIs e.g., 0.1, 1, and 10
  • Western blot on samples of the transduced cells are run and confirms a dose-dependent expression of CD19 CAR and for construct 9, increasing IL-15 protein levels corresponding to the increasing MOIs used as well as increased expression when a reverse orientation relative to CD19CAR transgene is used.
  • the RNV-transduced cells are labeled with a proliferation monitoring dye (e.g., cell trace violet).
  • a proliferation monitoring dye e.g., cell trace violet
  • the cells are then mixed with CD19-expressing cells (e.g., NALM-6; primary B cells, or modified cells that recombinantly express CD19) in in vitro culture experiments.
  • CD19-expressing cells e.g., NALM-6; primary B cells, or modified cells that recombinantly express CD19
  • a time course of 2, 6, 12, 24, and 48 hours of media and cells were taken from the mixed cell reactions
  • An ELISA confirms increased production of interferon gamma across MOIs when IL-12p70 is co-expressed by the CD19CAR RNV similar to the assays described above.
  • immune modulators can be engineered to be transcribed in the reverse orientation for increased transgene expression.
  • IL2supmut IL12p70 cytokines
  • IL-7Ra-CPT IL-7Ra-CPT
  • cJun as described in Example 13a-f can be used in place of IL-15.
  • pBA-9B RNV backbone can be replaced with a self-inactivating (SIN) LTR (constructs 27; SEQ ID NO:18). With destruction of RNV LTR promoter activity, replacement promoters are used. Intron less EFla or CMV promoters may be used to express transgenes from the pSIN-RNV. In this example, the human or mouse CD19CAR are expressed from the EFla promoter encoded within the RNV while still encoding an IRES or 2A sequence to express a kill switch (e.g., yeast cytosine deaminase, thymidine kinase or mutants thereof) (constructs 27).
  • a kill switch e.g., yeast cytosine deaminase, thymidine kinase or mutants thereof
  • transgenes may be expressed from CMV promoter downstream of the kill switch.
  • constructs 27 and 29 IL-15 cytokine is expressed from a CMV promoter.
  • the CMV promoter and IL-15 gene may be placed in two orientations relative to the transcription direction of CD19CAR (constructs 9 and 10). Similar studies as described above are used to characterize the expression and activity of all transgenes in these constructs.
  • the pBA-9B RNV backbone may be replaced with a self-inactivating (SIN) lentiviral backbone (construct 28; SEQ ID NO:19). All transgene designed including CAR, IRES-kill switch, immune modulators, miRNA target sequences, shRNA, and promoters may be used as described herein.
  • SI self-inactivating
  • woodchuck post-transcriptional regulatory element is used to enhance expression of transgenes (Zufferey et al., "Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes Delivered by Retroviral Vectors," Journal of Virology, 73(4):2886-2892, 1999).
  • the same in vitro and in vivo assays described herein to characterize transgene expression and activity is used for this non-replicating SIN lentiviral vector(s).
  • Non-replicating retroviral vectors of the disclosure can be used to modulate the activity of immune cells with expression of engineered siRNA, shRNA or miRNA that switches off or lowers expression of key genes that govern the activity of cells including immune cells.
  • targets include genes like PD-1 a central checkpoint in regulating T cell activity, and a key protein in preventing or reducing anti-cancer activity.
  • the in vitro and in vivo assays to measure shRNA-PD-1 expression and transcript knockdown ability is described in US patents US20150273029A1. To demonstrate efficient knockdown of PD-1 in primary T cells infected with construct 19 vector at an MOI Of 10, total RNA is extracted from T cells harvested at d3 post infection.
  • Gene expression of PD-1 is measured by qRT-PCR using RNA polIII promoter transcripts as an internal control for normalization.
  • the relative expression level of PD-1 to naive primary T cells is calculated using hhC(t) method. The data show that at day 3 post-infection, more than 70% of PD-1 is downregulated.
  • the infected cells are cultured in the presence of IL-2 up to 10 days and observe sustained knockdown of PD-1.
  • similarly transduced primary T cells are characterized for CD19CAR activity using methods and experiments described above.
  • IL-12 is composed of two subunits, IL-12A (p35) and IL-
  • IL-12p70 is naturally produced by antigen presenting cells, including dendritic cells and macrophage, in response to antigenic stimulation.
  • IL-12p70 is a proinflammatory cytokine, enhancing IFNg production and cytotoxic effector function of NK and T cells, promoting Thl phenotype in CD4 T cells and ADCC activity, and acting as a chemoattractant for dendritic cells and macrophage. These IL-12 activities together can stimulate host anti-tumor activity.
  • IL-12p70 as a single chain construct (construct 16) to a CAR-expressing construct supports sustained and potent T cell activity of the CAR and promotes anti-cancer immune activity of non- CAR transduced immune cells.
  • construct 16 The functional effect of IL-12p70 expressed from the same viral vector expressing a CD19-targeted CAR is tested in vivo by intravenous injection of the recombinant retroviral vectors (pBA9b- CD19CAR, construct 6 or 8, and pBAb-CD19CAR-IL-12p70, construct 16 (human CAR, IL12)
  • construct 41 is mouse CD19CAR & IL12] in 8-wk-old NSG mice implanted intravenously with luciferized Nalm6 acute lymphoblastic leukemia cells and human PBMC.
  • a dose of 1E6 to 5E8 TU of each vector stock or vehicle control is administered by intravenous injection to each treatment group composed of 10 animals per group for efficacy assessment (tumor burden and survival) and 5 animals per group for immune assessment (flow cytometry). Tumor burden is measured over the duration of the study by periodic imaging of luciferase signal. Survival of control and treatment animals is assessed. The results show treatment with viral vector expressing the CD19CAR (construct 6) reduces tumor burden as measured by luciferase signal and prolongs survival in comparison to control treated animals.
  • CD19CAR-IL-12p70 reduces tumor burden as measured by luciferase signal, prolongs survival in comparison to control and CD19CAR (construct 6) treated animals, and leads to complete absence of detectable tumor in some cases.
  • Flow cytometric analysis is carried out on Nalm6-affected lymph nodes shortly after the initiation of treatment. Results show enhanced T cell activation and degranulation in animals treated with CD19CAR- IL-12p70 (construct 16) compared to CD19CAR (construct 6).
  • IL-12p70 expressed from the same viral vector expressing a CD19-targeted CAR is tested in vivo by intravenous injection of the recombinant retroviral vectors (pBA9b- mCDl9CAR, construct 40 (SEQ ID NO:16) and pBAb-mCD19CAR-IL-12p70, construct 41; SEQ ID NO:13) in immune competent, 8-wk-old Balb/c mice implanted intravenously with luciferized A20 B cell lymphoma cells.
  • a dose of 1E6 to 5E8 TU of each vector stock or vehicle control is administered by intravenous injection to each treatment group composed of 10 animals per group for efficacy assessment (tumor burden and survival) and 5 animals per group for immune assessment (flow cytometry). Tumor burden is measured over the duration of the study by periodic imaging of luciferase signal. Survival of control and treatment animals is assessed. The result show treatment with viral vector expressing the mCD19CAR (construct 40) reduces tumor burden as measured by luciferase signal and prolongs survival in comparison to control treated animals.
  • mCD19CAR-IL-12p70 reduces tumor burden as measured by luciferase signal, prolongs survival in comparison to control treated animals, and leads to complete absence of detectable tumor in some cases.
  • Flow cytometric analysis is carried out on A20- affected lymph nodes shortly after the initiation of treatment. Results show enhanced T cell activation and degranulation in animals treated with mCD19CAR-IL-12p70 (construct 41) compared to mCD19CAR (construct 40).
  • mCD19CAR-IL-12p70-treated animals with no detectable tumor are able to resist rechallenge with A20 cells without any additional treatment.
  • the immune memory associated with resistance to rechallenge is in part associated with the persistence of the mCD19CAR-IL-12p70-transduced T cells, but also with immune learning associated with enhanced inflammation and activation of antigen presentation mediated by IL-12p70 expression.
  • the contribution of immune learning is confirmed by the killing of CAR- expressing cells by activation of the CD kill switch, present in both pBA9b-mCD19CAR and pBAb-CD19CAR-IL-12p70 viral vectors, with flucytosine prior to rechallenge. Following flucytosine IP administration, and confirmation of complete absence of cells expressing CD19CAR-IL-12p70, surviving A20 cured animals were resistant to A20 rechallenge.
  • IL12p70 is replaced with IL-2 (construct 42; SEQ ID NO:14) in the human and mouse CD19CAR- expressing RNV constructs 14 and 42 respectively).
  • IL-2 is highly inflammatory and acts in the periphery to promote the differentiation of naive T cells into effector and memory T cells.
  • IL-2 is naturally produced by activated CD4+ and CD8+ T cells.
  • IL-2 has been shown to have antitumor activity in several settings, but systemic administration of IL-2 is associated with severe side effects.
  • IL-2 wild-type also acts on Treg cells to suppress immune response.
  • IL-2F42A which does not act on Treg cells
  • IL-2F42A which does not act on Treg cells
  • a CAR-expressing construct supports sustained and potent T cell activity of the CAR and promotes anti-cancer immune activity of non-CAR transduced immune cells.
  • the natural homing capability of CAR expressing T cells to the tumor site of high antigen density for the specific CAR limits IL-2 expression to primarily within the tumor.
  • results show enhanced T cell activation and degranulation, and increased T cell numbers in Nalm6 tumor-bearing, PBMC-engrafted NSG mice treated with CD19CAR-IL-2F42A(v) (construct 42) compared to CD19CAR.
  • results show enhanced T cell activation and degranulation, and increased T cells in mice treated with mCD19CAR-IL-2F42A compared to mCD19CAR.
