FOXP3 engineered CD4+ T cells for use in treg-based immunotherapy
AssigneesThe Board of Trustees of the Leland Stanford Rinior University, Ospedale San Raffaele S.R.L., Fondazione Telethon
InventorsRosa Bacchetta, Maria Grazia Roncarolo, Yohei Sato, Luigi Naldini, Laura Passerini
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Abstract
Engineered Treg-like cells, CD4LVFOXP3 T cells, and their use in cellular therapy to promote immune tolerance are disclosed. In particular, CD4LVFOXP3 T cells are produced by transduction of CD4+ T cells with a lentiviral vector expressing FOXP3 under the control of a constitutive promoter. Transduced cells express FOXP3 at high and persistent levels and acquire immune suppressive characteristics resembling naturally occurring Treg cells.
Claims4
A pharmaceutical composition comprising a unit dose of CD4LVFOXP3 T cells produced by the method comprising:
- i) obtaining a biological sample comprising CD4+T lymphocytes from a subject;
- ii) activating the CD4+ T cells for up to 24 hours in medium comprising IL-2, IL-7, and anti-CD3 and anti-CD28 coated nanomatrix in the absence of antigen-presenting cells;
- iii) transducing the CD4+T lymphocytes in the absence of polybrene with a recombinant lentiviral vector comprising full-length FOXP3 cDNA wherein the recombinant lentiviral vector is a bidirectional lentiviral vector of not more than 10,500 bp comprising full-length FOXP3 cDNA expressed under an EF1a promoter and NGFR expressed under minimal CMV promoter from the opposite strand vector, comprising:
- a) a polynucleotide encoding forkhead box protein 3 (FOXP3);
- b) an elongation factor 1 a (EF1 a) promoter, wherein the EF1 a promoter is operably linked to the polynucleotide encoding FOXP3;
- c) a polynucleotide encoding NGFR;
- d) a modified Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) modified to remove a WHx start codon;
- e) human immunodeficiency virus (HIV)-derived elements comprising a 5′ long terminal repeat (5′ LTR), a Y packaging signal, a truncated Gag sequence, a rev response element (RRE) sequence, a central polypurine tract (cPPT), a central termination sequence (CTS), a truncated negative regulatory factor (NEF) sequence, and a SIN 3′ long terminal repeat (3′ LTR);
- f) a polyadenylation sequence;
- g) an SV40 origin of replication;
- h) a bacterial high copy origin of replication (Ori);
- and
- i) kanamycin resistance gene;
- iv) culturing the CD4+ T lymphocytes in medium comprising IL-2, IL-15 and anti-CD3 and anti-CD28 coated nanomatrix in the absence of antigen-presenting cells for a period of from 5 to 15 days, wherein the CD4+ T lymphocytes are converted into the Treg-like cells (CD4LVFOXP3 T cells);
- and
- v) purifying CD4LVFOXP3 T cells following step iv) by positive immunomagnetic selection to obtain a cell population of greater than 85% purity;
- and a pharmaceutically acceptable excipient.
Dependencies are inferred from the claim text.