TCR and uses thereof

Abstract

The present invention relates to a TCR or functional variant thereof having antigenic specificity for CD1c molecules associated with a self-lipid, preferably (mLPA) or derivative thereof, to relative polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population of cells, antibody and pharmaceutical composition. The present invention also relates to the uses of said TCR and relative products, in particular for use in the treatment and/or prevention of an hematological disorder.

Claims

1. An isolated or purified T-cell receptor (TCR) having antigenic specificity for a self-lipid associated to CDIc molecule, wherein the self-lipid is a methyl-lysophosphatidic acid or derivative thereof wherein the TCR is in monomeric form and comprises: a complementary determining region (CDRa1) amino acid sequence of SEQ. ID NO: 25; and a complementary determining region (CDRa2) amino acid sequence of SEQ. ID NO: 26; and a complementary determining region (CDRa3) amino acid sequence of SEQ. ID NO: 27; and a complementary determining region (CDRb1) amino acid sequence of SEQ. ID NO: 28; and a complementary determining regions (CDRb2) amino acid sequence of SEQ. ID NO: 29; and complementary determining regions (CDRb3) amino acid sequence of SEQ. ID NO: 30.

2. The isolated or purified T-cell receptor (TCR) according to claim 1 wherein the methyl-lysophosphatidic acid is selected from the group consisting of: methyl-lysophosphatidic acid (mLPA), C16-methyl-lysophosphatidic acid (C16-mLPA) or C18-methyl-lysophosphatidic acid (C18-mLPA) or a derivative thereof.

3. An isolated or purified nucleic acid comprising a nucleotide sequence encoding the TCR as defined in claim 1.

4. The isolated or purified nucleic acid according to claim 3, wherein the nucleotide sequence comprises a) a nucleotide sequence of SEQ ID NOs: 15 and b) a nucleotide sequence of SEQ ID NOs: 16.

5. A recombinant expression vector comprising the nucleic acid according to claim 3.

6. An isolated host cell comprising the recombinant expression vector of claim 5.

7. A pharmaceutical composition comprising a T cell comprising a recombinant expression vector including a nucleic acid encoding for a TCR and a pharmaceutically acceptable carrier; wherein the TCR has antigenic specificity for a self-lipid associated to CDIc molecule, wherein the self-lipid is a methyl-lysophosphatidic acid or derivative thereof; wherein the TCR is in monomeric form and comprises; a complementary determining region (CDRa1) amino acid sequence of SEQ. ID NO: 25; and a complementary determining region (CDRa2) amino acid sequence of SEQ. ID NO: 26; and a complementary determining region (CDRa3) amino acid sequence of SEQ. ID NO: 27; and a complementary determining region (CDRb1) amino acid sequence of SEQ. ID NO: 28; and a complementary determining regions (CDRb2) amino acid sequence of SEQ. ID NO: 29; and complementary determining regions (CDRb3) amino acid sequence of SEQ. ID NO: 30.

8. The pharmaceutical composition according to claim 7 further comprising methyl lysophosphatidic acid (mLPA).

9. A method of treating a hematological disorder in a subject, the method comprising administering the pharmaceutical composition of claim 7, wherein the hematological disorder is characterized by mLPA+ CD1c+ cancer cells.

10. The method of claim 9 wherein the hematological disorder is characterized by blood and bone marrow accumulation of immature and abnormal cells derived from hematopoietic precursors.

11. The method of claim 9, wherein the hematological disorder is acute leukemia.

12. The method of claim 9, further comprising administering at least one of asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and/or azacytidine.

13. The method of claim 9, wherein the method further comprises preventing hematological disorder relapse following hematopoietic stem cell transplantation (HSCT).

14. The method of claim 9, wherein the pharmaceutical composition further comprises methyl lysophosphatidic acid (mLPA).

15. A method for the production of a host cell as defined in claim 6 comprising the step of transducing said host cell with a nucleic acid encoding for a TCR in monomeric form, wherein the TCR comprises: a complementary determining region (CDRa1) amino acid sequence of SEQ. ID NO: 25; and a complementary determining region (CDRa2) amino acid sequence of SEQ. ID NO: 26; and a complementary determining region (CDRa3) amino acid sequence of SEQ. ID NO: 27; and a complementary determining region (CDRb1) amino acid sequence of SEQ. ID NO: 28; and a complementary determining regions (CDRb2) amino acid sequence of SEQ. ID NO: 29; and complementary determining regions (CDRb3) amino acid sequence of SEQ. ID NO: 30.

16. The method according to claim 15 further comprising expanding said host cell.

17. The isolated or purified nucleic acid according to claim 3, wherein the nucleotide sequence is codon optimized.

18. The isolated or purified T-cell receptor (TCR) according to claim 1, wherein the TCR further comprises a murine, a human or a humanized amino acid sequence of a constant region.

19. The isolated host cell according to claim 6, wherein the host cell is a T cell.

20. An isolated or purified T-cell receptor (TCR) having antigenic specificity for a self-lipid associated to CDIc molecule, wherein the self-lipid is a methyl-lysophosphatidic acid or derivative thereof, wherein the TCR is in monomeric form and comprises: the amino acid sequence of SEQ ID NO: 1; and the amino acid sequence of SEQ ID NO: 2.

21. An isolated or purified T-cell receptor (TCR) having antigenic specificity for a self-lipid associated to CD1c molecule, wherein the self-lipid is a methyl-lysophosphatidic acid or derivative thereof, wherein the TCR is in monomeric form and comprises: the amino acid sequence of SEQ ID NO: 1; and the amino acid sequence of SEQ ID No: 2, and wherein the TCR further comprises a murine, a human or a humanized amino acid sequence of a constant region.

22. The method of claim 11 wherein the acute leukemia is primary acute myeloid leukemia or B-cell acute leukemia.

23. The method according to claim 16 wherein said host cell is expanded in the presence of methyl lysophosphatidic acid (mLPA).

24. The isolated or purified T-cell receptor (TCR) according to claim 18, wherein the murine, human or humanized amino acid sequence of the constant region comprises: the amino acid sequence of SEQ ID NO: 11; the amino acid sequence of SEQ ID NO: 12; or the amino acid sequence of SEQ ID NO: 13; or the amino acid sequence of SEQ ID NO: 14.

25. The isolated or purified T-cell receptor (TCR) according to claim 21, wherein the murine, human or humanized amino acid sequence of the constant region comprises: the amino acid sequence of SEQ ID NO: 11; the amino acid sequence of SEQ ID NO: 12; or the amino acid sequence of SEQ ID NO: 13; or the amino acid sequence of SEQ ID NO: 14.

Description

(1) The present invention will be illustrated by means of non limiting examples in reference to the following figures.

(2) FIG. 1. Peripheral blood-derived T cells transduced with the CD1c self-reactive TCR DN 4.99 are efficiently retargeted against the CD1c-expressing acute leukemia cell line THP1. FIG. 1A Map of the bi-cistronic lentivirus vector pHR-SINEGFP carrying the chimeric TCR V-(D)-J segments from the mLPA-specific T cell clone DN4.99, fused with the mouse TCR C or C cDNAs. Both chains are expressed from the internal Spleen Focus Forming Virus (SSFV) promoter.

(3) FIG. 1B Generation of mLPA-specific T cell lines in vitro upon transduction of polyclonal T cells with the lentivirus described in (A). TCR expression by transduced (TrT, blue line) and untransduced (UTrT, red line) T cells at day 0 and day 15, and after sorting. TCR expression was detected using mouse C-specific (mTCR) mAbs. Data are representative of three experiments.

(4) FIG. 1C Recognition C1R-CD1c LCL by T cells transduced with the DN4.99 TCR (TrT) but not by untransduced ones (UTrT). T cells were cultured without (no APCs) or with C1R-CD1c cells in the presence (-CD1c) or absence (ctrl) of anti-CD1c blocking mAbs. Bars indicate means.d. of IFN- production of triplicates.

(5) FIG. 2. CD4 and CD8 Peripheral blood-derived T cells transduced with the CD1c self-reactive TCR DN 4.99 efficiently recognize CD1c-expressing acute leukemia cell line THP1. T cells were activated in vitro with anti-CD3/CD28 beads plus IL-2 and IL-7 and transduced with a lentivirus encoding the chimeric humanV-mouseC TCR DN 4.99. FIG. 2A. The transduced T cells expressing or not-expressing the chimeric TCR (mTCRbeta) were sorted based on the expression of CD4 or CD8 co-receptor. FIG. 2B. The sorted CD4+ T cells were assayed for the recognition of CD1c+ or WT THP1 cells, as determined by the specific release of IFNg by ELISA after 12 h of co-culture. FIG. 2C. The sorted CD8+ T cells were assayed for the recognition of CD1c+ or negative (WT) THP1 cells, as determined by the specific release of IFNg by ELISA after 12 h of co-culture. FIG. 2D. The sorted CD8+ T cells were tested for specific killing of CD1c+ or WT THP1 cells in a 72 h co-culture assay. Arrows indicate quadrants containing THP1 cells, detected by the expression of CD33. CD1c+ THP1 cells, but not WT THP1 ones, disappear only in the co-culture with TCR-transduced CD8+ T cells, indicating specific killing by T cells transduced with the CD1c self-reactive TCR.