  • IL12p70 is replaced with IL-15-
  • IL-15Ra constructs 49 & 54 in the human and mouse CD19CAR- expressing RNV. IL-15 is expressed primarily by dendritic cells, monocytes, and macrophage and supports the activation, proliferation, and survival of T and NK cells. IL-15 exists in a membrane bound form complexed with the IL 15Ra receptor. This potent signaling complex can be recapitulated in a soluble form by single chain expression of IL-15 linked to the sushi domain of IL-15Ra (IL- 15-IL15Ra).
  • IL-15-IL-15Ra as a single chain construct to a CAR-expressing construct supports sustained and potent T cell activity of the CAR, differentiation of CAR and non- CAR T cell to long-lived memory T cells, and anti-cancer immune activity of non-CAR transduced immune cells.
  • results show enhanced T cell activation and degranulation in mice treated with mCD19CAR-IL-15-IL-15Ra (v) (construct 49 compared to mCD19CAR Treatment of A20 tumor-bearing immune competent mice with mCD19CAR- IL-2F42A(v), but not mCD19CAR(v) alone or control treated animals, also leads to complete cures along with immune learning similar to above.
  • IL12p70 is replaced with IL-7Ra- CPT (constructs 15 & 55 - human and mouse CD19CARs) in the CD19CAR- expressing RNV.
  • Interleukin-7 receptor subunit alpha IL7Ra
  • CD antigen CD127 which belongs to the type I cytokine receptor family and type 4 subfamily.
  • IL7Ra/CD127 is expressed on various cell types, including naive and memory T cells and many others.
  • the IL7Ra forms a heterodimer with Iinterleukin-2 receptor subunit gamma (IL2RG) to transmit IL7 signal.
  • IL2RG Iinterleukin-2 receptor subunit gamma
  • IL7Ra can acquire cysteine and/or noncysteine mutations to form a homodimer, leading to ligand (IL7) independent signaling events.
  • This ligand independent IL7Ra homodimer supports continued proliferation and long-term persistence of T cells.
  • the addition of mutant IL7Ra construct (construct 15) to a CAR-expressing construct supports sustained proliferation, persistence and potency while promoting anti-cancer immune activity of reprogrammed T cells in vivo.
  • results show enhanced T cell activation and degranulation, and increased T cells in mice treated with mCD19CAR-IL-7Ra-CPT (construct 55 compared to mCD19CAR Treatment of A20 tumor-bearing immune competent mice with mCD19CAR-IL-7Ra-CPT(v), but not mCD19CAR(v) alone or control treated animals, also leads to complete cures along with immune learning similar to above.
  • IL12p70 is replaced with c-Jun (Construct 12; SEQ ID NO:7) in the human and mouse CD19CAR- expressing RNV.
  • c-Jun is a protein encoded by the JUN gene.
  • c-Jun in combination with c-Fos, forms the AP-1 early response transcription factor, c-jun transcription is autoregulated by its own product thus it prolongs its signals from extracellular stimuli.
  • Expression of c-Jun supports cellular proliferation while overexpression accelerates it. T cells' dysfunction or exhaustion have been associated with decreased c-Jun levels and overexpression of it restored T effector functions while supporting continued cell cycle progression and long-term persistence.
  • c-Jun construct construct xx
  • construct xx construct xx
  • a CAR-expressing construct supports effector functions, sustained proliferation, persistence and potency while promoting anti-cancer immune activity of reprogrammed T cells in vivo sustained proliferation, persistence and potency while promoting anti-cancer immune activity of reprogrammed T cells in vivo.
  • results show enhanced T cell activation and degranulation, and increased T cell numbers in Nalm6 tumor-bearing, PBMC-engrafted NSG mice treated with CD19CAR-C-Jun compared to CD19CAR.
  • results show enhanced T cell activation and degranulation, and increased T cells in mice treated with mCD19CAR-c-Jun compared to mCD19CAR.
  • IL12p70 is replaced with a short hairpin RNA against checkpoint inhibitor PD-1 (construct 19; SEQ ID NO:9) in the CD19CAR-expressing RNV.
  • PD-1 is expressed by T cells after activation and is an indicator of T cell exhaustion.
  • PD-1 suppresses T cell inflammatory activity and promotes T cell apoptosis.
  • T cells' dysfunction or exhaustion can be reversed by blocking PD-1 activity.
  • PD-1 checkpoint blockade with therapeutic antibodies has been shown clinically efficacy in certain cancer indications by supporting T cell killing of cancer cells.
  • shPD-1 (construct 19) supports sustained activity of the CAR-expressing T cell, including survival, proliferation, and cancer cell killing.
  • results show enhanced T cell activation and degranulation, and increased T cell numbers in Nalm6 tumor-bearing, PBMC-engrafted NSG mice treated with CD19CAR-shPD-l (construct 19) compared to CD19CAR.
  • results show enhanced T cell activation and degranulation, and increased T cells in mice treated with mCD19CAR-shPD-l (construct m- shPD-1) compared to mCD19CAR (construct 40; SEQ ID NO:16).
  • Treatment of A20 tumor-bearing immune competent mice with mCD19CAR-shPD-l(v), but not mCD19CAR(v) alone or control treated animals also leads to complete cures along with immune learning similar to above.
  • BCMA is a mature plasma cell marker that is expressed minimally elsewhere, and has been used successfully as a CAR recognized target in the treatment of multiple myeloma (Kochenderfer JN. et al. N Engl J Med., 380(18):1726-1732, May 2, 2019).
  • Anti-BCMA CARS are used to show the efficacy of the in vivo CAR Delivery system for antigens other than CD19.
  • the structure of the vector is as for anti-CD19 CARs with the substitution of known anti-BCMA CAR sequences such as the humanized Vh only FHVH33-CD8BBZ (Lam et al., Nature Communic., 2020) to make pBA-9b-BCMACARl.FHVH33-CD8BBZ.miRT663A-223.
  • a corresponding codon optimized DNA nucleic acid sequence is:
  • ATCACCCTGT ACT G CAACCACAG G AACAAG AG G G G G AAG AAG CTG CTGTACAT CTT CAAG CAG CCCTT C
  • ACAT CT ACAT CTGGGCCCCCCTGGCCGGCACCTGCGGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTG
  • ACAT CT ACAT CTGGGCCCCCCTGGCCGGCACCTGCGGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTG
  • the resulting plasmids are used to generate the corresponding infectious RNV preparations.
  • Infectious vector is used to test activity of CARs in vitro and in vivo as described below.
  • First CAR T cells are created in vitro by transduction of PBMC with appropriate T cell stimulation as described herein.
  • the transduced cells are characterized and result in 20—80% T cell transduction. These cells are then used for in vitro testing [ 00251 ] Degranulation assay (CD107a mobilization)
  • T-cells are incubated in 96-well plates (40,000 transduced cells/well), together with an equal amount of cells expressing or not expressing the BCMA protein. Co-cultures are maintained in a final volume of 100 m ⁇ of X-VivoTM-15 medium (Lonza) for 6 hours at 37°C with 5% CO .
  • CD107a staining is performed during cell stimulation, by the addition of a fluorescent anti-CD107a antibody (APC conjugated, from Miltenyi Biotec) at the beginning of the co-culture, together with lg/ml of anti-CD49d (BD Pharmingen), 1 mg/ml of anti-CD28 (Miltenyi Biotec), and lx Monensin solution (eBioscience). After the 6h incubation period, cells are stained with a fixable viability dye (eFluor 780, from eBioscience) and fluorochrome-conjugated anti-CD8 (PE conjugated Miltenyi Biotec) and analyzed by flow cytometry.
  • APC conjugated from Miltenyi Biotec
  • the degranulation activity is determined as the % of CD8+/CD107a+ cells, and by determining the mean fluorescence intensity signal (MFI) for CD107a staining among CD8+ cells. Degranulation assays are carried out at east 24h after PBMC/T cell transduction.
  • Anti-BCMA CARs T cells are active against BCMA expressing cancer cells expressing BCMA (RPMI8226 and NCI-H929), while no activity is detected in CARs T cells wherein said CAR is against an irrelevant target (e.g., mouse CD19) or in T cells transduced with GFP. No/background activity is seen with either type of transduced T cells when the target is BCMA negative (K562 cells).
  • mice Female NOD-Cg-Prkdcscid IL2rgtmlWj1/SzJ (NSG; Jackson Laboratories) mice receive subcutaneous (s.c.) injections of 0.2mL of a 5E7 cells/mL suspension containing E7 BCMA+ RPMI-8226 MM tumor cells to establish s.c. xenografts. At approximately 10-15 days post tumor implantation, mice with xenografts receive a single intravenous (i.v.) injection of 0.2mL of a cell suspension containing E5 to E8 of human T cells.
  • i.v. intravenous
  • mice are randomized to groups of 10 mice, when mean tumor volumes of 96 +/-16 mm 3 and after confirming engraftment of human T cells.
  • Groups receive iv administration of CAR encoding vectors or control vectors at doses of E3, E4, E5, E6, E7, E8 or E9 TU's of vector.
  • An optional additional positive control group receives lmg/kg of bortezomib (Velcade) i.v. twice weekly, for 4 weeks.
  • Mice are monitored until approximately day 100 for tumor growth. Tumor growth inhibition, compared to controls, is observed in some groups of mice and not in others depending on the dose and vector preparation used, and the most effective dose and vector are identified.
  • Example 22 In vivo testing of CD8-targeted/pseudotyped particles containing non-replicating retroviral vector expressing a chimeric antigen receptor targeted against BCMA and expressing IL-12p70 [00257] Non-replicating retroviral particles encoding for aBCMA- CAR and single chain IL12p70 are pseudotyped for in vivo transduction of CD8+ T cells through incorporation of an anti-CD8a scFv into the measles envelop protein (construct 52; SEQ ID NO:20) and the Truncated measles F protein that causes fusion once the virus has "docked" via the "H” protein hybrid.
