CD5 SPECIFIC T CELL RECEPTOR CELL OR GENE THERAPY

Abstract

The present invention is directed to the field of immunotherapy, in particular, adoptive T cell therapy or T cell receptor (TCR) gene therapy of cancer. The invention provides nucleic acids encoding at least one TCR alpha or beta chain construct of a TCR construct capable of specifically binding to a peptide from the T-cell lineage specific antigen CD5, preferably SEQ ID NO: 1 or 33, in the context of a human MHC I such as HLA-A*02, in particular HLA-A*02:01. The invention also provides corresponding proteins and host cells, preferably, CD8+ T cells, expressing said TCR construct. Treatment optionally is in the context of allogeneic stem cell transplantation, in particular, mismatch-transplantation, or haploidentical transplantation, or in combination with an agent capable of inhibiting expression of HLA-A*02 in the TCR-transgenic T cells. The invention thus also provides compositions and kits comprising the nucleic acids of the invention in combination with an agent capable of inhibiting expression of HLA-A*02, and, as well as the medical use of such compositions and kits. The nucleic acids, compositions and kits, proteins or host cells may be for use in the diagnosis, prevention and/or treatment of a CD5-positive T-cell lymphoma or T-cell leukemia, no matter whether the antigen is expressed on the cell surface, intracytoplasmic or in both manners.

Claims

1. A nucleic acid encoding a TCR alpha chain construct (TRA) and/or a TCR beta chain construct (TRB) of a TCR construct specific for an epitope in complex with a human MHC I molecule, wherein the epitope is an epitope from human CD5.

2. The nucleic acid of claim 1, wherein the MHC I is HLA-A*02 and the epitope is SEQ ID NO: 33, wherein the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 36, and/or the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 39.

3. The nucleic acid of claim 2, wherein the TRA comprises a CDR3 of SEQ ID NO: 36.

4. The nucleic acid of any of claim 2 or 3, wherein the TRB comprises a CDR3 of SEQ ID NO: 39.

5. The nucleic acid of any of claims 2-4, wherein the TRA comprises a CDR1 having at least 85% sequence identity to SEQ ID NO: 34 and a CDR2 having at least 87% sequence identity to SEQ ID NO: 35, and/or the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 37 and a CDR2 having at least 83% sequence identity to SEQ ID NO: 38, wherein, preferably, the TRA has a variable region having at least 70% sequence identity to SEQ ID NO: 40 and/or the TRB has a variable region having at least 70% sequence identity to SEQ ID NO: 41.

6. The nucleic acid of any of claims 2-4, wherein the TRA comprises a CDR1 having SEQ ID NO: 34, a CDR2 having SEQ ID NO: 35 and a CDR3 having SEQ ID NO: 36, and/or the TRB comprises a CDR1 having SEQ ID NO: 37, a CDR2 having SEQ ID NO: 37 and a CDR3 having SEQ ID NO: 38.

7. The nucleic acid of any of claims 2-6, wherein the TRA has a variable region having at least 80% sequence identity to SEQ ID NO: 40 and/or the TRB has a variable region having at least 80% sequence identity to SEQ ID NO: 41, wherein, optionally, the nucleic acid encoding the TRA has at least 80% sequence identity to SEQ ID NO: 42 and/or the nucleic acid encoding the TRB has at least 80% sequence identity to SEQ ID NO: 43.

8. The nucleic acid of claim 1, wherein the MHC I is HLA-A*02 and the epitope is SEQ ID NO: 1, wherein the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 4, and/or the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 7, wherein, preferably, the TRB comprises a CDR3 having SEQ ID NO: 7.

9. The nucleic acid of claim 8, wherein the TRA comprises a CDR3 of SEQ ID NO: 4.

10. The nucleic acid of any of claim 8 or 9, wherein the TRA comprises a CDR1 having at least 85% sequence identity to SEQ ID NO: 2 and a CDR2 having at least 87% sequence identity to SEQ ID NO: 3, and/or the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 5 and a CDR2 having at least 83% sequence identity to SEQ ID NO: 6, wherein, preferably, the TRA has a variable region having at least 70% sequence identity to SEQ ID NO: 10 and/or the TRB has a variable region having at least 70% sequence identity to SEQ ID NO: 11.