(6) FIG. 3. Jurkat 76 cells transduced with CD1c self-reactive TCRs recognize K562-CD1c AML cell line. FIG. 3A expression of the chimeric CD1c self-reactive TCRs on transduced and untransduced Jurkat 76 determined by flow cytometry with anti m TCR mAb FIG. 3B Assessing the reactivity of Jurkat 76 cells transduced with the CD1c self-reactive TCRs. Transduced Jurkat 76 cells were plated in 96 U bottomwells containing 4105 CD1c.sup.+ K562-CD1c cells, loaded or not with mLPA (black bars) at 2.5:1 Effector T cell: leukemia Target cell (E:T=effector T cell: Target cell (ie leukemioa cell) ratio. K562 WT cells were used as negative control (white bars). After ON in vitro culture CD69 expression on Jurkat 76 cells was measured by flow citometry.

(7) FIG. 4. Jurkat 76 2m.sup. cells transduced with CD1c self-reactive TCRs recognize K562-CD1c AML cell line. FIG. 4A expression of the chimeric CD1c self-reactive TCRs on transduced and untransduced Jurkat 76 2m.sup. determined by flow cytometry with anti-mTCR mAb. FIG. 4B Assessing the reactivity of Jurkat 76 cells transduced with the CD1c self-reactive TCRs. Transduced Jurkat 76 cells were plated in 96 u bottom-wells containing 4105 CD1c.sup.+ K562-CD1c cells loaded or not with mLPA (black bars) at E:T=2.5:1 ratio. K562 WT cells were used as negative control (white bars). After ON culture CD69 expression on Jurkat 76 cells was measured by flow citometry.

(8) FIG. 5. TCR affinity for mLPA. FIG. 5A Expression of the CD1c self-reactive TCRs on the transduced and untransduced Jurkat 76 2m.sup. cells utilized for the mLPA titration reported in (B), as determined by flow cytometry with anti-mTCR mAb. FIG. 5B Lipid titration assay. Interpolation curve of CD69 expression on TCRtransduced Jurkat 76 2m.sup. cells in response to K562-CD1c cells loaded with indicated mLPA dilutions.

(9) FIG. 6. Primary T cells transduced with mLPA-specific TCRs recognize leukemia cell lines. FIG. 6A TCRs expression on transduced and untransduced primary T cells as determined by flow cytometry with anti-mouse TCRb mAb, gating on CD3.sup.+ cells. FIG. 6B Reactivity of primary T cells transduced with the mLPA-specific TCRs. Transduced primary T cells were plated in 96 U bottom-wells containing 410.sup.5 THP1 either expressing (black bars) or not expressing (white bars) CD1c at E:T=2.5:1 ratio. After 24 h, IFN secreted in the supernatant was measured by ELISA assay. FIG. 6C Reactivity of primary T cells transduced with the mLPA-specific TCRs. Transduced primary T cells were plated in 96 U bottom-wells containing 410.sup.5 K562 either expressing (black bars) or not expressing (white bars) CD1c at E:T=2.5:1 ratio. After 24 h, IFN secreted in the supernatant was measured by ELISA assay.

(10) FIG. 7. Primary T cells transduced with the lead mLPA-specific TCR DN4.99 recognize and kill THP1-CD1c AML cell line. FIG. 7A CD8.sup.+ T cells were sorted at day 10 post TCR-transduction and restimulated with anti-CD3/CD28 immunomagnetic beads and cytokines. TCR transduced or mock transduced T cells were plated in 96 u bottom-wells with 10.sup.5 THP1-CD1c or THP1 WT cells at E:T=1:1 ratio. After 24 h, supernatant was collected and IFN production was measured by ELISA assay. FIG. 7B CD4.sup.+ T cells were sorted at day 10 post TCR-transduction and restimulated with anti-CD3/CD28 immunomagnetic beads and cytokines. TCR transduced or mock transduced T cells were plated in 96 u bottom-wells with 10.sup.5 THP1-CD1c or THP1 WT cells at E:T=1:1 ratio. After 24 h, supernatant was collected and IFN production was measured by ELISA assay. FIG. 7C Killing of THP1-CD1c or THP1 WT AML cells by TCR-transduced or mock-transduced CD8.sup.+ T cells was evaluated by flow cytometry after 72 hours of coculture. FIG. 7D Killing of THP1-CD1c or THP1 WT AML cells by TCR-transduced or mock-transduced CD4.sup.+ T cells was evaluated by flow cytometry after 72 hours of coculture.

(11) FIG. 8. Primary T cells transduced with the lead mLPA-specific TCR DN4.99 kill primary CD1c.sup.+ AML blasts. FIG. 8A CD1c expression on primary circulating Acute Myeloid Leukemia blasts (AML-04). FIG. 8B killing of primary CD1c.sup.+ AML-04 blasts (CD33.sup.+, arrows) by TCR DN4.99-transduced primary T cells after 72 h of co-culture at 10:1 E:T ratio.

(12) FIG. 9. the lead mLPA-specific T cell clone DN4.99 recognizes CD1c expressing blasts from a relapsing patient. FIG. 9A CD1c expression on primary circulating AML blasts (AML-34) at first diagnosis (left) and at post-transplant disease relapse (right). FIG. 9B IFN production by DN 4.99 T cells upon 24 h coculture with primary blasts at first diagnosis (left) and relapse (right).

(13) FIG. 10. Structure of the phospholipids tested for recognition by mLPA-specific TCRs.

(14) FIG. 11. FIG. 11A Quantification of CD69 expression on Jurkat 76 cells transduced with mLPA specific TCR DN4.99 after ON co-culture with K562-CD1c AML cells loaded with 4 g/ml of the indicated lipids. FIG. 11B Quantification of CD69 expression on Jurkat 76 cells transduced with mLPA specific TCR DN4.2 after ON co-culture with K562-CD1c AML cells loaded with 4 g/ml of the indicated lipids.

DETAILED DESCRIPTION OF THE INVENTION

Material and Methods

TCR Constructs and Cloning in Lentiviral Vectors

(15) cDNA coding for the TCR and chains of the CD1c self-reactive T cell clones were synthetized by GeneArt and codon optimized for expression in eukaryotic cells. The sequence coding the TCR V portions, fused in frame to the mouse C sequence (SEQ NO. 11), are followed by the 2A peptide (EGRGSLLTCGDVEENPGP, SEQ ID NO. 57) and by the sequence coding the V regions of the TCR chain fused in frame with the murine C segment (SEQ NO. 12) which ends with a stop codon. The chimeric constructs were cloned into the pHR-SIN-BX-IRES-Em LV vector (a gift from E. Cerundolo, University of Oxford and Lepore M. et al J Exp Med. 2014 Jun. 30; 211(7):1363-77. doi: 10.1084/jem.20140410), in which the IRES-GFP cassette allows the identification of cells expressing the construct and can be easily excised by NotI/NotI digestion.

Production of Lentivirus in 293T Cells

(16) 910.sup.6 293T cells (HEK-293, ATCC CRL-1573) were seeded 24 h before transfection in a 15 cm petri dish with 20 ml of IMDM with L-glutamine, 10% FBS, and 100 U/ml Penicillin-Streptomycin. The medium was changed 2 h before transfection. The DNA mix was prepared by mixing 32 g of transfer vector plasmid plus 9 g pMD2.G-ENV (Addgene plasmid #12259), 16.25 g of CMVR8.74 (Addgene plasmid #22036) and 6.25 g of pRSV-REV (Addgene plasmid #12253, Dull T, Zufferey R, Kelly M, Mandel R J, Nguyen M, Trono D, Naldini L. J Virol. 1998 November 72(11):8463-71.) plasmids in a final volume of 1125 l of TE 0.1/H.sub.2O (2:1; H.sub.2O). 125 l of 2.5M CaCl.sub.2 were added to the plasmid mix and the precipitate was formed by drop wise addition of 1250 l of HBS 2 solution to the DNA-TE-CaCl.sub.2 mixture. The precipitate was immediately added to the 293T cells. After 14 h, the media in each dish was replaced with 16 ml of fresh complete medium and after further 30 h, the supernatant containing virus was collected, concentrated by ultracentrifugation and stored at 80 C.