  • CD8+ T cells This allows for extremely transduction of mainly CD8+ T cells. This avoids potential transduction of other proliferating cell types, including tumor cells, non-CD8+ immune cells, and proliferating liver and endothelial cells. Efficient targeting of CD8+ cells also increases the relative number of transduced CD8+ cells since untargeted cells no longer act a sink for vector deposition.
  • CD8-targeted/pseudotyped particles containing non-replicating retroviral vector expressing a chimeric antigen receptor targeted against BCMA and expressing IL- 12p70 is tested in vivo by intravenous injection of the recombinant retroviral vectors BCMA-CAR-IL-12p70, made from the amphotropic producer line and the producer line with the hybrid envelopes, in 8- wk-old NSG mice implanted subcutaneous with a BCMA positive tumor cell line and intravenously with human PBMC.
  • a dose of 1E6 to 5E8 TU of each vector stock or vehicle control is administered by intravenous injection to each treatment group composed of 10 animals per group for efficacy assessment (tumor volume). Tumor volume is measured over the duration of the study.
  • the results show treatment with viral vector expressing the BCMA-CAR-IL-12p70 reduces tumor volume in comparison to control treated animals.
  • CD8-PSMA-CAR-IL- 12p70 reduces tumor volume in comparison to control and non-targeted PSMA-CAR-IL-12p70 treated animals, and leads to complete absence of measurable tumor in some cases.
  • Flow cytometric analysis of peripheral blood 2 days after intravenous injection of the recombinant retroviral vectors confirms that -CAR-IL-12p70 transduction is limited to CD8+ cells while non-targeted BCMA-CAR- IL-12p70 transduction is detectible across a broad range of immune cell populations, including CD8+, CD4+, B cells and monocytes.
  • the percent of CD8+ cells transduced is higher with targeted vector- compared to non-targeted vector.
  • miRNA expression levels were quantified from primary biopsies from non-Hodgkin's lymphoma (represented by DLBCL) and multiple myeloma patients in addition to CD4+ and CD8+ peripheral blood mononuclear cells from healthy donors. Samples were extracted for total RNA and then processed for miRNA isolation using Illumina Small RNA-Seq library construction. Libraries were sequenced via Illumina NextSeq sequencing at approximately 10,000,000 reads per sample, using single end reads, minimum read length 1x75 bp. Sequencing results were processed and analyzed in R to determine miRNA expression across samples. Target miRNAs and their corresponding Target Sequence were identified by two methods differential and ranked analysis of the resulting miRNA expression profiles from sequencing.
  • Non-Hodgkin's Lymphoma (NHL) miRNA candidate analysis [00260] Tables 2 and 3 show the results of two different methods (differential or ranked) to calculate and identify the miRNA expressed in DLBCL that are not expressed well in T cells. Top candidates are highlighted in both tables. Table 5 shows common targets identified by both methods.
  • Table 4 Rank Percentile Comparison Method (Based on the RPKM matrix tables, all miRNA expression grouped by cell types was ranked ordered); Median RPKM expression values for each miRNA are shown in the table below for the two groups together with the log2FC of these values.
  • MM Multiple Myeloma
  • Tables 6 and 7 show the results of two different methods (differential or ranked) to calculate and identify the top miRNA expressed in MM that are not expressed well in T cells. Top candidates are highlighted in both tables. Table 6 shows common targets identified by both methods.
  • Figure 12 shows an example box plot of ideal miRNA characteristics whose corresponding target sequences can be used in gene therapy constructs to reduce off target expression of transgenes.
  • Candidate miRNAs and miRNA target sequences encoded in RNVs were confirmed to have biological activity in vitro.
  • FIG. 13 shows the effect of including a miRNA target sequence in an RNV vector.
  • the target sequence for the miR223-3p was inserted into a GFP vector to give the sequence pBA-9B-GFPmiR223-3pB- 4TX (construct 7; SEQ ID NO:2) and used to make infectious vector.
  • miR223-3p is a microRNAs which is produced at significant concentrations only in monocytic or myeloid cells.
  • FIG. 13 shows that in the U937 monocytic cell line, a 100 fold reduction in GFP expression in the GFPmiR223 infected cell line, compared to the two other vectors. All three vectors produced equivalent amounts of GFP in HT1080 fibrosarcoma cells or other non- monocytic cells.
  • Retroviral vector constructs The original N2-derived retroviral vector pKT-1 (Patent Applications WO 91/06852 and WO 92/05266), and all its safety modifications, are summarized in FIG. 4A-C.
  • Retroviral vector rOBb-gal is derived from pKT-1 and codes for the b galactosidase and neo r genes.
  • the reduced homology vector, pBA-5b is a result of several safety modifications incorporated into pKT-1.
  • Vector pKT-1 which already contained the modification ATT in place of the normal ATG start site of gag, is modified to contain two stop codons in the extended packaging signal (Y+); the ATT modified start site was changed to the stop codon TAA, and an additional TGA stop codon was inserted 21 nt downstream. All extraneous MLV-derived retroviral sequences upstream of the 5' LTR, downstream of the 3' LTR, and between the polypurine tract and the stop codon of env, are eliminated, creating the vector pBA-9b.
  • MoMLV-derived gag/pol constructs Safety-modifications on the original MoMLV-derived gag/pol plasmid pSCVIO (patent applications WO 91/06852, WO 92/05266) were carried out to reduce sequence homology to the retroviral vector and env expression constructs (Fig. 4B).
  • the expression cassette pCI-WGPM contains degenerate code in approximately the first 400 nt of the coding region for gag, as well as deletions of all 5' and 3' untranslated sequences. In addition, the sequence coding for the last 28 amino acids of the pol gene is deleted, resulting in a truncated integrase gene. Plasmids pCI-GPM and pSCVlO/5',3'tr. contain the same gag/pol cDNA as pCI-WGPM except that the 5' area of gag contains the native sequence.
  • Envelope constructs To reduce sequence overlap in the gag/pol and retroviral vector plasmids, the original 407OA-derived amphotropic expression plasmid pCMVenv am Dra (Patent Application WO 91/06852) was used to generate two plasmids (Fig. 4C) with either all 3' untranslated sequences deleted after the env stop codon (pCMVenv am DraLBGH), or all 3' and 5' untranslated sequences deleted (pCMV- /env am ).
  • the xenotropic retroviral envelope expression cassette pCMV xeno was derived from NZB9-1 and the amphotropic envelope expression cassette pMLPenvTM was derived from 4070A.
  • Parent cell lines used to generate clinical vector producing cell lines are banked and tested in accordance with FDA guidelines for origin (i.e., isoenzyme analysis and karyotyping), absence of expressed retroviral sequences and adventitious agents including mycoplasma, bacteria, fungus and viruses.
  • VSV-G Pseudotyped Supernatant Large-scale production of concentrated VSV-G (vesicular stomatitis virus glycoprotein) pseudotyped vector supernatant (G-supernatant) is performed as outlined by Yee et al. with some minor modifications. Briefly, HA-LB packaging cells (Table 9) are plated into T225 flasks at 1 x 10 7 cells/flask. After 12 to 20 hours the cells are CaPOi- transfected with the VSV-G coding plasmid pMLP-G and the respective retroviral vector using the ProFection kit (Promega Corp., WI).
  • ProFection kit ProFection kit
  • the DNA suspension is removed and fresh media added. After 12 to 20 hours, the supernatant are collected and fresh media applied. Four to five repeat collections are made and the G-supernatant pooled, filtered (0.45 pm) and concentrated by centrifugation at 9000g and 8°C for 8- 18 hours. Pellets are resuspended in a small volume of fresh media, aliquoted, frozen under liquid nitrogen, and stored at -70°C. This concentrated viral supernatant is then evaluated for titer by transfer of expression (TOE, see below) and PCR titer analysis before carrying out high m.o.t. generation of producer pools and clones.
  • TOE transfer of expression
  • PCLs DA, 2A, HX, and 2X are described in detail (Patent Nos. WO 91/06852 and WO92/05266) and the procedure further refined for the generation of the PCLs 2A-LB, HA-LB, HAII, DAII, and DAwob.
  • retroviral gag/pol and env expression plasmids are sequentially introduced into cells by CaPOi-mediated co-transfection with a phleomycin or methotrexate marker plasmid followed by the appropriate selection for 2 weeks.
  • gag/pol intermediate pools are analyzed for p30 expression and subsequently dilution cloned into 96-well plates according to standard protocols.
  • Gag/pol intermediate clones are analyzed for p30 expression in a Western blot (polyclonal goat anti- p30 antibodies, kindly provided by J. Elder) as well as for titer potential by transduction with a retroviral vector encoding amphotropic env plus a selectable marker and titering the vector produced.
  • Clones with the highest titer potential are co-transfected with a retroviral env expression plasmid and a marker, transfected cells selected, dilution cloned and PCL clones are analyzed for gp70 expression in a Western blot (polyclonal goat anti-gp70 antibodies; Quality Biotech, MD) and for titer potential.
  • the titer potential is tested by several rounds of transduction using several retroviral vector constructs into PCLs at a high ratio of vector to PCL in order to test the limits of the packaging capacity.
  • the amphotropic env can be engineered by modification of the proline rich region of 4070A env sequence.
  • GFP reporting marker to track along env expression
  • scFV anti CD8 sequences presented in both orientations
  • L Leucine
  • the Measles H protein can be used in conjunction with various targeting protein scaffold moieties to achieve targeting using single change fragment variable (scFv) monoclonal antibodies, designed ankyrin repeat proteins (DARPins) and bispecific antibodies that recognize 2 separate receptor epitopes.
  • scFv single change fragment variable
  • DARPins ankyrin repeat proteins
  • bispecific antibodies that recognize 2 separate receptor epitopes.