11. The nucleic acid of any of claims 8-10, wherein the TRA comprises a CDR1 having SEQ ID NO: 2, a CDR2 having SEQ ID NO: 3 and a CDR3 having SEQ ID NO: 4, and/or the TRB comprises a CDR1 having SEQ ID NO: 5, a CDR2 having SEQ ID NO: 6 and a CDR3 having SEQ ID NO: 7.

12. The nucleic acid of any of claims 8-11, wherein the TRB comprises a CDR3 having SEQ ID NO: 8, wherein preferably, the TRA has a variable region having at least 80% sequence identity to SEQ ID NO: 10 and/or the TRB has a variable region having at least 80% sequence identity to SEQ ID NO: 11, wherein, optionally, the nucleic acid encoding the TRA has at least 80% sequence identity to SEQ ID NO: 14 and/or the nucleic acid encoding the TRB has at least 80% sequence identity to SEQ ID NO: 15.

13. The nucleic acid of any of claims 8-11, wherein the TRB comprises a CDR3 having SEQ ID NO: 9, wherein preferably, the TRA has a variable region having at least 80% sequence identity to SEQ ID NO: 12 and/or the TRB has a variable region having at least 80% sequence identity to SEQ ID NO: 13, wherein, optionally, the nucleic acid encoding the TRA has at least 80% sequence identity to SEQ ID NO: 16 and/or the nucleic acid encoding the TRB has at least 80% sequence identity to SEQ ID NO: 17.

14. The nucleic acid of any of the preceding claims, encoding at least one TCR alpha and beta chain construct of the TCR construct, wherein the TCR alpha chain construct and the TCR beta chain construct preferably further comprise a constant region selected from the group comprising a human constant region, a murine constant region or a chimeric constant region.

15. The nucleic acid of any of the preceding claims, which is selected from the group comprising a viral vector, a transposon or a vector suitable for CRISPR/CAS based recombination.

16. A protein encoded by the nucleic acid of any of the preceding claims.

17. A composition or kit comprising a nucleic acid of any of claims 1-15 and an agent for inhibiting expression of HLA-A*02 selected from the group comprising silencing RNA, siRNA, shRNA, miRNA, a nucleic acid encoding a silencing RNA, siRNA, shRNA, miRNA, a ribonucleoprotein complex comprising CRISPR and a guide RNA suitable for targeting CRISPR to suppress HLA-A*02 expression, a Transcription Activator-like Effector Nuclease suitable for suppressing HLA-A*02 expression, and a Zincfinger nuclease suitable for suppressing HLA-A*02 expression, wherein, if the miRNA is encoded by a nucleic acid, said nucleic acid can be on the same nucleic acid as the nucleic acid of any of claims 1-15.

18. A composition or kit comprising a nucleic acid of any of claims 1-15 and an agent for inhibiting expression of CD5 selected from the group comprising silencing RNA, siRNA, shRNA, miRNA or a nucleic acid encoding a silencing RNA, siRNA, shRNA, miRNA, or a ribonucleoprotein complex comprising CRISPR and a guide RNA suitable for targeting CRISPR to suppress CD5 expression, a Transcription Activator-like Effector Nuclease suitable for suppressing CD5 expression, and a Zincfinger nuclease suitable for suppressing CD5 expression, wherein, if the miRNA is encoded by a nucleic acid, said nucleic acid can be on the same nucleic acid as the nucleic acid of any of claims 1-15.

19. A host cell comprising a nucleic acid of any of claims 1-15 and/or a protein of claim 16, wherein the host cell preferably is a human CD8.sup.+ T cell.

20. A host cell of claim 19, wherein the host cell does not express HLA-A*02 or comprises an agent for inhibiting expression of HLA-A*02 selected from the group comprising silencing RNA, siRNA, shRNA, miRNA or a nucleic acid encoding a silencing RNA, siRNA, shRNA, miRNA, or a ribonucleoprotein complex comprising CRISPR and a guide RNA suitable for targeting CRISPR to suppress HLA-A*02 expression.