Lentivirus Titration in Jurkat 76 Cells

(17) Lentivirus was titrated by transducing Jurkat 76 cells that do not express the endogenous TCR. 2510.sup.5 Jurkat 76 cells were seeded in 48 wells plate and transduced with 7 serial dilution of supernatant from the packaging 293T cells containing the produce lentivirus (10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8) in a final volume of 200 l of RPMI medium 1640+GlutaMAX, 10% FBS, 100 U/ml Penicillin-Streptomycin, 1 mM Sodium Pyruvate and 0.1 mM MEM NEAA. Fresh media was added 24 h after transduction to a final volume of 1 ml per well. After 4 days from the transduction, the expression of chimeric TCRs were detected by flow cytometry with anti-mouse TCR C mAb (Biolegend) or GFP. To calculate viral titer (transducing unit (TU)/ml) the following equation was used:

(18) $\mathrm{TU}/\mathrm{ml}=\frac{\begin{array}{c}\left(\mathrm{infected}\mathrm{cells}\mathrm{number}\right)\\ \left(\frac{\mathrm{positive}\mathrm{cells}\mathrm{percentage}}{100}\right)\left(\mathrm{dilution}\mathrm{factor}\right)\end{array}}{\mathrm{Inoculum}\mathrm{Volume}\left(\mathrm{ml}\right)}$

(19) To have a reliable estimation of lentivirus titer, the range of positive cells between 5-30% was considered.

Jurkat 76 Cells Transduction with Lentiviral Vectors

(20) 510.sup.5 Jurkat 76 cells (Hart D P, Xue S A, Thomas S, Cesco-Gaspere M, Tranter A, Willcox B, Lee S P, Steven N, Morris E C, Stauss H J. Gene Ther. 2008 April; 15(8):625-31) were seeded in 48 wells plate in a final volume of 200 l RPMI medium 1640+GlutaMAX, 10% FBS, 100 U/ml Penicillin-Streptomycin, 1 mM Sodium Pyruvate and 0.1 mM MEM NEAA. Jurkat 76 cells were transduced with MOI 100 and 24 hs after the transduction fresh media was added to a final volume of 1 ml per well. The expression of chimeric TCRs was detected 4 days after the transduction by flow cytometry analysis with anti mouse TCR C mAb.

Jurkat 76 2m.SUP. Cells Transduction with Lentiviral Vectors

(21) 510.sup.5 Jurkat 76 2m.sup. cells were seeded in 48 wells plate in a final volume of 200 l RPMI medium 1640+GlutaMAX, 10% FBS, 100 U/ml Penicillin-Streptomycin, 1 mM Sodium Pyruvate and 0.1 mM MEM NEAA, and transduced with MOI 100. The expression of chimeric TCRs was detected 4 days after the transduction by flow cytometry analysis staining the cells with anti-mouse TCR C mAb.

Primary Human T Cells Transduction with Lentiviral Vector

(22) Peripheral blood mononuclear cells were purified from healthy donors. CD3.sup.+ cells were activated and expanded with Dynabeads human T-Activator CD3/CD28, using the beads:T cells ratio=3:1. After 48 h in culture, 510.sup.5 T cells were seeded in 48 wells plate in a final volume of 200 l RPMI medium 1640+GlutaMAX, 10% FBS, 100 U/ml Penicillin-Streptomycin, 1 mM Sodium Pyruvate, 0.1 mM MEM NEAA, 100 U/ml rhIL2 and 10 ng/ml hrIL-7. T cells were transduced with MOI 100 and 24 h after fresh media was added to a final volume of 1 ml per well. The expression of the transduced TCRs was verified after 4 days by flow cytometry using anti-mouse TCR C mAb. CD4.sup.+ and CD8.sup.+ T cells expressing or not expressing the transduced TCRs were sorted 14 days after the transduction. Sorted T cells were re-expanded with Dynabeads human T-Activator CD3/CD28 at a beads:T cells ratio=1:10 and 100 U/ml rhIL2 and 10 ng/ml hrIL-7.

Recognition of Leukemia Cell Lines by TCR-Transduced Jurkat 76 Cells

(23) 410.sup.4 THP1-CD1c, or K562-CD1c (a gift of Dr. D. Branch Moody, Harvard University, Boston, USA de Jong A, Pea-Cruz V, Cheng T Y, Clark R A, Van Rhijn I, Moody D B. Nat Immunol. 2010 December; 11(12):1102-9) target cells or THP1 (ATCC TIB-202) or K562 (ATCC CCL243) WT cells, were plated complete medium (RPMI 10% FCS+1% P/S+1% NEAA+1% NaPyr, 2-ME) in 96 U bottom-wells. When indicated, THP1-CD1c or K562-CD1c cells were also pre-loaded with 4 g/ml of mLPA for 4 h at 37 C. 105 Jurkat 76 cells expressing the transduced TCRs were added to the target cells in a final volume of 100 l of complete medium. After overnight (ON) at 37 C., the expression of CD69 were analyzed by flow cytometry with anti-CD69 mAb (Biolegend).

Recognition Assay of THP1 Cells by Transduced Jurkat 76 Cells

(24) 410.sup.4 THP1-CD1c target cells, or negative control THP1 WT cells, were plated in 50 ul of complete medium (RPMI 10% FCS+1% P/S+1% NEAA+1% NaPyr, 2-ME) in 96 U bottom-wells. When indicated, THP1-CD1c cells were also pulsed with 4 g/ml of mLPA for 4 h at 37 C. 10.sup.5 Jurkat 76 cells expressing the transduced TCRs were added to the target cells in a final volume of 100 ul of complete medium. After ON at 37 C., the expression of CD69 were analyzed by flow cytometry with anti-CD69 mAb (Biolegend).

Recognition of Leukemia Cell Lines by TC-Transduced Primary Human T Cells

(25) For primary T cell recognition, 10.sup.5 THP1-CD1c or K562-CD1c target cells, or negative control THP1 and K562 WT cells, were plated in complete medium (RPMI 10% FCS+1% P/S+1% NEAA+1% NaPyr, 2-ME) in 96 U bottom-wells. 10.sup.5 T cells expressing or not expressing the transduced TCRs were added to target cells in 100 l of complete medium and incubated at 37 C. ON. When indicated, TCR-transduced CD4.sup.+ or CD8.sup.+ T cells were separated by cell sorting before the assay. A 50 l aliquot of supernatant from each condition was collected and the secreted IFN was measured by ELISA. After additional 4 days of coculture, the killing of target cells was evaluated by flow cytometry analysis, by staining cells with DAPI, anti-mTCR C and anti-CD33 to determine the elimination of CD33.sup.+ leukemia cells.

Recognition Assay of THP1 Cells by Transduced Human T Cells

(26) For primary T cell recognition, 10.sup.5 THP1-CD1c target cells, or negative control THP1 WT cells, were plated in 50 ul of complete medium (RPMI 10% FCS+1% P/S+1% NEAA+1% NaPyr, 2-ME) in 96 U bottom-wells. 10.sup.5 CD4.sup.+ or CD8.sup.+ T cells expressing or not expressing the transduced TCRs were added to target cells in 100 ul of complete medium and incubated at 37 C. A 50 l aliquot of supernatant from each condition was collected after ON and the secreted IFN was measured by ELISA. After additional 4 days of coculture, the killing of target cells was evaluated by flow cytometry analysis staining cells with DAPI, anti-mTCR C and anti-hCD33.

Killing Assay

(27) Killing assays were performed using primary AML blasts (obtained from the San Raffaele Biobank) incubated either alone or with T cells (210.sup.4 cells/ml) at 10:1 Effector T cell:Target leukemia cell, in the presence or absence of the CD1c blocking antibody anti-CD1c mAb (clone M241 Santa Cruz, Calif.). After 72 h residual viable target cells were identified and quantitated as DAPI.sup./CD33.sup.+/CD3.sup. by flow cytometry.

T Cell Activation Assay

(28) T cell activation assay was performed by co-culturing the indicated numbers of target cells with T cells (2010.sup.4/well) in the presence or in the absence of anti-CD1c (clone M241, Santa Cruz, Calif.) blocking monoclonal antibody. Supernatants were collected after 48 h and IFN- was measured by ELISA.

(29) In FIG. 11, 410.sup.4 THP1-CD1c target cells were plated in 50 ul of complete medium (RPMI 10% FCS+1% P/S+1% NEAA+1% NaPyr, 2-ME) in 96 U bottom-wells. THP1-CD1c cells were also pulsed with 4 g/ml of the indicated lipids for 4 h at 37 C. Synthetic PS18:1, LPE16:0, LPE18:0, LPE18:1 were obtained by Dr Matthew Skaley, University of Oklahoma Health Sciences Center Oklahoma City, USA. Synthetic mLPA was obtained from Prof. Gennaro De Libero, University of Basel, CH) 10.sup.5 Jurkat 76 cells expressing the transduced TCRs were added to the target cells in a final volume of 100 ul of complete medium. After ON at 37 C., the expression of CD69 were analyzed by flow cytometry with anti-CD69 mAb (Biolegend).