  • the typical location of the H protein to engineer the targeting moiety is the H-Noose-Epitope of the Edmonston Measles strain.
  • point mutations are engineered to destroy the recognition sequence.
  • the CD34 targeting Anti-HPCA-1 monoclonal antibody is the preferred scFV moiety to use and engineer into the measles H protein sequence because receptor-mediated endocytosis is triggered on CD34+ hematopoietic cells after stimulation with the anti-HPCA-1 antibody.
  • the use of the chimeric measles H protein with the anti- HPCA-1 scFV together with the use of the measles fusion (F) protein, will allow targeting and fusion MuLV pseudotyped viral vectors to CD34+ cells.
  • the Sindbis virus envelope proteins can also be used to pseudotype and target lentiviral and MuLV vectors to CD34+ cells by conferring CD34+ specificity at the level of cell entry.
  • the ZZ domain of protein A has been incorporated into the Sindbis envelope, and vector transduction is achieved through antibody binding to specific antigens on the surface of the targeted cells.
  • the 2.2 vector has successfully targeted human leukocyte antigen (HLA) class
  • CD34+ cells L, CD4, CD19, CD20, CD45, CD146, P-glycoprotein of melanoma cells, and prostate stem cell antigen with CD34, CD133 and C-kit used for targeting human hematopoietic progenitor cells.
  • the preferred antibody for targeting CD34+ cells is the anti-HPCAl, clone MylO.
  • bi-specific antibody that allows the use of both anti-HPCAl along with anti C-kit+ moiety will enhance targeting.
  • Nipah chimeric envelope membrane bound G protein and F protein variants have been used for cell specific targeting with several advantages such as (1) shown not to have pre-existing immunity in the population, (2) have higher viral pseudotyped titers and (3) have a higher surface density.
  • Producer cell lines encoding mouse or human CD19 CAR retroviral vectors.
  • the safety-modified pBA-9b retroviral vector is also similarly engineered to encode either mouse or human anti CD19CAR vector constructs in its basic construct form consisting of a scFV mAB-Hinge-TM domain region linked to a 4-1BB costimulatory signal domain with the anti-CD3z chimeric antigen receptor (CAR) genetic sequence (FIG. 15A-B).
  • Retroviral non-clonal producer pools as well as clones are established (as referenced in FIG. 6) from clonal PCLs using a high multiplicity of transduction "high m.o.t.” approach with m.o.t.'s of >20 using a single or multiple back-to-back rounds of transductions.
  • the multiplicity of transduction is defined as the number of infectious viral particles used per PCL cell for the production of VPCL non-clonal pools.
  • a PCL culture is seeded at 1 X 10 5 cells/well in a 6-well plate one day prior to transduction.
  • the appropriate volumes of vector supernatants are then added to PCLs (in the presence of 8 pg/ml polybrene) corresponding to m.o.t.'s of 0.1, 0.5, 5, 25, and 125. After 20-24 h the vector supernatant is replaced with 2 ml of fresh media. To increase the m.o.t.'s, the transduction procedure can be repeated for a second day using the same volume of vector supernatant. Producer pools are grown to confluence and supernatants collected daily at 24, 48, and 72 h post-confluence to determine PCR titers and confer transfer of expression.
  • Selected non-clonal pools are cloned using limited dilution seeding into 96 well plates that result in a single cell per well that are analyzed in several rounds of titer determination and transfer of expression assays as individual groups of clones expand.
  • the basic pBA-9B-CD19 CAR sequence can be modified to also encode microRNA (miR) target sequences as an effective method of down regulating vector expression in cell type specific manner to increase the safety profile of the vector and prevent expression in non-intended cell types.
  • FIG. 16 shows example miR target sequences used to impede expression in myeloid, B and NK cell types.
  • FIG. 17 shows an example design of pSIN-BA-9B with a hCD19 CAR gene cloned into the multiple cloning site.
  • PCR titer analysis of vector samples is carried out.
  • MLV-specific primers (5'-GCG-CCT-GCG-TCGGTA-CTA-G-3' (SEQ ID NO:26), 5'- GAC-TCA-GGT-CGG-GCC-ACA-A-3' (SEQ ID NO:27)
  • probe (5'-AGT-TCG- GAA-CAC-CCG-GCC-GC-3' (SEQ ID NO:28)) are used to amplify a 80-bp product.
  • the amplification reaction is carried out in 50 m ⁇ with 200-400mM dNTPs, 900 nM primers, and 100 nM probe oligonucleotide.
  • the resulting fluorescence is detected and titer based on provector copy number expressed as transduction units/ml (TU/ml).
  • Transduction units are defined as the provector copy number per genome equivalent relative to a known copy number standard, and represent a true reflection of vector integration units.
  • This general titering assay utilizes HT-1080 target cells seeded one day prior to transduction at 3 X 10 5 cells per well in a six-well plate (Corning Costar, NY). Polybrene (8 pg/ml) is added 2 h before transduction with serial dilutions of vector supernatants. After 20- 24 h, the supernatant is replaced with 1-2 ml of fresh media. Cells are allowed to grow for an additional 24-48 h before supernatants or genomic DNA are assayed for transfer of expression of the gene product (TOE titer) or the number of provector copies present (PCR titer).
  • the b-gal TOE titers are determined by two independent methods. The first method is a biochemical staining procedure using X-gal staining following a standard protocol. The second procedure is a chemiluminescent detection method utilizing the Galacto-light Plus Kit (Tropix, Inc., MA).
  • VPCLs Two procedures are used to determine the presence or absence of RCR in the VPCL or the vector product, respectively, i) The first procedure tested post-production VPCLs. These cells are seeded into culture with an equal number of the replication permissive cell line M. dunni. VPCLs are seeded into flasks at a small scale (1 X 10 7 cells) or roller bottles at a large scale (1 X 10 8 cells). Cells are co-cultured for several passages and finally harvested. Cell free culture supernatant is tested using a marker rescue or PG4S + L- assay. An RCR producing cell line generated by infection of M. dunni cells with a hybrid murine leukemia virus served as a positive control for the cocultivation procedure. Naive M.
  • dunni cells served as the negative control, ii)
  • the second procedure tests the vector preparations directly. Unprocessed production harvest or purified bulk product is applied to M. dunni cells using 100 ml inoculation volume per roller bottle. After a brief inoculation period, 150 ml of additional media is added to the culture and cells are passaged four to five times before a portion of the culture supernatant is harvested, filtered, and assayed for RCR by a marker rescue or PG4S+ L- test. As per recent FDA guidance documents ([www.]FDA.gov), 300 ml of crude vector from clinical production lots are assayed for RCR. The M. dunni amplification (large scale) and PG4S + L- detection method used for product release has been validated for single unit RCR detection.
  • MuLV murine leukemia virus
  • VPCL produced from the parental HT1080 cells (ATCC CCL-121) is described however the method can also be used for VPCLs produced from HEK 293T cells (CRL-1573), D-17 (ATCC CCL-183) and Cf2Th (ATCC CRL-1430) under the preferred conditions of 37°C under 5% CO2 conditions.
  • VPCLs are cryopreserved under liquid nitrogen conditions, stored in cryoprotectant plastic vials containing 1 X 10 7 cells frozen in a cryoprotectant cell culture media solution containing 10% DMSO, 50- 90% fetal bovine serum in cell culture growth media solution.
  • cells are expanded by initially seeding into a T-75 flask with subsequent expansions into two T-175's and then subsequently cultured to ten T-175 flasks with the following growth medium:
  • cells are harvested with TrpZean® (Sigma) and neutralized with the same growth medium using standard cell culture methods.
  • the cells are seeded into three 10- layer Cell Stacks (Corning) at a preferred seeding density of 3.1 x 10 L 4 viable cells/cm 2 in the same medium previously described above, to produce the virus.
  • Each Cell Stack contained 1.1L of the growth medium.
  • the Cell Stacks are incubated at 37°C and 5% CO2.
  • the Cell Stack cultures will approach or reach confluence.
  • the medium in each culture is replaced with fresh medium.
  • the medium, containing produced virus is harvested (Harvest #1), and the cultures re-fed with the same volume (1.1L) of fresh medium.
  • a second harvest is conducted and the cell cultures re fed with same volume (1.1 L) of fresh growth medium.
  • a 3 rd harvest (Harvest #3) is performed. The 3 harvests are then pooled for purification. Viral titers for the 3 harvests and the pool are listed in the following table.
  • Viral production by another cell line with the same technique can be used to produce any of the disclosed viruses, in the human cell line HEK 293 cells (ATTC# CRL-1573) and canine cell lines Cf2TH (ATCC# CRL-1430) or D-17 (ATCC # CCL-183).
  • Three harvests, as described in Example 32, can be collected and pooled.
  • the viral titer in the harvest pool using Cf2TH cells can be 4.9 x 10 L 6 TU/mL.
  • a central mechanism that regulates HSPC-directed migration from blood to bone marrow and retention in the bone marrow niches involves activation of the CXCR4 receptor on HSPCs by chemokine CXCL12 (also known as Stromal cell-Derived Factor-1 (SDF-1) (Nagasawa et al. 1996; Oberlin et al. 1996) expressed on different subsets of stromal cells including reticular Nestin+ mesenchymal stem and progenitor cells (MSPCs) (Mendez-Ferrer et al. 2010), human reticular CD146+ MSPCs (Sacchetti et al. 2007) and perivascular reticular leptin receptor ⁇ cells (Ding et al.
  • SDF-1 Stromal cell-Derived Factor-1
  • CXCL12 secreted by these cells and adsorbed to the extracellular matrix inducing a CXCL12 gradient and induction of HSPC adhesion to the bone marrow niches (Sugiyama et al. 2006). Interference with CXCL12/CXCR4 interaction, such as by conditional deletion of CXCR4 or CXCL12 in mice, resulted in a reduced retention of HSPCs in the bone marrow and in dramatically increased migration of HSPC numbers into the peripheral blood and into spleen (Tzeng et al. 2011). CXCL12 (SDF-1) itself activates cell-surface integrins VLA-4, VLA-5, and LFA-1 (Peled et al. 2000).