21. A host cell of any of claim 19 or 20, wherein the host cell does not express CD5 or comprises an agent for inhibiting expression of CD5 selected from the group comprising silencing RNA, siRNA, shRNA, miRNA or a nucleic acid encoding a silencing RNA, siRNA, shRNA, miRNA, a ribonucleoprotein complex comprising CRISPR and a guide RNA suitable for targeting CRISPR to suppress CD5 expression.

22. A pharmaceutical composition comprising a) a nucleic acid of any of claims 1-15 encoding a TCR construct capable of specifically binding to a peptide of SEQ ID NO: 1 or 33 in the context of HLA-A*02; or b) a protein of claim 16 comprising a TCR construct capable of specifically binding to a peptide of SEQ ID NO: 1 or 33 in the context of HLA-A*02; or c) a composition or kit of any of claim 17 or 18; or d) a host cell of any of claims 19-21 expressing a TCR construct capable of specifically binding to a peptide of SEQ ID NO: 1 or 33 in the context of HLA-A*02; wherein the host cell preferably is a host cell of any of claim 20 or 21, wherein, optionally, the composition in addition to said host cell of claim 20 or 21 comprises further T cells not expressing HLA-A*02 and/or CD5, or comprising an agent for inhibiting expression of HLA-A*02 and/or CD5.

23. The pharmaceutical composition of claim 22 for use in the treatment of a patient having a T cell lymphoma or T cell leukemia, wherein the patient expresses HLA-A*02, wherein, optionally, said treatment is in the context of allogeneic transplantation of T cells not expressing HLA-A*02, wherein said patient preferably does not express HLA-C*12.

Description

FIGURE LEGENDS

[0103] FIG. 1: Immunisation of ABabDII mice with the CD5 epitope, CD5.sub.51-59 (YLKDGWHMV), SEQ ID NO: 1. [0104] a) Alignment of human CD5 and mouse CD5 sequences spanning the CD5.sub.51-59 epitope that is underlined. The mouse sequences strongly differ with regard to the sequence corresponding to the epitope. [0105] b) Intracellular cytokine staining (ICS) of peripheral blood cells to detect IFN-γ secreting CD8.sup.+-T cells after prime-boost immunization. Cells were stimulated with anti-CD3/CD28 antibodies as positive control (left panel). An irrelevant peptide was used as a negative control (middle panel). [0106] c) IFN-γ capture assay was done to detect and sort IFN-γ secreting CD8 .sup.+-T cells from in vitro-expanded splenocytes. Populations in the gates were sorted to isolate the RNA for identification of TCR variable chain rearrangements- [0107] d) Identified TCR α and β pairs were used to construct a TCR cassette as shown.

[0108] FIG. 2: Re-expression of identified TCRs in HLA-A2.sup.− human peripheral blood lymphocytes [0109] a) FACS analysis of HLA-A2.sup.− human peripheral blood lymphocytes (hPBLs) after transduction with T-20109 and T-20332 TCRs. The transduction rate varied between 30-80% depending on the virus titer. [0110] b) Co-culture with T2 cells loaded with decreasing concentration of YLK peptide (SEQ ID NO: 1) to deduce TCR affinity. A representative of peptide titration is shown here. [0111] c) IFN-γ release by effector cells against CD5.sup.+-HLA-A2.sup.+ target cells. Effector cells secreted IFNγ only when HLA-A2 molecule was present on the CD5 .sup.+ cells (H9/HLA-A2 and CCRF-CEM/HLA-A2), showing HLA restriction. [0112] d) IFN-γ release by effector cells against CD5.sup.+-HLA-A2.sup.−. We did not detect any IFN-γ by ELISA, showing HLA-A2 dependency of killing. [0113] e) Recognition of primary cells from blood donors by T-20109. Only CD5.sup.+ fraction of HLA-A2+ donors induced CD137 upregulation on TCR transduced effector cells indicating T20109 TCR can recognize primary T cells isolated from human blood.