(30) TABLE-US-00001 Sequences CLONES Vsegment Nregion(bold) Jsegment(lowercase) Underlined:CDRregions CLONEDN4.99 TRAV38.2-Ja44 CDR1 MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQ CDR2CDR3 MILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYRSPLntgtaskltfgtgtrl qvtl(SEQIDNO.1) TRBV28-Jb2.7 CDR1 MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLR CDR2CDR3 LIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSPWVssyeqyfgpgtrltvt (SEQIDNO.2) CLONECD4P8E3 TRAV38-1-J31 CDR1 MTRVSLLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTSENNYYLFWYKQPPSR CDR2CDR3 QMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFAnnarlmfgdgtqlvvk p(SEQIDNO.3) TRBV28-J1.1 CDR1 MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLR CDR2CDR3 LIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASTDTGnteaffgqgtrltyv (SEQIDNO.4) CLONEDN4.2 TRAV26-2-J53 CDR1 MKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVI CDR2CDR3 HGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILRLRggsnykltfgkgtlltvnp(SEQID NO.5) TRBV4-1-J2.1 CDR1 MGCRLLCCAVLCLLGAGPIDTEVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPP CDR2CDR3 ELMFVYSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSPIMGLAATHneqffgpg trltvl(SEQIDNO.6) CLONEPZ-CD8P8A6 TRAV26-2-J44 CDR1 MKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVI CDR2CDR3 HGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILRDvntgtaskltfgtgtrlqvtld(SEQ IDNO.7) TRBV4-1-J2-3 CDR1 MGCRLLCCAVLCLLGAGPIDTEVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPP CDR2CDR3 ELMFVYSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSRLGLstdtqyfgpgtrltvl (SEQIDNO.8) CLONEDN7.6.16 TRAV13.1-J28 CDR1 MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKGPQL CDR2CDR3 IIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAAPRglgvtnslsgrgpnsrsyqn (SEQIDNO.9) TRBV27-J2.1 CDR1 MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLR CDR2CDR3 QIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLVWGTyneqffgpgtrltv l(SEQIDNO.10) Murineconstantregionalpha DIQNPEPAVYQLKDPRSQDSTLCL FTDFDSQINVPKTMKSGTFITDKT VLDMKAMDSKSNGAIAWSNQTSFT CQDIFKETNATYPSSDVPCDATLT EKSFETDMNLNFQNLSVMGLRILL LKVAGFNLLMTLRLWSS(SEQIDNo.11) CodonoptimizedmCanucleotidesequence atCcagaacccagaacctgctgtgtaccagttaaaagatcctcggtctcaggacagcaccctctgcctgttcaccgactttgactcc caaatcaatgtgccgaaaaccatggaatctggaacgttcatcactgacaaaactgtgctggacatgaaagctatggattccaaga gcaatggggccattgcctggagcaaccagacaagcttcacctgccaagatatcttcaaagagaccaacgccacctaccccagttc agacgttccctgtgatgccacgttgaccgagaaaagctttgaaacagatatgaacctaaactttcaaaacctgtcagttatgggact ccgaatcctcctgctgaaagtagcgggatttaacctgctcatgacgctgaggctgtggtccagt(SEQIDNO.58) Murineconstantregionbeta1 EDLRNVTPPKVSLFEPSKA EIANKQKATLVCLARGFFP DHVELSWWVNGKEVHSGVS TDPQAYKESNYSYCLSSRL RVSATFWHNPRNHFRCQVQ FHGLSEEDKWPEGSPKPVT QNISAEAWGRADCGITSAS YHQGVLSATILYEILLGKA TLYAVLVSGLVLMAMVKKK NS(SEQIDNo.12) CodonoptimizedmCb1nucleotidesequence Gaggatctgagaaatgtgactccacccaaggtctccttgtttgagccatcaaaagcagagattgcaaacaaacaaaaggctaccc tcgtgtgcttggccaggggcttcttccctgaccacgtggagctgagctggtgggtgaatggcaaggaggtccacagtggggtcagc acggaccctcaggcctacaaggagagcaattatagctactgcctgagcagccgcctgagggtctctgctaccttctggcacaatcc tcgaaaccacttccgctgccaagtgcagttccatgggctttcagaggaggacaagtggccagagggctcacccaaacctgtcaca cagaacatcagtgcagaggcctggggccgagcagactgtggaatcacttcagcatcctatcatcagggggttctgtctgcaaccat cctctatgagatcctactggggaaggccaccctatatgctgtgctggtcagtggcctggtgctgatggccatggtcaagaaaaaaa attcctga(SEQIDNO.59) HumanConstantregionalphasequence: IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAW SNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNL LMTLRLWSS(SEQIDNo.13) CodonoptimizedhCanucleotidesequence ATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGT GCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACTCCGACGTGTACAT CACCGACAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACTCCGCCGTGGCCTGG TCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCT TTTTCCCCAGCCCCGAGAGCAGCTGCGACGTGAAACTGGTGGAGAAGAGCTTCGAGACCGACAC CAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCAGAATCCTGCTGCTGAAGGTGGCCGGC TTCAACCTGCTGATGACCCTGCGGCTGTGGAGCAGC(SEQIDNO.60) HumanConstantbetasequence: EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLK EQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWG RADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQIDNo.14) CodonoptimizedhCbnucleotidesequence GAGGACCTGAAGAACGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCA GCCACACCCAGAAAGCCACCCTGGTCTGCCTGGCCACCGGCTTCTACCCCGACCACGTGGAGCTG TCTTGGTGGGTGAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAG AGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGAGAGTGAGCGCCACCTT CTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGAC GAGTGGACCCAGGACAGAGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGCAGAG CCGACTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGTCCGCCACCATCCTGTACGA GATCCTGCTGGGCAAGGCCACACTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCTATG GTGAAGCGGAAGGACAGCCGGGGCTGA(SEQIDNO.61) Inbold= Nregion Inunderlined= Jregion TCRfromcloneDN4.99 SequenceoftheTCRVaregionofTcellcloneDN4.99 TRAV38-2(Va14.1)-Ja44 1/131/1161/21 | | TRAV38-2(Va14.2)| GAATTCGCCCTTCTGCAGCAGGGACCTGTGAGCATGGCATGCCCTGGCTTCCTGTGGGCACTTGTGATCTCCACCTGTCTTGAATTTAGC MACPGFLWALVISTCLEFS 91/31121/41151/51 | | | ATGGCTCAGACAGTCACTCAGTCTCAACCAGAGATGTCTGTGCAGGAGGCAGAGACCGTGACCCTGAGCTGCACATATGACACCAGTGAG MAQTVTQSQPEMSVQEAETVTLSCTYDTSE 181/61211/71241/81 | | | AGTGATTATTATTTATTCTGGTACAAGCAGCCTCCCAGCAGGCAGATGATTCTCGTTATTCGCCAAGAAGCTTATAAGCAACAGAATGCA SDYYLFWYKQPPSRQMILVIRQEAYKQQNA 271/91301/101331/111 | | | ACAGAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCTCAAGATCTCAGACTCACAGCTGGGGGATGCCGCGATG TENRFSVNFQKAAKSFSLKISDSQLGDAAM 361/121N-region391/131421/141 | | | Ja44| | TATTTCTGTGCTTATAGGAGCCCCTTAAATACCGGCACTGCCAGTAAACTCACCTTTGGGACTGGAACAAGACTTCAGGTCACGCTC(SEQIDNo.15) YFCAYRSPLNTGTASKLTFGTGTRLQVTL(SEQIDNO.1) SequenceoftheTCRVbregionofTcellcloneDN4.99 TRBV28(Vb3.1)-Jb2.7 1/131/1161/21 | TRBV28(Vb3.1)| | cccaccatgggaatcaggctcctctgtcgtgtggccttttgtttcctggctgtaggcctcgtagatgtgaaagtaacccagagctcgaga MGIRLLCRVAFCFLAVGLVDVKVTQSSR 91/31121/41151/51 | | | tatctagtcaaaaggacgggagagaaagtttttctggaatgtgtccaggatatggaccatgaaaatatgttctggtatcgacaagaccca YLVKRTGEKVFLECVQDMDHENMFWYRQDP 181/61211/71241/81 | | | ggtctggggctacggctgatctatttctcatatgatgttaaaatgaaagaaaaaggagatattcctgaggggtacagtgtctctagagag GLGLRLIYFSYDVKMKEKGDIPEGYSVSRE 271/91301/101331/111Nregion | | | | | Jb2.7 aagaaggagcgcttctccctgattctggagtccgccagcaccaaccagacatctatgtacctctgtgccagcagtCCATGGGTAAGCTCC KKERFSLILESASTNQTSMYLCASSPWVSS 361/121391/131 | | TACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACASEQIDNo.16 YEQYFGPGTRLTVTSEQIDNO.2 TCRfromcloneCD4-P8E3 TCRValfachaincloneCD4P8E3TRAV38-1(Va14.2)-Ja31 1/131/1161/21 | | | atgacacgagttagcttgctgtgggcagtcgtggtctccacctgtcttgaatccggcatggcccagacagtcactcagtctcaaccagag MTRVSLLWAVVVSTCLESGMAQTVTQSQPE 91/31121/41151/51 | | | atgtctgtgcaggaggcagagactgtgaccctgagttgcacatatgacaccagtgagaataattattatttgttctggtacaagcagcct MSVQEAETVTLSCTYDTSENNYYLFWYKQP 181/61211/71241/81 | | | cCCAGCAGGCAGATGATTCTCGTTATTCGCCAAGAAGCTTATAAGCAACAGAATGCAACGGAGAATCGTTTCTCTGTGAACTTCCAGAAA PSRQMILVIRQEAYKQQNATENRFSVNFQK 271/91301/101331/111NregionJa31 | | | GCAGCCAAATCCTTCAGTCTCAAGATCTCAGACTCACAGCTGGGGGACACTGCGATGTATTTCTGTGCTTTCGCAAACAATGCCAGACTC AAKSFSLKISDSQLGDTAMYFCAFANNARL 361/121391/131 | | ATGTTTGGAGATGGAACTCAGCTGGTGGTGAAGCCC(SEQIDNo.17) MFGDGTQLVVKP(SEQIDNo.3) TCRVbetacloneCD4P8E3(= P11D10)TRBV28(Vb3)-Jb1.1 1/1TRBV2831/1161/21 | | | actgcctggtcctgggagaagacctattctttcttcaaagcagccatgggaatcaggctcctctgtcgtgtggccttttgtttcctggct MGIRLLCRVAFCFLA 91/31121/41151/51 | | | gtaggcctcgtagatgtgaaagtaacccagagctcgagatatctagtcaaaaggacgggagagaaagtttttctggaatgtgtccaggat VGLVDVKVTQSSRYLVKRTGEKVFLECVQD 181/61211/71241/81 | | | atggaccatgaaaatatgttctggtatcgacaagacccaggtctggggctacggctgatctatttctcatatgatgttaaaatgaaagaa MDHENMFWYRQDPGLGLRLIYFSYDVKMKE 271/91301/101331/111 | | | aaaggagatattcctgaggggtacagtGTCTCTAGAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCCAGCACCAACCAGACA KGDIPEGYSVSREKKERFSLILESASTNQT 361/121N-region391/131Jb1.1421/141 | | | TCTATGTACCTCTGTGCCAGCACCGACACTGGGAACACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTA(SEQIDNo.18) SMYLCASTDTGNTEAFFGQGTRLTVV(SEQIDNO.4) TCRfromcloneDN4.2 TCRalfachaincloneDN4.2TRAV26-2(Va4.1)-Ja53 1/131/11TRAV2661/21 | | | cacagagtctgagttctggggcctggaacctcaatgtgcacttgaacaATGAAGTTGGTGACAAGCATTACTGTACTCCTATCTTTGGGT MKLVTSITVLLSLG 91/31121/41151/51 | | | ATTATGGGTGATGCTAAGACCACACAGCCAAATTCAATGGAGAGTAACGAAGAAGAGCCTGTTCACTTGCCTTGTAACCACTCCACAATC IMGDAKTTQPNSMESNEEEPVHLPCNHSTI 181/61211/71241/81 | | | AGTGGAACTGATTACATACATTGGTATCGACAGCTTCCCTCCCAGGGtccagagtacgtgattcatggtcttacaagcaatgtgaacaac SGTDYIHWYRQLPSQGPEYVIHGLTSNVNN 271/91301/101331/111 | | | agaatggcctctctggcaatcgctgaagacagaaagtccagtaccttgatcctgcaccgtgctaccttgagagatgctgctgtgtactac RMASLAIAEDRKSSTLILHRATLRDAAVYY 361/121391/131421/141 | N-regionJa52| | tgcatcctgagattaagagGAGGTAGCAACTATAAACTGACATTTGGAAAAGGAACTCTCTTAACCGTGAATCCA(SEQIDNo.19) CILRLRGGSNYKLTFGKGTLLTVNP(SEQIDNO.5) TCRbetachaincloneDN4.2TRBV4-1(Vb7)-Jb2.1 1/1TRBV4-131/1161/21 | | | atgggctgcaggctgctctgctgtgcggttctctgtctcctgggagcaggtcccatagacactgaagttacccagacaccaaaacacctg MGCRLLCCAVLCLLGAGPIDTEVTQTPKHL 91/31121/41151/51 | | | gtcatgggaatgacaaataagaagtctttgaaatgtgaacaacatatggggcacagggctatgtattggtacaagcagaaagctaagaag VMGMTNKKSLKCEQHMGHRAMYWYKQKAKK 181/61211/71241/81 | | | ccaccggagctcatgtttgtctacagctatgagaaactctctataaatgaaagtgtgccaagtcgcttctcaCCTGAATGCCCCAACAGC PPELMFVYSYEKLSINESVPSRFSPECPNS 271/91301/101331/111N-region | | | TCTCTCTTAAACCTTCACCTACACGCCCTGCAGCCAGAAGACTCAGCCCTGTATCTCTGCGCCAGCAGCCCAATCATGGGACTAGCGGCG SLLNLHLHALQPEDSALYLCASSPIMGLAA 361/121Jb2.1391/131 | | ACCCACAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTA(SEQIDNo.20) THNEQFFGPGTRLTVL(SEQIDNO.6) TCRfromclonePZ-CD8P8A6 TCRalfachainclonePZ-CD8P8A6TRAV26-2(Va4.1)-Ja44 1/1TRAV26-231/1161/21 | | | atgaagttggtgacaagcattactgtactcctatctttgggtattatgggtgatgctaagaccacacagccaaattcaatggagagtaac MKLVTSITVLLSLGIMGDAKTTQPNSMESN 91/31121/41151/51 | | | gaagaagagcctgttcacttgccttgtaaccactccacaatcagtggaactgattacatacatTGGTATCGACAGCTTCACTCCCAGGGT EEEPVHLPCNHSTISGTDYIHWYRQLHSQG 181/61211/71241/81 | | | CCAGAGTACGTGATTCATGGTCTTACAAGOAATGTGAACAACAGAATGGCCTCTCTGGCAATCGCTGAAGACAGAAAGTCCAGTACCTTG PEYVIHGLTSNVNNRMASLAIAEDRKSSTL 271/91301/101331/111Ja44 | | | ATCCTGCACCGTGCTACCTTGAGAGATGCTGCTGTGTACTACTGCATCCTGAGAGACGTAAATACCGGCACTGCCAGTAAACTCACCTTT ILHRATLRDAAVYYCILRDVNTGTASKLTF 361/121391/131 | | GGGACTGGAACAAGACTTCAGGTCACCCTCGAT(SEQIDNo.21) GTGTRLQVTLD(SEQIDNO.7) TCRbetachainclonePZ-CD8P8A6TRBV4-1(Vb7.1)Jb2.3 1/1TRBV4-131/1161/21 | | | atgggctgcaggctgctctgctgtgcggttctctgtctcctgggagcaggtcccatagacactgaagttacccagacaccaaaacacctg MGCRLLCCAVLCLLGAGPIDTEVTQTPKHL 91/31121/41151/51 | | | gtcatgggaatgacaaataagaagtctttgaaatgtgaacaacatatggggcacagggctatgtattggtacaagcagaaagctaagaag VMGMTNKKSLKCEQHMGHRAMYWYKQKAKK 181/61211/71241/81 | | | ccaccggagctcatgtttgtctacagctatgagaaactctctataaatgaaagtgtgccaagtcgcttctcaCCTGAATGCCCCAACAGC PPELMFVYSYEKLSINESVPSRFSPECPNS 271/91301/101331/111 | | | N-regionJb2.3 TCTCTCTTAAACCTTCACCTACACGCCCTGCAGCCAGAAGACTCAGCCCTGTATCTCTGCGCCAGCAGCCGGCTAGGACTATCCACGGAT SLLNLHLHALQPEDSALYLCASSRLGLSTD 361/121391/131 | | ACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTC(SEQIDNo.22) TQYFGPGTRLTVL(SEQIDNO.8) TCRfromcloneDN7.6.16 TCRalfachaincloneDN7.6.16TRAV13.1(Va8.1)-Ja28 1/131/1161/21 | | | ATGacatccattcgagctgtatttatattcctgtggctgcagctggacTtggtgaatggagagaatgtggagcagcatccttcaaccctg MTSIRAVFIFLWLQLDLVNGENVEQHPSTL 91/31121/41151/51 | | | agtgtccaggagggagacagcgctgttatcaagtgtacttattcagacagtgcctcaaactacttcccttggtataagcaagaacttgga SVQEGDSAVIKCTYSDSASNYFPWYKQELG 181/61211/71241/81 | | | aaaggacctcagcttattatagacattcgTTCAAATGTGGGCgAAAAGAAAGACCAACGAATTGCTGTTACATTGAACAAGACAGCCAAA KGPQLIIDIRSNVGEKKDQRIAVTLNKTAK 271/91301/101331/111 | | | CATTTCTCCCTGCACATCACAGAGACCCAACCTGAAGACTCGGCTGTCTACTTCTGTGCAGCCCCCCGCGGGCTGGGAGTTACCAACTCA HFSLHITETQPEDSAVYFCAAPRGLGVTNS 361/121391/131 | | CTTTCGGGAAGGGGACCAAACTCTCGGTCATACCAAAAT(SEQIDNo.23) LSGRGPNSRSYQN(SEQIDNO.9) TCRbetachaincloneDN7.6.16TRBV27(Vb14)Jb2.1 1/131/1161/21 | | | ATGccccagctccttggctatgtggtcctttgccttctaggagcaggccccctggaagcccaagtgacccagaacccaagatacctcatc MPQLLGYVVLCLLGAGPLEAQVTQNPRYLI 91/31121/41151/51 | | | acagtgactggaaagaagttaacagtgacttgttctcagaatatgaaccatgagtatatgtcctggtatcgacaagacccagggctgggc TVTGKKLTVTCSQNMNHEYMSWYRQDPGLG 181/61211/71241/81 | | | ttaaggcagatAtactattcaatgaatgttgaggtgactgataagggagatgttcctgaagggtacaaagtCTCTCGAAAAgAGAAGAGG LRQIYYSMNVEVTDKGDVPEGYKVSRKEKR 271/91301/101331/111 | | | N-regionJb2.1 AATTTCCCCCTGATCCTGGAGTCGCCCAGCCCCAACCAGACCTCTCTGTACTTCTGTGCCAGCAGTTTGGTCTGGGGAACCTACAATGAG NFPLILESPSPNQTSLYFCASSLVWGTYNE 361/121391/131 | | CAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTA(SEQIDNo.24) QFFGPGTRLTVL(SEQIDNO.10) Inbred= Nregion Inblue= Jregion Ingreenmurineconstantregion TCRfromcloneDN4.99 SequenceoftheTCRVaregionofTcellcloneDN4.99 TRAV38(Va14.2)-Ja44fusedwithmCa 1/131/1161/21 | | TRAV38(Va14.2)| GAATTCGCCCTTCTGCAGCAGGGACCTGTGAGCATGGCATGCCCTGGCTTCCTGTGGGCACTTGTGATCTCCACCTGTCTTGAATTTAGC MACPGFLWALVISTCLEFS 91/31121/41151/51 | | | ATGGCTCAGACAGTCACTCAGTCTCAACCAGAGATGTCTGTGCAGGAGGCAGAGACCGTGACCCTGAGCTGCACATATGACACCAGTGAG MAQTVTQSQPEMSVQEAETVTLSCTYDTSE 181/61211/71241/81 | | | AGTGATTATTATTTATTCTGGTACAAGCAGCCTCCCAGCAGGCAGATGATTCTCGTTATTCGCCAAGAAGCTTATAAGCAACAGAATGCA SDYYLFWYKQPPSRQMILVIRQEAYKQQNA 271/91301/101331/111 | | | ACAGAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCTCAAGATCTCAGACTCACAGCTGGGGGATGCCGCGATG TENRFSVNFQKAAKSFSLKISDSQLGDAAM 361/121N-region391/131421/141 | | | JA44| | mCA TATTTCTGTCCTTATAGGAGCCCCTTAAATACCGGCACTGCCAGTAAACTCACCTTTGGGACTGGAACAAGACTTCAGGTCACGCTCGAT YFCAYRSPLNTGQASKLTFGTGTRLQVTLD 451/151481/161511/171 | | | ATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAA IQNPRPAVYQLKDPRSQDSTLCLPTDFDSQ 541/181571/191601/201 | | | ATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGG INVPKTMESGTFITDKTVLDMKAMDSKSNG 631/211662/221691/231 | | | GCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGT AIAWSNQTSFTCQDIFKETNATYPSSDVPC 721/241751/251781/261 | | | GATGCCACGTTGACCGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTG DATLTEKSFETDMNLNPQNLSVMGLRILLL 811/271841/281871/291 | | | AAAGTAGCGGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGTTGAGGATCCCGAAGGGCGAATTC(SEQIDNO:55) KVAGFNLLMTLRLWSS* SequenceoftheTCRVbregionofTcellcloneDN4.99 TRBV28(Vb3.1)-Jb2.7fusedwithmCb 1/131/1161/21 | TRBV28(Vb3.1)| | cccaccatgggaatcaggctcctctgtcgtgtggccttttgtttcctggctgtaggcctcgtagatgtgaaagtaacccagagctcgaga MGIRLLCRVAFCFLAVGLVDVKVTQSSR 91/31121/41151/51 | | | tatctagtcaaaaggacgggagagaaagtttttctggaatgtgtccaggatatggaccatgaaaatatgttctggtatcgacaagaccca YLVKRTGEKVFLECVQDMDHENMFWYRQDP 181/61211/71241/81 | | | ggtctggggctacggctgatctatttctcatatgatgttaaaatgaaagaaaaaggagatattcctgaggggtacagtgtctctagagag GLGLRLIYFSYDVKMKEKGDIPEGYSVSRE 271/91301/101331/111Nregion | | | |Jb2.7| Jb2.7 aagaaggagcgcttctccctgattctggagtccgccagcaccaaccagacatctatgtacctctgtgccagcagtCCATGGGTAAGCTCC KKERFSLILESASTNQTSMYLCASSPWVSS 361/121391/131421/141 | | mCb| TACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAgaggatctgagaaatgtgactccacccaaggtctccttgtttgagcca YEQYFGPGTRLTVTEDLRNVTPPKVSLPEP 451/151481/161511/171 | | | tcaaaagcagagattgcaaacaaacaaaaggctaccctcgtgtgcttggccaggggcttcttccctgaccactgtgagctgagctggtgg SKAEIANKQKATLVCLAEGFFFDHVELSWW 541/181571/191601/201 | | | gtgaatggcaaggaggtccacagtggggtcagcacggaccctcaggcctacaaggagagcaattatagctactgcctgagcagccgcctg VNGKEVHSGVSTDPQAYKESNYSYGLSSRL 631/211661/221691/231 | | | agggtctctgctaccttctggcacaatcctcgaaaccacttccgctgccaagtgcagttccatgggctttcagaggaggacaagtggcca RVSATFWHNPRNHFRCQVQFHGLSEEDKWP 721/241751/251781/261 | | | gagggctcacccaaacctgtcacacagaacatcagtgcagaggcctggggccgagcagactgtggaatcacttcagcatcctatcatcag EGSPKPVTQNISAEAWGRADCGITSASYHQ 811/271841/281871/291 | | | ggggttctgtctgcaaccatcctctatgagatcctactggggaaggccaccctatatgctgtgctggtcagtggcctggtgctgatggcc GVLSATILYEILLGKATLYAVLVSGLVLMA 901/301 | atggtcaagaaaaaaaattcctgagac(SEQIDNO:56) MVKKKNS * SEQIDNO.25YDTSESDYY SEQIDNO.26RQEAYK SEQIDNO.27RSPLNTGTASKLT SEQIDNO.28QDMDHENMF SEQIDNO.29SYDVKMK SEQIDNO.30SPWVSSYEQY SEQIDNO.31YDTSENNYY SEQIDNO.32RQEAYK SEQIDNO.33FANNARLM SEQIDNO.34QDMDHENMF SEQIDNO.35SYDVKMK SEQIDNO.36TDTGNTEAF SEQIDNO.37HSTISGTDY SEQIDNO.38GLTSNV SEQIDNO.39RLRGGSNYKLT SEQIDNO.40QHMGHRAMY SEQIDNO.41FVYSYEK SEQIDNO.42SPIMGLAATHNEQF SEQIDNO.43HSTISGTDYI SEQIDNO.44GLTSNV SEQIDNO.45RDVNTGTASKLT SEQIDNO.46QHMGHRAMY SEQIDNO.47FVYSYEK SEQIDNO.48SRLGLSTDTQY SEQIDNO.49YSDSASNYF SEQIDNO.50DIRSNV SEQIDNO.51PRGLGVTNSL SEQIDNO.52QNMNHEYMS SEQIDNO.53SMNVEV SEQIDNO.54SLVWGTYNEQF