  • CD34(+) cells with CXCL12 led to firm adhesion and transendothelial migration, which was dependent on LFA-l/ICAM-1 and VLA-4/VCAM-1 interactions, and furthermore, CXCL12 (SDF-1)-induced polarization and extravasation of CD34(+) / CXCR4(+) HSPC through the extracellular matrix underlining the endothelium was dependent on both VLA-4 and VLA-5 (Peled et al. 2000).
  • CXCL12 SDF-1 also activates the adhesion molecule CD44 and thereby rapidly and potently stimulates HSPC adhesion to immobilized hyaluronic acid and homing of HSPC to bone marrow, which could be blocked by anti-CD44 monoclonal antibodies or by soluble hyaluronic acid, and significantly impaired after intravenous injection of hyaluronidase (Avigdor et al. 2004).
  • inhibition of CXCL12 (SDF-1)/CXCR4 interaction has downstream effects on multiple HSPC-stromal adhesion interactions.
  • AMD3100 (Plerixafor) is a synthetic organic molecule of the bicyclam class, originally developed as an anti-HIV drug (De Clercq 2019). By antagonizing the CXCR4 receptor, thus interfering with the CXCR4 / CXCL12 (SDF-1) interaction which tethers stem cells to the bone marrow stroma, AMD3100 was found to rapidly mobilize HSPCs in various animal models (Broxmeyer et al. 2005).
  • AMD3100/Plerixafor (trade name Mozobil) was approved in the United States for use in combination with G-CSF to mobilize HSPCs to the peripheral blood for collection and subsequent autologous transplantation in patients with Non-Hodgkin's lymphoma or multiple myeloma, and is used as a supportive measure in cases where G-CSF- mediated HSPC mobilization fails to induce sufficient numbers of HSPCs (De Clercq 2019).
  • the rapid mobilization by AMD3100 does not only regulate CXCL12 levels, but also induces activation of proteases such as matrix metalloprotease-9 (MMP-9) and urokinase- type plasminogen activator (uPA) (Dar et al. 2011).
  • MMP-9 matrix metalloprotease-9
  • uPA urokinase- type plasminogen activator
  • Activated bone marrow stromal and endothelial cells also play a role in secreting CXCL12 (SDF
  • T- 140 (4F-benzoyl-TN14003) is another CXCR4 inhibitor that mobilizes HSPCs and also erythroblasts in murine models and displays synergy with G-CSF (Abraham et al. 2007).
  • Firategrast an a4b1 and a4b7 integrin inhibitor, has been shown to disrupt HSPC retention in the postnatal hematopoietic niche and to synergistically interact with the CXCR4 inhibitor AMD3100 (Kim et al. 2016).
  • Vedolizumab a humanized monoclonal antibody against the a4b7 integrin, is available and has been tested in clinical trials for Crohn's disease and ulcerative colitis, and more recently for prevention of graft-vs.-host disease after allogeneic HSPC transplantation (Chen et al. 2019). Reduction in plasma CXCL12 levels, accompanied by reduced HSPC egress, has been observed in mice treated with vedolizumab.
  • BOP N-(benzenesulfonyl)-L-prolyl-L-O-(1- pyrrolidinylcarbonyl)tyrosine
  • integrins a9b1/a4b1. It has been reported that a single dose of this small molecule antagonist rapidly mobilizes long-term multi-lineage reconstituting HSPC, and also enhanced AMD3100-induced HSPC mobilization (Cao et al. 2016).
  • RhoA activity The Rac small GTPases expressed in hematopoietic progenitors and HSPCs have been found to be intricately involved in HSPC mobilization, and application of a specific inhibitor of Rac activity (but not Cdc42 or RhoA activity) may be used for rapid HSPC mobilization (Cancelas et al. 2005). Recently, it was shown that Racl activation leads to reversible conformational change in human CXCR4 that potentiates CXCL12 / CXCR4 signaling, implying reciprocal cross-talk between these signaling pathways (Zoughlami et al. 2012).
  • Hematopoietic growth factors especially granulocyte colony stimulating factor (G-CSF, filgrastim, lenograstim) and granulocyte-macrophage colony stimulating factor (GM-CSF, molgramostim, sargramostim) have been shown to be effective at mobilizing HSPC into the peripheral blood, to levels up to 60-fold over baseline using mobilization with hematopoietic growth factors alone (Peters et al. 1993; Gazitt 2002).
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • G-CSF Endothelial cells have been shown to be the main constitutive source of infection-induced expression of G-CSF in bone marrow (Boettcher et al. 2014). It is therefore likely that these signals are related to or part of mobilization of HSPCs.
  • neutrophils permeabilize the sinusoid endothelial barrier in the bone marrow, as shown by transmission electron microscopy studies (Lee et al. 2009).
  • disruption of the bone marrow endothelial barrier is prominent during repetitive G-CSF stimulations, indicating that the endothelial cells do function as a target of G-CSF during HSPC mobilization (Szumilas et al. 2005).
  • osteopontin In osteoblasts, SCF/c-kit ligand, IL-7, and VCAM-1 and, as a counter-regulator of HSPC maintenance, osteopontin are all selectively down-regulated during mobilization by G-CSF treatment or after b3 adrenoreceptor activation (Mendez-Ferrer, Battista, and Frenette 2010). Since G-SCF stimulates b-adenergic sympathetic nervous activity, this provides evidence for a signaling chain inducing HSPC mobilization.
  • G-CSF treatment was shown to induce a robust expansion of neutrophils within the bone marrow, and they are the first cells to egress from the bone marrow during mobilization after G-CSF administration (Day and Link 2012).
  • All three classical types of HSPC mobilization by external stimuli i.e. G-CSF, chemotherapy and chemokines, are disrupted if the G-CSF receptor is absent in neutrophilic granulocytes, but not if it is absent in HSPC despite normal hematopoiesis (Liu, Poursine-Laurent, and Link 2000).
  • G-CSF receptor has been shown to be required for cyclophosphamide- or IL-8-induced, but not FLT3L-induced mobilization (Liu, Poursine-Laurent, and Link 2000).
  • Serine proteases and metalloproteinases released by neutrophilic granulocytes have been shown to cleave VCAM-1, c-kit, SCF, and CXCL12 (SDF-1) from stromal niche cells or HSPCs and include neutrophil elastase, cathepsin G and MMP-9 (Levesque et al. 2002; Heissig et al. 2002).
  • G-CSFR Granulocyte colony-stimulating factor receptor
  • Myelopoietin Myelopoietin (MPO), a multifunctional agonist of interleukin-3 (IL-3) and G-CSF receptors, has also been reported to be an effective and efficient mobilizer of hematopoietic colony-forming cells (CFC) and CD34+ HSPC relative to control cytokines in normal nonhuman primates (MacVittie et al. 1999).
  • MPO Myelopoietin
  • IL-3 interleukin-3
  • G-CSF receptors G-CSF receptors
  • VEGF Growth factor-stimulated progenitor cells produce large quantities of cytokines (e.g. vascular endothelial growth factor, VEGF) which may act on endothelial cells to modify their growth, motility, permeability, and fenestration. Increased vascular leakage as well as 2- to 3-fold increases in HSC numbers in the blood occurred within 15 min of an intravenous administration of rhVEGF164 or histamine (Smith-Berdan et al. 2015).
  • cytokines e.g. vascular endothelial growth factor, VEGF
  • VEGF vascular endothelial growth factor
  • VEGF may also be specifically involved in the mobilization and homing of hematopoietic progenitor cells as indicated by murine models in which hematopoiesis did not develop in the absence of VEGF or VEGF- receptors (Shalaby et al. 1995). Notably, however, administration of VEGF followed by treatment of mice with AMD3100 resulted in mobilization of both endothelial progenitor cells and stromal progenitor cells, but suppressed HSPC mobilization (Pitchford et al. 2009).
  • SCF Cytokine receptors such as c-kit, the receptor for the cytokine kit-ligand (stem cell factor, SCF) are also downregulated on circulating HSPC. Because membrane-bound cytokines such as kit-ligand (SCF) are expressed on bone marrow stromal and endothelial cells, c-kit may also act as an adhesion molecule and play a role in progenitor cell mobilization and homing. Downregulation of CXCL12 (SDF-1) in the bone marrow was also observed upon administration of SCF and FLT3-L (Christopher et al.
  • SDF-1 CXCL12
  • osteoblastic cells secrete b-AR agonists to down-regulate expression of CXCL12, VCAM-1, and SCF (Katayama et al. 2006; Mendez-Ferrer, Battista, and Frenette
  • Complement factors Monocytic cells are involved through the complement cascade, which is activated by radio- and chemotherapy, thereby liberating the complement factors C3 (C3a, desArgC3a) and C5 (C5a and desArgC5a) cleavage fragments, which act as anaphylatoxins (Ratajczak et al. 2013).
  • Complement factor 3 (C3) knockout mice under steady-state conditions, are hematologically normal but display a significant delay in hematopoietic recovery subsequent to irradiation or transplantation of wild-type HSPCs, and C3 complement factors increase responsiveness of HSPCs to CXCR4 / CXCL12 axis (Ratajczak et al. 2013), also suggesting an involvement of the classical complement activation pathway in HSPC mobilization. Moreover, complement factor 5(C5)-deficient mice showed impaired mobilization of HSPCs (Ratajczak et al. 2013).