[0114] FIG. 3: HLA-A2 downregulation by RNAi on TCR- transduced HLA-A2.sup.+hPBLs. [0115] a) hPBLs were isolated from HLA-A2.sup.+ blood donors and transduced with vectors carrying CD5 TCRs with or without the HLA-A2 targeting miRNA sequences. Transduced T cells were expanded for 11 days following transduction. Both viability (DAPI-negative cells) and fraction of HLA-A2.sup.− cells were analyzed by FACS every other day. [0116] b) Percentage of alive cells for T cells transduced with CD5 TCRs decreased in time due to fratricide (dashed lines). Introduction of HLA-A2-targeting miRNA sequence (RNAi-T20109 and RNAi-T-20332) to the vector rescued the viability of the after day 8. [0117] c) HLA-A2.sup.− cells expanded in culture over time due to the selective pressure applied by the fratricide. The percentage of HLA-A2.sup.− cells remained the same when the cells received RNAi and a control TCR. [0118] d, e) RNAi-CD5 TCR transduced T cells from HLA-A2.sup.+ donors were co-cultured with peptide loaded T2 cells to assess any changes in TCR-affinity due to introduction of HLA-A2 targeting miRNA. HLA-A2.sup.−-TCR-transduced T cells served as a control (dashed lines). The Kd values increased by 2.24 and 1.48-fold for T-20109 (d) and T-20332 (e), respectively, indicating slight but non-significant decrease in TCR affinities to μMHC complex.

[0119] FIG. 4: CRISPR/Cas9 mediated HLA-A2-editing on TCR-transduced T cells. [0120] hPBLs were isolated from HLA-A2.sup.+ blood donors, electroporated with RNP complexes targeting HLA-A2 genomic sequence and transduced with T-20109 and T-20332 TCRs. Transduced T cells were expanded for 18 days following transduction. Viability and fraction of HLA-A2.sup.− cells were analyzed by FACS every other day. [0121] a) hPBLs from HLA-A2.sup.+ blood donors were electroporated with RNP complexes targeting HLA-A2 genomic sequence. HLA-A2 expression on the surface was analyzed by FACS 72h after electroporation. crRNA A2-5 yielded highest KO efficiency, therefore, was selected for downstream experiments. [0122] b) Electroporation was coupled to transduction with T-20109 or T-20332 TCRs. The viability of cells transduced with T-20109 and receiving A2-5 RNPs (triangle data points, solid line) recovered slightly after d8, while the ones receiving Cas9 only (triangle data points, dashed line) had decreasing viability in time. [0123] c) Fratricide-induced selective pressure resulted in dramatic increase in the percentage of HLA-A2-edited cells by day 8 when cells received A2-5 RNPs and were transduced with both CD5 TCRs, but not control TCR. [0124] d, e) HLA-A2-edited cells modified with T-20109 (d, solid line) or T-20332 TCRs (e, solid line) were used in a co-culture with peptide loaded T2 cells for a peptide titration assay. HLA-A2-edited cells with TCR performed similar to TCR transduced cells from an HLA-A2.sup.− donor (d, e, dashed lines), showing that knocking out HLA-A2 does not have any effect on TCR functionality(see table 2 for Kd values).

[0125] FIG. 5: Comparison of RNAi mediated HLA-A2 knock down and CRISPR mediated HLAA2-editing on functionality of CD5-TCR transduced T cells.

[0126] RNAi-TCR cells and CRISPR-TCR cells were co-cultured with cell lines expressing CD5 and/or HLA-A2 molecule. Activation was analyzed by FACS via CD137 upregulation on the effector cell surface. Cells from an HLA-A2.sup.− donor performed slightly better for T-20109 (left pane, white bar). RNAi-TCR cells (left pane, black bar) and CRISPR-TCR cells (left pane, patterned bar) did not exhibit any difference in terms of CD137 upregulation. We did not observe any difference in case of T-20332 among HLA-A2.sup.− donor (right pane, white bar), RNAi-TCR cells (right pane, black bar) or CRISPR-TCR cells (right pane, patterned bar)

[0127] FIG. 6: Re-expression of T-7378 TCR in HLA-A2.sup.− human peripheral blood lymphocytes.