Example 1

T Cell Transduction with Lentivirus Encoding the mLPA-Specific TCR Genes

(31) To assess whether it was possible to generate primary T cell lines endowed with CD1c self-reactivity, the inventors transduced PBMCs from healthy donors with a lentivirus expressing the TCR and chains of a CD1c self-reactive mLPA-specific T cell clone. The inventors generated a lentivirus vector pHR-SIN-EGFP (provided by V. Cerundolo, Oxford, UK) encoding the TCR and genes from the mLPA specific T cell clone DN4.99, linked by 2A peptide. To improve the homologous pairing of the CD1c-restricted TCR and minimize mispairing with endogenous TCR chains, the inventors generated chimeric genes with the human V and the mouse C regions, as described (Canderan et al., 2010 Canderan G, Gruarin P, Montagna D, Fontana R, Melloni G, Traversari C, Dellabona P, Casorati G. An efficient strategy to induce and maintain in vitro human T cells specific for autologous non-small cell lung carcinoma. PLoS One. 2010 Aug. 9; 5(8):e12014.) (FIG. 1A). To generate the chimeric TCR chains, the TRAV38-TRAJ44 (SEQ ID NO: 15) and TRBV28-TRBJ2-7 (SEQ ID NO: 16) segments were fused with the mouse TRAC and TRBC cDNA, respectively, by PCR.