  • Sphingolipids and Nucleotides In addition to CXCL12 (SDF-1) and its receptor CXCR4 a number of other chemoattractants, inducing migration of HSPCs are known. These include the sphingolipids sphingosine-l-phosphate (SIP) (Golan et al. 2012; Ratajczak et al. 2014) that couple to G protein-coupled sphingosine 1-phosphate receptor 1 (S1P1), and ceramide-l-phosphate (C1P)
  • SIP sphingolipidsphingosine-l-phosphate
  • S1P1P1P1 G protein-coupled sphingosine 1-phosphate receptor 1
  • C1P ceramide-l-phosphate
  • extracellular nucleotides such as adenosine triphosphate (ATP) or uridine triphosphate (UTP) (Rossi et al. 2007) and the divalent cation Ca2+ and its receptor (CaR) (Adams et al. 2006), and H+ (Krewson et al. 2020) regulate adhesion molecules on the surface of the HSPCs as well as on certain cells of the stem cell niches, such as endothelial cells. Whereas the cells of origin of these molecules are in many cases not clear as yet, their action includes the mediation of engraftment (after transplantation), adhesion (under steady state), and mobilization (after its induction).
  • SIP SIP and the receptor S1P1 are suggested to regulate HSPC steady-state egress and mobilization from the bone marrow.
  • SIP is produced by mature red blood cells and activated platelets, leading to micromolar SIP concentrations in blood, mostly bound to albumin and high-density lipoproteins (HDL) (Pappu et al. 2007; Liu et al. 2011). Since only low concentrations of SIP are detectable in solid tissues, a constant SIP concentration gradient between the bone marrow and blood important for the constant steady-state release of HSPCs has been suggested.
  • HDL high-density lipoproteins
  • a second SIP receptor, S1PR3, expressed on HSPCs is suggested to be involved in the retention of HSPCs in the bone marrow niche. Inhibition or knockout of S1PR3 results in mobilization of HSPCs into blood circulation, whereby S1PR3 antagonism suppresses bone marrow and plasma CXCL12 (SDF-1) concentrations (Ogle et al. 2017). On the other hand, S1PR3 antagonism increases AMD3100 induced mobilization, indicating a synergy between S1PR3 and CXCR4 pathways.
  • C1P After lethal irradiation, levels of SIP and C1P have been seen to increase in the bone marrow microenvironment; moreover, peripheral circulating HSPCs are exposed to relatively high levels of SIP and C1P present in the circulation. Both these mechanisms may desensitize responses to potential homing gradients of these bioactive lipids (Ratajczak et al. 2014).
  • ATP and UTP 5-nucleotide triphosphates, particularly ATP and UTP, engage P2 nucleotide receptor-mediated regulation of proliferation, differentiation, cell death, and chemotaxis on hematopoietic cells including HSPCs (Lemoli et al. 2004).
  • UTP is thought to represent an endogenous danger signal, which is rapidly released into the extracellular environment due to tissue injury and cell death.
  • UTP and other nucleotides induce migration of leukocytes into the injured tissue, stimulate tissue recovery by induction of cell proliferation, and promote resolution of the immune response by activating anti-inflammatory pathways (Di Virgilio, Boeynaems, and Robson 2009).
  • UDP-glc has been shown to mobilize long-term repopulating HSPCs, and co-administration of UDP-glc and G-CSF led to greater HSPC mobilization than G-CSF alone (Kook et al. 2013).
  • HSPCs mobilized with UDP-glc plus G-CSF repopulated better than HSPCs mobilized with G-CSF alone.
  • UDP-glc-mobilized HSPCs exhibited a more lymphoid-biased differentiation capacity, indicating that UDP-glc mobilizes a functionally distinct subset of HSPCs (Kook et al.
  • ROS reactive oxygen species
  • Ca2+ and CaR HSPCs express the seven-transmembrane- spanning CaR which is strongly involved in retention and liberation of HSPCs in/from the stem cell niche.
  • Newborn mice deficient in CaR revealed a reduced cellularity in the bone marrow and a lack of primitive HSPCs, whereas increased numbers of HSPCs were found in the circulation and spleen.
  • the fetal liver of CaR-/- mice had normal numbers of HSPCs with normal proliferation, differentiation, and migrational capacity. But these HSPCs revealed an adhesion defect to collagen I, leading to a defective lodgment to the endosteal niche (Adams et al. 2006).
  • Ca2+ treatment increased transcription and expression of CXCR4 of bone marrow cells and SDF-l-mediated CXCR4 internalization, while Ca2+ influx inhibitors or blocking of CaR with antibodies inhibited Ca2+- induced CXCR4 expression, indicating that Ca2+-induced changes on HSPCs are partially modulated by an increased expression of CXCR4 (Wu et al. 2009).
  • CAMPs Cationic antimicrobial peptides
  • Cathelicidin LL-37 1-2 ng/ml
  • 2-defensin 2-defensin
  • GTPases small guanine nucleotide triphosphatases
  • Rac-1 and Rac-2 small guanine nucleotide triphosphatases
  • Cathelicidin LL-37 is an antimicrobial peptide expressed by bone marrow stromal cells and is upregulated after bone marrow irradiation. It was shown that LL-37 enhances chemotactic migration and adhesiveness of HSPCs and that short-time pre-incubation of HSPCs with LL-37 prior to transplantation accelerates recovery of platelet and neutrophil counts in mice (Wu et al. 2012). Its effect on mobilization remains to be determined.
  • Prostaglandin E2 Murine and human HSPCs express prostaglandin E2 (PGE2) receptors.
  • PGE2 prostaglandin E2
  • short-term ex vivo exposure to PGE2 enhances homing and proliferation of HSPCs and increases the number of primitive, long- term repopulating cells, indicating that PGE2 supports self-renewal of HSPCs (North et al. 2007; Hoggatt et al. 2009).
  • CFU-GM colony-forming units-granulocyte macrophage
  • CFU-M macrophage
  • NSAID nonsteroidal anti-inflammatory drugs
  • PEG2 in the bone marrow niche is supported by the findings of i) an increased PGE2 production by irradiated bone marrow stromal cells, and ii) a C1P- and SlP-induced, increased expression of cyclooxygenase 2 in stromal cells, whereby C1P and SIP is released from lethally irradiated, damaged bone marrow cells, suggesting that conditioning for HSPC transplantation by lethal irradiation induces PGE2 production in bone marrow (Ratajczak et al. 2014).
  • PGE2 treatment increases cellular CXCR4 mRNA concentration and the expression of CXCR4 on the HSPC surface, leading to an enhanced migration to SDF-1/CXCL12 in vitro and homing to bone marrow in vivo. Furthermore, PGE2 enhances HSPC survival due to an increased expression of survivin and reduction in intracellular active caspase-3 (Hoggatt et al. 2009). Another mechanism, which is important for PGE2-mediated modulation of HSPC retention and egress of HSPCs into the blood, is the regulation of osteopontin expression.
  • osteopontin is a negative regulator of the size of the stem cell pool, and down-regulation or absence of osteopontin is associated with an increased number of primitive HSPCs (Stier et al. 2005).
  • osteopontin expressed by stromal cells may be associated with a superior repopulating ability and long-term engraftment of G-CSF plus NSAID mobilized stem cell graft compared to grafts mobilized with G-CSF alone (Hoggatt et al. 2013).
  • EPI-X4 is a 16 amino acid peptide isolated from human blood filtrate and plasma and generated from albumin under acidic conditions by aspartic proteases cathepsin D and E, which are released from immune cells during inflammatory processes (Zirafi et al. 2015). Neutrophils were shown to produce EPI-X4 from albumin (Zirafi et al. 2015). Since neutrophilic granulocytes in the bone marrow are strongly activated during HSPC mobilization with G-CSF, which leads to a highly proteolytic environment (Levesque et al.
  • GRO a truncated form of CXCR2 agonist
  • Sildenafil A factor which is likely dependent on monocytes and which regulates HSPC mobilization is nitric oxide. Mobilization studies in inducible nitric oxide synthase (iNOS) -/- mice indicated that iNOS is a negative regulator of hematopoietic cell migration and prevents egress of HSPCs into peripheral blood during mobilization (Adamiak et al. 2017). Sildenafil citrate (Viagra), a phosphodiesterase type 5 (PDE5) inhibitor, blocks degradation of cyclic GMP in the smooth muscle cells lining blood vessels, resulting in vasodilation.
  • Viagra Sildenafil citrate
  • PDE5 a phosphodiesterase type 5
  • Cyclophosphamide-mobilized CD34+cells have few pre-B lymphocytes, as determined by low CD19 expression, together with low CD71 expression, suggesting a lack of active proliferation, and variation in the incidence of the subpopulations between different patients.
  • CD38 antigen CD38-
  • G-CSF-mobilized PBPC contain a significantly greater proportion of the primitive progenitors (only 88% of cells expressing CD38) than those mobilized by chemotherapy plus G-CSF (97% express CD38), chemotherapy plus GM-CSF (96.4% express CD38) or high-dose chemotherapy alone (99.1% express CD38).
  • LTCIC long-term culture-initiating cells
  • Chemotherapeutic drug regimens for mobilization [00346]
  • the number of circulating HSPC can be significantly increased during the recovery period after myelosuppressive chemotherapy, as compared to steady-state levels.
  • a single dose of 4 g/m 2 cyclophosphamide can result in up to 25-fold increase in the number of CFU-GM.
  • CD34+ cell yields on the order of 3.62 c 10 6 cells/kg can be achieved following cyclophosphamide doses of 4-7 g/m 2 ).
  • High-dose etoposide (2 g/m 2 ) may also be used as a safe and effective method to mobilize HSPC.
  • Chemotherapy-induced mobilization may avoid delay in the administration of effective anti-cancer treatment and allows in vivo purging in addition to HSPC mobilization.
  • G-CSF G-CSF alone can effectively mobilize HSPC from the bone marrow into the peripheral blood.
  • a dose of filgrastim 10 pg/kg/day is usually adequate, but some studies have used 16 pg/kg/day.
  • G-CSF The capacity of G-CSF to increase the circulating number of progenitor cells was first noted by Durhsen et al34, and further studies have shown that G-CSF mobilizes progenitor cells from the bone marrow into peripheral blood in patients with various malignant diseases.
  • Sheridan et al. investigated the use of G-CSF (12 pg/kg/day for 6 days) alone to mobilize PBPC in 17 patients with non-myeloid malignant disorders of poor prognosis. A mean total of 33 x 10 4 CFU-GM/kg were harvested.
  • G-CSF (10 pg/kg/day) adequately mobilized PBPC in 34 patients with Hodgkin's disease or NHL, allowing the harvest of a median 32.6 c 10 4 CFU-GM cells/kg and 2.8 x 10 6 CD34+ cells/kg.
  • De Arriba et al. reported a mean yield of 0.77 x 10 6 CD34+ cells/kg and 1.42 c 10 6 CD34+ cells/kg after the first and second leukapheresis, respectively, in 10 breast cancer patients following mobilization with G-CSF alone (0.84 ⁇ 0.1 pg/kg/day).
  • G-CSF dose-response effect A mobilization dose-response effect is apparent in healthy adults and patients.
  • the characterization of the PBPC mobilized may also be affected by the size of the administered G-CSF dose. Increased mobilization of less mature progenitors into the circulation (i.e. mixed colony-forming cells, CD34+ CD33- cells and CD34+ HLA-DR- cells) may occur when the dose of G-CSF is raised from 100 to 200 pg/m 2 .
  • G-CSF Practical example: Based on current dosage recommendations for HSPC mobilization,10 pg/kg/day of filgrastim or lenograstim are administered as de novo mobilization, or 5 g/kg/day for filgrastim or lenograstim when given in combination with chemotherapy (Amgen; Chugai Pharma UK Ltd/Rhone-Poulenc Rorer), for at least 4 days. Virus vector infusion can commence on day 5 and continue for 3 consecutive days.
  • GM-CSF is also effective for mobilizing HSPC, with doses of 4-64 pg/kg/day by continuous intravenous infusion for up to 7 days in cancer patients resulting in 4- to 18-fold increased HSPC in the peripheral blood.
  • HSPC mobilization can be achieved using GM-CSF alone, although it should be noted that, while the feasibility of using GM- CSF for progenitor cell mobilization has been confirmed, there is no evidence of superiority over mobilization with G-CSF alone.
  • the CFU-GM content of HSPC populations mobilized by GM- CSF appears to be dose responsive with a 250 pg/m2 dose producing a superior yield to 125 pg/m2.
  • the objective of the introduction of a hematopoietic growth factor into a chemotherapy-based mobilization regimen is to enhance HSPC yield while supporting early anti-cancer treatment.
  • the addition of a hematopoietic growth factor after chemotherapy significantly increases the number of HSPC mobilized in comparison with chemotherapy alone.
  • the inclusion of chemotherapy in the mobilization regimen may be important for the prompt initiation of the treatment of some tumors. However, when this is not required, the adverse effects of chemotherapy should be carefully considered, and a hematopoietic growth factor-only mobilization regimen may be more appropriate. Also, repeated chemotherapy plus G-CSF may lead to induction of fewer long-term culture initiating cells (LTC-IC) than following mobilization with G-CSF alone.
  • LTC-IC long-term culture initiating cells
  • G-CSF plus cyclophosphamide The combination of G-CSF with high-dose cyclophosphamide (4-7 g/m 2 ) has been shown to improve the yield of CD34+ HSPC levels in the blood.
  • cyclophosphamide may be used to improve the yield of HSPC.
  • patients with multiple myeloma can be mobilized with high-dose cyclophosphamide 7 g/m 2 plus G-CSF (300 pg/day), resulting in significantly higher yields of >2.5 c 10 6 CD34+ cells/kg, than could be obtained with cyclophosphamide 4 g/m 2 plus G-CSF.
  • G-CSF plus etoposide has been shown to be effective in the treatment of NHL, Hodgkin's disease and, to a lesser extent, breast cancer. Inclusion in the mobilization regimen of patients with such malignancies is logical to provide treatment during the mobilization phase.
  • HSPC high-dose etoposide (2 g/m 2 ) by continuous intravenous infusion over 24 hours plus G-CSF (5 pg/kg/day) beginning 48 hours after the completion of the etoposide infusion.
  • the mobilization regimen was effective, as evidenced by median yields of 24 * 10 6 CD34+ cells/kg (range: 9.5-27.7 c 10 6 /kg), 28.0 x 10 6 CD34+ cells/kg (range: 1.7-81.8 c 10 6 /kg), 22.7 c 10 6 CD34+ cells/kg (range: 2.7-79.3 c 10 6 /kg) mobilized in the patients with Hodgkin's disease, NHL, and breast cancer, respectively, measured starting from the first day that the white blood cell count recovered to 1 * 10 9 /L (median 10 days post-etoposide, range 7-16 days).
  • G-CSF plus cyclophosphamide plus etoposide HSPC mobilization can also be achieved following high-dose cyclophosphamide (4 g/m 2 ) plus etoposide (600 mg/m 2 ), with or without G-CSF, in patients with breast cancer, myeloma and other malignancies.
  • the inclusion of G-CSF in the mobilization regimen led to a yield with nearly 5-fold more CD34+ cells than following mobilization with cyclophosphamide and etoposide alone.
  • G-CSF plus combination chemotherapy Combination chemotherapy with 5-fluorouracil (500 mg/m 2 ), epirubicin (120 mg/m 2 ) and cyclophosphamide (500 mg/m 2 ) intravenously on day 1, followed by G-CSF (10 pg/kg/day) from day 2, resulted in a median 17.7 * 10 6 (range: 9.4-50.6 c 10 6 ) CD34+ cells/kg in patients with breast cancer.
  • chemotherapeutic drugs including but not restricted to, e.g., paclitaxel, to regimens combining cyclophosphamide plus G-CSF may produce further improvement in mobilization.
  • the vector encodes CRISPR/CAS9 and guide RNAs to knockout CCR5 (FIG. 18A). In another example, the vector encodes CRISPR/CAS9 and guide RNAs to knockout CCR2 (FIG. 18B). In additional examples, the vector encodes CRISPR/CAS9 and guide RNAs to knockout both CCR5 and CCR2 (FIG.
  • the vector encodes a single chain antibodies, including camelid and shark antibodies or other protein binding molecules such as darpins, affimers, hikamers and the like U.H.Wiedle et al. Cancer Genomics & Proteomics 10: 155-168, 2013;
  • Further embodiments include the addition of lineage specific promoters so that CCR5 and CCR2 are knocked out in the desired T cell population.
  • CRISPR/CAS9 is driven by a CD3 promoter so that expression of CRISPR/CAS9 occurs in the T cell lineage from post-HSC transduction by vector and subsequent HSC maturation (FIG. 18D).
  • Additional T cell promoters may be substituted including CD4 and CD8 promoters.
  • the vector encodes a single chain antibodies, including camelid and shark antibodies or other protein binding molecules such as darpins, affimers, hikamers and the like (U.H. Wiedle et al. Cancer Genomics & Proteomics 10: 155-168, 2013;
  • CRISP/CAS 9 is replaced with shRNA or microRNA or silencing RNA (all referred to as siRNA) that knockdown expression of CCR5 and or CCR2 using the same configuration as for the CRISPR/CAS9 system (FIG. 20A-D).
  • shRNA or microRNA or silencing RNA all referred to as siRNA
  • the vectors described in FIG. 21 is used to treat patients diagnosed with multiple sclerosis.
  • CRISPR/CAS9 or siRNAs are used to remove or reduce CCR5 and CCR2 expression from T cells using the HSC linage specific CD3 promoter.
  • the vector contains a cellular kill switch, thymidine kinase (TK) that allows for the termination of treatment or to stop acute inflammatory episodes whereby patients are given TK prodrug that is converted into a toxic chemical that kills the TK expressing cells (FIG. 21).
  • TK thymidine kinase
  • a patient viremic with HIV is taken off antivirals that are known or suspected to inhibit retroviral vectors for 2—10 days, then administered a dose of E6, E7, E8 E9, E10, Ell or E12 of a clinically acceptable (GMP) preparation of the retroviral vector that contains transgenes for CRISPR/CAS9 and guide RNAs to knockout CCR5.
  • GMP clinically acceptable
  • Vector infusion is complimented with an appropriate adjuvant that mobilizes HSC from the relatively inaccessible bone marrow to the accessible periphery, as described in the examples herein.
  • the patient's T cells show loss of CCR5 expression over time and subsequent reduced levels of HIV DNA positive T cells in the positive cells in the blood and increased levels of transduced HIV-free T cells.
  • the majority of patients T cells are converted to CCR5 negative and are resistant to HIV infection within 1 -3 months. Over time, HIV is cleared from the patient's body and the patients goes into long term remission with long-term resistance to HIV.
  • a multiple sclerosis patient at the early, inflammatory phase of the disease undergoes dosing with a GMP preparation of retroviral vector with a titer of E8 TU/ml or greater on PC3 Cells designed to silence CCR2/5 in HSC and descendant T cells post-vector transduction.
  • Dosing parameters are the same or similar to those used for the HIV patient with pretreatment by an appropriate, mobilizing agent.
  • the loss of CCR2 and CCR5 prevents T cells from migrating to MS-related inflammation and prevents damage to tissues.
  • ganciclovir is given to the MS patient during acute phase of MS and leading to depletion of marked T cells and reduction in MRI-measured lesions in the patient.