(32) T cells were purified form healthy donors and activated with anti-CD3/CD28 immunomagnetic beads (Dynal) in RPMI-FCS complete medium supplemented with 100 U/ml hrIL-2 and 10 ng/ml IL-7 (R&D Systems). Two days after activation, 410.sup.5 T cells were infected by lentivirus in 500 l complete medium at a MOI of 5. After 15 days, transduced T cells were sorted for mouse TCRP expression. Both positive and negative cells were stimulated with anti-CD3/CD28 immunomagnetic beads and cytokines and tested for antigen recognition after 10 days. Only the T cells displaying surface expression of the transduced TCR chains (i.e. only the mouse C expressing cells), and not the untransduced ones sorted from the same cell line (FIG. 1B), recognized a CD1c expressing tumor cell line (FIG. 1C). Anti-CD1c-mAbs inhibited recognition, underscoring the specificity of the immune reaction.

(33) These results indicated that mLPA-specific T cells, able to recognize leukemia blasts in CD1c-dependent manner, could be generated in vitro upon transduction of mLPA-specific TCR genes. This strategy might represent a relatively safe approach for immunotherapy of acute leukemia. Indeed allogeneic T cells from stem cell donors might be transduced and their eventual alloreactivity might be prevented by editing the endogenous TCR genes (Canderan et al., 2010 Canderan G, Gruarin P, Montagna D, Fontana R, Melloni G, Traversari C, Dellabona P, Casorati G. An efficient strategy to induce and maintain in vitro human T cells specific for autologous non-small cell lung carcinoma. PLoS One. 2010 Aug. 9; 5(8):e12014; Provasi et al., 2012 Provasi E, Genovese P, Lombardo A, Magnani Z, Liu PQ, Reik A, Chu V, Paschon D E, Zhang L, Kuball J, Camisa B, Bondanza A, Casorati G, Ponzoni M, Ciceri F, Bordignon C, Greenberg P D, Holmes M C, Gregory P D, Naldini L, Bonini C. Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer. Nat Med. 2012 May; 18(5):807-15.). An issue that deserves careful evaluation is that mLPA-specific T cells killed also normal monocytes in vitro. In the case of mLPA-specific adoptive T cell therapy in the course of HSCT, this side effect might be tolerated by patients during the peritransplant period, when the control of minimal residual leukemia is paramount.

(34) To determine the capacity to recognize leukemic cells by the two main T cell subsets present in transduced T cells, CD4.sup.+ and CD8.sup.+ T cells were sorted from a PBMC transduced with the lentiviral vector expressing the DN4.99 TCR (FIG. 2A) and tested for their ability to recognize CD1c.sup.+ leukemia cells. Both CD4.sup.+ (FIG. 2B) and CD8.sup.+ (FIG. 2C) subsets were able to recognize the THP1-CD1c.sup.+ target cell line. TCR-transduced CD8.sup.+ T cells were also assessed for killing leukemia target cells. Only the TCR-transduced T cells specifically killed the leukemia cell line THP1 expressing CD1c, and not the THP1 WT cells (FIG. 2D). The arrows indicate the CD33.sup.+ THP1 leukemia cell line that disappears after 72 hs of co-culture with CD8.sup.+ T cells transduced with DN4.99 TCR.

(35) These results show that both CD4+ and CD8.sup.+ T cells can be successfully retargeted against CD1c+ leukemia cell lines by transducing CD1c self-reactive TCR, and confirm that transduced CD8.sup.+ T cells are strongly cytotoxic against malignant cells

Generation of a Library of CD1c Self-Reactive TCRs to Target Leukemia Cells

(36) To investigate the anti-leukemia efficacy of additional mLPA-specific TCRs, the inventors cloned the cDNA coding the TCRs from five different CD1c self-reactive T cell clones isolated from either healthy donors or leukemia patients. Table 1 reports the sequences of the TCRs.

(37) TABLE-US-00002 TABLE1 SequencesoftheCDR3regionsoftheTCRVaandVbportionsoftheCD1c selfreactiveTcellclones. Va N Vb clone TRAV seq reg Jaseq TRBV seq Nreg Jbseq DN4.99 TRAV38-1 CAYRS PL NTGTASKLTFGTGTRLQVTLJ44 TRBV28 CASS PWV SSYEQYFGPGTRLTVTJ2-7 CD4P8E3 TRAV38-1 CAF A NNARLMFGDGTQLVVKPJ31 TR13V28 CAS TDTG NTEAFFGTRLTVVJ1-1 DN4.2 TRAV26-1 CILR LR GGSNYKLTFGKGTLLTVNPJ53 TRBV4-1 CASSP IMGLAATH NEQFFGPGTRLTVLJ2-1 PZP8A6 TRAV26-2 CILRD VNTGTASKLTFGTGTRLQVTLDJ44 TRBV4-1 CASS RLGL STDTQYFGPGTRLTVLJ2.3 DN7.6.16 TRAV13.1 CAA PR GLGVTNSLSGRGPNSRSYQNJ28 TRBV27 CASS LVWGT YNEQFFGPGTRLTVLJ2-1

(38) For each TCR is reported the name of the clone, the name of the Va (TRAV) and Vb (TRBV) gene segments, the sequence of the last aa of the V regions (Va and Vb seq) starting from the conserved Cystein, the N-regions (N reg) and the sequences of the J segment (Ja seq and Jb seq).

(39) As shown in Table 1, CD1c self-reactive T cell clones preferentially express either TRAV38 paired with TRBV28 or TRAV26 paired with TRBV4, even though they were isolated from different donors. The TCR Valpha chains are characterized by a very short N region sequence, while the Vbeta chains utilize very similar Jbeta segments. Both TCR Valpha and Vbeta chains contain diverse N regions, each characterizing a specific T cell clone (underlined in the sequence).

(40) For each TCR the inventors generated a lentiviral vector in which the cDNA coding the TCR Valpha and Vbeta chains were joined with mouse Calpha and Cbeta, respectively, and linked together using the 2A peptide. In the lentivector, the chimeric TCRs genes are followed by an IRES sequence to allow the transcription of the eGFP reporter marker. The GFP gene can be excised by a simple digestion in order to obtain the same constructs devoid of the fluorescent marker.

(41) To verify whether the cloned TCRs could be expressed on the surface of T cells, each construct has been transduced into Jurkat 76 cells, which is a T-cell acute leukemia cell line that does not express the endogenous TCR, but allows the membrane expression of any exogenous TCR. Moreover, the TCR-signaling machinery is intact, therefore Jurkat 76 cells can be utilized to investigate the antigen recognition by the exogenous TCRs. FIG. 3A shows the expression of transduced TCRs detected by mAb specific for mouse Cbeta. Four out of five TCRs are expressed well on the cell surface. Only the TCR cloned from the self-reactive T cell clone DN 7.6.16 is weakly expressed. In initial experiments, Jurkat 76 cells expressing the DN4.99 and CD4P8E3 TCRs were also challenged with THP1-CD1c cells, in the absence or in the presence of the synthetic lipid antigen mLPA. As shown in FIG. 3B, upon recognition of CD1c.sup.+ THP1 cells, transduced Jurkat 76 cells upregulate the activation marker CD69, which is an indicator of TCR signaling. The upregulation of CD69 by the TCR-transduced Jurkat 76 cells is further enhanced by the addition of mLPA to the assay, confirming the antigen specificity of the two clones TCRs.

Example 2

Identification of the Lead CD1c-Restricted TCR to Engineer T Cells for Adoptive Immunotherapy

(42) The inventors have cloned into lentiviral vectors five different mLPA-specific TCRs from five independent CD1c self-reactive T cell clones bearing seemingly different affinities. To assess expression and relative Ag-affinities of these TCRs, the inventors transduced them into Jurkat 76 cells and tested their ability to recognize leukemia target cells by analyzing CD69 upregulation on the cell surface. As shown in FIG. 3A, the 5 TCRs are expressed at variable level: 4 out of 5 (TCR DN4.99, DN4.2, PZ-P8A6 and P8E3) are well expressed; one (TCR DN7.6.16) is retained inside the Jurkat 76 cells (data not shown)

(43) Further, 3 out of the 4 expressed TCRs (TCR DN4.99, DN4.2, PZ-P8A6) recognize the K562 acute myeloid leukemia cell line expressing CD1c and the recognition is further increased by the addition of synthetic mLPA antigen to the target cells (FIG. 3B). These data confirm the lipid Ag-specificity of the TCRs of the present invention.

(44) The inventors noticed that Jurkat 76 cells express CD1c and reasoned that this might result in a tonic stimulation of the Jurkat 76 cells transduced with the mLPA-specific TCRs, increasing their threshold of responsiveness to subsequent Ag stimulation and ultimately impacting their functional response. To avoid this confounding factor, the inventors hence generated Jurkat 76 cells devoid of all CD1 surface expression by deleting 2m gene with CRISP/Cas9 technology. Jurkat 76 cells wee electroporated with a plasmid encoding Cas9 recombinase together with guide RNA targeting 2m gene. After 45 days, electroporated cells that lost MHC class I expression were isolated by cell sorting and further cultured until day 85, when CD1c was also found downregulated from the cell surface) Jurkat 76 2m.sup. cells were transduced with the 5 CD1c self reactive TCRs, obtaining surface expression similar to those displayed by Jurkat 76 cells (FIG. 4A), and challenged with the leukemia cell line K562-CD1c with or without mLPA. As shown in FIG. 4B, TCR-transduced Jurkat 76 2m.sup. cells displayed a comparable pattern of recognition observed with Jurkat 76 cells, although with a markedly increased CD69 RFI suggesting a much increased Ag responsiveness despite overall similar surface TCR expression compared to Jurkat 76 cells.

(45) Jurkat 76 2m.sup. cells expressing the TCR DN4.99, TCR DN4.2 and TCR PZP8A6 were challenged with mLPA lipid titration to assess the affinity of the transduced TCRs.

(46) The level of expression of the transduced TCRs on the very same Jurkat cells utilized for the antigen recognition is shown in FIG. 5A.

(47) This assay provides Ag affinities independent of the surface expression of tested TCR. As shown in FIG. 5B, TCR DN4.99 displayed the highest TCR affinity (EC50=100 ng/ul), suggesting that this could represent the lead TCR to arm T cells for adoptive immunotherapy protocols.

(48) To confirm that large amount of T cells expressing the transduced mLPA-specific TCRs can be generated, viruses coding for TCR DN4.99, TCR DN4.2 and TCR PZP8A6 were transduced into polyclonal T cells isolated from the peripheral blood of healthy donors. As shown in FIG. 6A, more than 60% of T cells express the transduced chimeric TCR. Furthermore, T cells engineered with each of the three mLPA-specific TCRs specifically recognized two different AML cell lines (THP1, K562) expressing CD1c (FIG. 6B).

(49) This supports the feasibility of the approach to retarget large numbers of T cells against CD1c-expressing leukemia cells by the transduction of mLPA-specific TCRs.

(50) The inventors also assessed whether both major CD4.sup.+ and CD8.sup.+ T cell subsets expressing the transduced mLPA-specific TCRs recognized CD1c-expressing AML cell cell lines. Indeed, either TCR-transduced CD4.sup.+ or CD8.sup.+ T cells specifically recognized (FIGS. 7A-B) and killed THP1-CD1c.sup.+ leukemia cells, as shown by the disappearance of CD33+ cells after overnight of co-culture with transduced T cells (FIG. 7C-D).

(51) The transfer of mLPA-specific TCR into primary polyclonal CD4+ and CD8+ T cells generates effector cells that can kill a CD1c-expressing leukemia cell line, which is the pre-requisite for their clinical use.

Example 3

Assessing the Anti-Leukemia Efficacy of T Cells Engineered with the Lead mLPA-Specific TCR DN4.99

(52) The inventors first tested whether total primary T cells engineered with the lead TCR DN4.99 killed also primary blasts freshly isolated from an AML patient. As shown in FIG. 8, TCR DN 4.99 transduced T cells, and not mock transduced one, specifically killed primary AML blasts supporting the potential efficacy of this strategy.

(53) FIG. 8A confirms that primary AML blasts express CD1c, making them targettable by mLPA-specific T cells. FIG. 8B shows that the transfer of mLPA-specific TCR into primary polyclonal cells generates effector T cells that can kill the primary CD1c-expressing leukemia blast, which is the ultimate target for the therapeutic application of these TCRs.

(54) Currently, it is envisaged to apply adoptive cell therapy with leukemia-specific T cells at disease relapse after stem cell transplantation or induction chemotherapy. This could rescue relapsing patients. In this therapeutic scenario, one crucial point to be verified for the proposed leukemia targeting by CD1c self reactive T cells is the expression of CD1c on relapsed leukemia blasts and their recognition by mLPA-specific T cells. To address this critical issue, freshly isolated leukemia blasts were assessed for CD1c expression and for their ability to activate the DN4.99 mLPA-specific T cells both at diagnosis and at post-transplant relapse. FIG. 9 shows representative results.

(55) The expression of CD1c on primary blasts at diagnosis is maintained at prost-transplant relapse and both primary and relapsed leukemia cells are recognized at similar extent by mLPA-specific T cells.

(56) Collectively, the results shown in FIGS. 9A and B support the application of adoptive cell therapy with redirected mLPA-specific T cells at disease relapse after stem cell transplantation or induction chemotherapy.

Example 4

Assessing the Safety of T Cells Engineered with the Lead mLPA-Specific TCR DN4.99

(57) The inventors are also assessing the safety of the adoptive cell therapy approach with T cells engineered with the lead mLPA-specific TCR DN4.99. The main issue is to rule out possible detrimental on-target off-tumor recognition by the transduced T cells. mLPA is highly enriched in leukemia cells and in primary activated dendritic cells derived from circulating monocytes by culture with GM-CSF and IL-4, whereas it is very low in circulating monocytes and B cells at steady state. In principle, this Ag expression pattern should selectively target the reactivity of T cells engineered with the lead mLPA-specific TCR DN4.99 against the leukemia cells and a selected subset of normal mature DCs. Moreover, CD1c expression is absent in hematopoietic precursors (Lepore, de Lalla et al J Exp Med 2014 Lepore M, de Lalla C, Gundimeda S R, Gsellinger H, Consonni M, Garavaglia C, Sansano S, Piccolo F, Scelfo A, Hiussinger D, Montagna D, Locatelli F, Bonini C, Bondanza A, Forcina A, Li Z, Ni G, Ciceri F, Jen6 P, Xia C, Mori L, Dellabona P, Casorati G, De Libero G. A novel self-lipid antigen targets human T cells against CD1c+ leukemias. J Exp Med. 2014 Jun. 30; 211(7):1363-77), ruling out the recognition of essential cells for the maintenance of normal hematopoiesis. However, as a further measure to assess safety in vitro, the inventors have investigated a possible cross-reactivity of the lead mLPA-specific TCR DN4.99 with common and abundant phospholipids that share structural features with mLPA and may trigger off-tumor T cell recognition. Phosphatidilserine (PS) is a component of all cells and blood platelet membranes, while lysophosphatidylethanolamine (LPE) is a phospholipid found at high concentration in circulation as well as in biological membranes (FIG. 10).

(58) As shown in FIG. 11, Jurkat 76 cells expressing the mLPA-specific TCRs DN4.99 (A) or TCR DN4.2 (B) did not recognize PS and three different LPE species. By contrast, cells were activated by mLPA, as suggested by the up regulation of CD69 expression on TCR transduced Jurkat 76 cells upon co-culture with lipid-loaded K562-CD1c cells.

(59) This supports the safety of the adoptive cell therapy with T cells engineered with mLPA-specific TCRs, because they will selectively recognize the leukemia-enriched mLPA antigen, and not the other widely distributed and abundant phospholipids with analog structures.

(60) The ability of the TCR transduced T cells to control leukemia cells in NSG mice is also assessed. CD1c-expressing acute leukemia cell lines or primary blasts are transplanted into immunodeficent NSG mice, followed by the transfer of polyclonal allogeneic and, also patient-autologous T cells transduced or not transduced with the mLPA-specific TCRs. To follow leukemia progression in vivo, the inventors have generated THP-1 cells expressing the luciferase with or without CD1c (data not shown). Progression of the primary leukemia is monitored by flowcytometry on blood samples.