  • a pediatric Adenosine DeAminase (ADA) deficient Severe Combined Immuno-Deficient (SCID) patient is dosed with a GMP preparation of retroviral vector with a titer of E8 TU/ml or greater on PC3 Cells that leads to expression of human ADA in transduced cells. Doses are between E6 to E12 total TU's and are preceded by appropriate dosing with an HSC mobilizing agent. The recovery to normal or pseudo-normal immune function is as described by A.Aiuti et al. NEJM 2009 after conventional transplantation of ex vivo transduced autologous stem cells into pediatric ADA SCID patients.
  • Plerixafor can be used to specifically mobilize CD34+ HSPCs, either used alone or as an adjunct to G-CSF.
  • the doses used are 160 pg/kg x 1 on day 5 for plerixafor, and 10 pg/kg on days 0,
  • a single dose of plerixafor at 240 pg/kg (subcutaneously) may provide a more rapid and possibly less toxic and cumbersome alternative to traditional G-CSF based mobilization.
  • G-CSF 10 pg/kg subcutaneously daily for up to eight days, together with plerixafor, beginning on the evening of day 4 and continuing daily for up to four days, subcutaneously at a (daily) dose of 240 pg/kg, has been approved by FDA and recommended for autologous stem cell mobilization and transplantation for patients with Non-Hodgkin's lymphoma or multiple myeloma.
  • the conventional dose of plerixafor is 240 pg/kg, but this dose could be safely increased (in healthy individuals) to 480 pg/kg.
  • CD34+ HSPC counts in the blood elevate from 1-2 cells/pL to a peak of approximately 25 cells/pL within 8-10 hours, and gradually diminish but are still at approximately 10 cells/pL by 24 hours post injection.
  • CD34+ HSPC counts in the blood elevate from 1-2 cells/pL to a peak of approximately 30 cells/pL within 8-10 hours, and gradually diminish but are still at approximately 20 cells/pL by 24 hours post-injection.
  • Viagra was administered via oral gavage (OG) (3 mg/kg) to mice, once, 1 h before a single subcutaneous (SQ) injection of AMD3100 (2.5 mg/kg).
  • Control mice received 5-day G-CSF treatment, administered once daily (250 mg/kg).
  • Blood was collected by perfusion 1 h after AMD3100 or 24 h after G-CSF and analyzed by flow cytometry and multilineage reconstitution of lethally irradiated recipients.
  • HSCs in the bloodstream in the rapid 2-hr (2,500 HSCs/mouse) and 3-day (2,800 HSCs/mouse) Viagra/AMD3100 combination were similar to the numbers present 1 day after 4 consecutive days of G-CSF injections (3,400 HSCs/mouse).
  • the HSPC were harvested from peripheral blood (PB), spleen(S) and bone marrow (BM) after transduction in vivo with high titer (>E8 TU/ml) purified RNV encoding GFP, and characterized by two different assays: colony formation; and FACS analyses.
  • the mobilization protocol was shown to work in the target animals (Balb/c mice).
  • Balb/c mice were used.
  • a first group (Group 1) was mobilized using cyclophosphamide + GCSF + AMD3100 as follows: o Cyclophosphamide + G-CSF + AMD3100 mobilization regimen o Day 0: Cyclophosphamide (CY) (ip) 4mg/100 pL per 20g mouse (200 mg/kg/day) o Day 1: G-CSF (sc) 5pg / pL per 20g mouse per day (250pg/kg/day) o Day 2: G-CSF (sc) 5pg/ pL per 20g mouse per day (250pg/kg/day) o Day 3: G-CSF (sc) 5pg/ pL per 20g mouse per day (250pg/kg/day) o Day 4: AMD3100 (sc) 100 mg/ pL
  • a second group (Group 2) was not mobilized.
  • Table 10 Summary of results from FACS analyses of mobilization of HSPC (Lin-, Scal+, Kitl+) in PB, spleen & bone marrow. Individual numbers represent values from individual mice
  • HSPC Hematopoietic Stem/Progenitor Cell
  • PB Peripheral blood
  • S Spleen
  • BM Bone Marrow
  • PB Peripheral blood
  • S Spleen
  • BM Bone Marrow
  • Table 11 Mobilization and transduction with RNV-GFP of HSPC from PB S, & BM; numbers below 0.1% were counted as zero as that is the limit of reliable detection of the FACS machine
  • Table 12 (Experiment A2) HPSC mobilization followed by RNV transduction in vivo for 2 hours: Peripheral Blood(PB); CFU: colony forming unit, M: monocyte/macrophage, G: granulocyte, GM: granulocyte/macrophage, GEMM: granulocyte/erythroid/macrophage/ megakaryocyte.
  • PB Peripheral Blood
  • CFU colony forming unit
  • M monocyte/macrophage
  • G granulocyte
  • GM granulocyte/macrophage
  • GEMM granulocyte/erythroid/macrophage/ megakaryocyte.
  • 1+2+3 pooled peripheral blood from mobilized but untransduced negative controls
  • 4 thru 9 peripheral blood from individual mobilized and RNV-transduced mice
  • 10 peripheral blood from non-mobilized untransduced control:
  • Table 12 shows the numbers of GFP + hematopoietic stem/progenitor cell (HSPC) derived colonies obtained after culturing 4xl0 4 peripheral blood (PB) cells per well in 12-well plate for 5 days in MethoCult GFM3434 (Stem Cell Technologies).
  • Table 13 (Experiment A2) HPSC mobilization followed by RV transduction in vivo for 2 hours: Spleen (S); CFU: colony forming unit, M: monocyte/macrophage, G: granulocyte, GM: granulocyte/ macrophage, GEMM: granulocyte/erythroid/macrophage/ megakaryocyte.
  • Table 13 shows the Numbers of GFP+ hematopoietic stem/progenitor cell (HSPC) derived colonies obtained after culturing 2xl0 5 spleen (S) cells per well in 12-well plate for 5 days in MethoCult GFM3434 (Stem Cell Technologies).
  • HSPC hematopoietic stem/progenitor cell
  • Table 14 (Experiment A2) HPSC mobilization followed by RV transduction in vivo for 2 hours: Bone Marrow (BM); CFU: colony forming unit, M: monocyte/macrophage, G: granulocyte, GM: granulocyte/macrophage, GEMM: granulocyte/erythroid/macrophage/ megakaryocyte.
  • 1+2+3 pooled bone marrow from mobilized but untransduced negative controls, 4 thru 9: bone marrow from individual mobilized and RV- transduced mice, 10: bone marrow from non-mobilized untransduced control:
  • Table 14 shows the numbers of GFP + hematopoietic stem/progenitor cell (HSPC)-derived colonies obtained after culturing 3xl0 4 bone marrow (BM) cells per well in 12-well plate for 5 days in MethoCult GF M3434 (StemCell Technologies).
  • HSPC hematopoietic stem/progenitor cell
  • Table 15 (Experiment A3) HPSC mobilization followed by RNV transduction in vivo for 2 days; CFU: colony forming unit, M: monocyte/macrophage, G: granulocyte, GM: granulocyte/macrophage, GEMM: granulocyte/erythroid/macrophage/megakaryocyte. 1+2+3: pooled peripheral blood from mobilized but untransduced negative controls,
  • peripheral blood from individual mobilized and RV- transduced mice (note: #5 died after RNV injection and was removed from analysis), 10: peripheral blood from non-mobilized untransduced control:
  • Table 15 shows the numbers of GFP + hematopoietic stem/progenitor cell (HSPC)-derived colonies obtained after culturing 4xl0 4 peripheral blood (PB) cells per well in 12-well plate for 7 days in MethoCult GF M3434 (StemCell Technologies).
  • Table 16 (Experiment A3) HPSC mobilization followed by RV transduction in vivo for 2 days: Spleen (S); CFU: colony forming unit, M: monocyte/macrophage, G: granulocyte, GM: granulocyte/ macrophage, GEMM: granulocyte/erythroid/macrophage/ megakaryocyte.
  • Table 16 shows the numbers of GFP + hematopoietic stem/progenitor cell (HSPC)-derived colonies obtained after culturing 2xl0 5 spleen (S) cells per well in 12-well plate for 7 days in MethoCult GF M3434 (StemCell Technologies.
  • HSPC hematopoietic stem/progenitor cell
  • Table 17 (Experiment A3) HPSC mobilization followed by RNV transduction in vivo for 2 days: Bone Marrow (BM); CFU: colony forming unit, M: monocyte/macrophage, G: granulocyte, GM: granulocyte/macrophage, GEMM: granulocyte/erythroid/macrophage/ megakaryocyte.
  • 1+2+3 pooled bone marrow from mobilized but untransduced negative controls
  • 4 thru 9 bone marrow from individual mobilized and RV-transduced mice (note: #5 died after RNV injection and was removed from analysis), 10: bone marrow from non- mobilized untransduced control.
  • Table 17 shows the numbers of GFP + hematopoietic stem/progenitor cell (HSPC)-derived colonies obtained after culturing 3xl0 4 bone marrow (BM) cells per well in 12-well plate for 7 days in MethoCult GF M3434 (StemCell Technologies).
  • HSPC hematopoietic stem/progenitor cell
  • Table 18 (Experiment Bl) Human CD34+ HSPC in vitro RNV transduction for 2 hours: transduction control study; CFU: colony forming unit, M: monocyte/macrophage, G: granulocyte, GM: granulocyte/macrophage, GEMM: granulocyte/erythroid/macrophage/ megakaryocyte, (untransduced: untransduced control CD34+ cells; RV TD 1, 2: RV transduction replicates):
  • Table 18 shows the numbers of GFP+ hematopoietic stem/progenitor cell (HSPC)-derived colonies obtained after culturing 5x102 human CD34+ cells per 35mm dish for 15 days in MethoCult Classic (GF H4434, StemCell Technologies).
  • HSPC hematopoietic stem/progenitor cell
  • This experiment (Bl) shows that the preparations of RNV that give in vivo transduction of mouse stem cells, are also capable of transducing human CD34+ HSPC.

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