[0128] The T-7378 TCR recognizing the SIC epitope (SEQ ID NO: 33) was generated and cloned following the same methods described for the TCRs specific to YLK epitope. [0129] a) FACS analysis of HLA-A2.sup.− human peripheral blood lymphocytes (hPBLs) after transduction with T-7378. The transduction rate varied between 11-40% depending on the virus titer. [0130] b) To deduce TCR affinity, T cells transduced with T-7378 TCR were co-cultured overnight with T2 cells loaded with decreasing concentration of SIC peptide (SEQ ID NO: 33) at an effector to target ratio of 1:1. IFN-γ release was detected by ELISA. A representative of peptide titration is shown here. [0131] c) IFN-γ release by T-7378 transduced T cells against CD5 .sup.+ target cell lines. The target cell lines did not have HLA-A2 allele; therefore, they were generated by retroviral delivery of HLA-A2. T cells were co-cultured with target cell lines at an effector to target ratio of 1:1. After overnight incubation, cell-free supernatant was collected and IFN-γ release was detected by ELISA. Effector cells secreted high level of IFN-γ only when HLA-A2 molecule was present on the CD5.sup.+ cells (H9/HLA-A2, CCRF-CEM/HLA-A2, Jurkat/HLA-A2 and Molt14/HLA-A2), showing HLA restriction. [0132] d) IFN-γ release by T-7378 transduced T cells against CD5.sup.−-HLA-A2.sup.+ after overnight co-culture at an effector to target ratio of 1:1. No IFN-γ was detected by ELISA, showing that T7378 induces CD5-dependent killing.

[0133] FIG. 7: Co-culture with a panel of LCLs to identify any potential alloreactivity of T-7378.

[0134] The T-7378-transduced T cells were co-cultured overnight with 14 different LCL lines with known HLA genotype to identify potential alloreactivity at an effector to target ratio of 1:1. The LCL lines do not express any CD5. No IFN-γ release by effector cells against any of the LCLs was detected, showing T-7378 does not have allo-reactive response to any of the HLA alleles covered by the LCL lines (Table 3).

[0135] FIG. 8 HLA-KO in T-7378 TCR-transfected T cells

[0136] PBLs from HLA-A2.sup.+ blood donors were electroporated with RNP complexes targeting HLA-A2 genomic sequence and transduced with T-7378 TCR. Transduced cells were expanded for 18 days and counted to analyze cell expansion. Viability and fraction of HLA-A2-cells were analyzed by FACS on time points indicated on graphs.

[0137] A) The cells in expansion were counted and total number of cells in culture was calculated. T7378 transduced T cells that received A2-5 gRNA (diamond data point, solid line) expand comparable to PBLs transduced with control TCR receiving either A2-5 gRNA or only Cas9. On the other hand, Cas9 receiving T cells transduced with T-7378 TCR cannot expand due to fratricide (diamond data point, dashed line).

[0138] B) Prevention of fratricidal killing by HLA-A2 knock out has effect on the viability. The viability of T-7378 transduced T cells receiving A2-5 recovers in time (as they lose HLA-A2 on the surface) while Cas9 receiving cells do not change.

[0139] C) Fratricide induced selective pressure resulted in rapid increase in the fraction of HLA-A2 knock out cell in the population of T cells transduced with T-7378 but not control TCR.

SEQUENCES

[0140] SEQ ID NO: 1 epitope from CD5

[0141] SEQ ID NO: 2 T-20109+T-20332 alpha chain CDR1

[0142] SEQ ID NO: 3 T-20109+T-20332 alpha chain CDR2

[0143] SEQ ID NO: 4 T-20109+T-20332 alpha chain CDR3

[0144] SEQ ID NO: 5 T-20109+T-20332 beta chain CDR1

[0145] SEQ ID NO: 6 T-20109+T-20332 beta chain CDR2

[0146] SEQ ID NO: 7 beta chain CDR3 consensus sequence

[0147] SEQ ID NO: 8 T-20109 beta chain CDR3

[0148] SEQ ID NO: 9 T-20332 beta chain CDR3

[0149] SEQ ID NO: 10 variable region T-20109 alpha chain (aa)

[0150] SEQ ID NO: 11 variable region T-20109 beta chain (aa)

[0151] SEQ ID NO: 12 variable region T-20332 alpha chain (aa)

[0152] SEQ ID NO: 13 variable region T-20109 beta chain (aa)

[0153] SEQ ID NO: 14 variable region T-20109 alpha chain (na)

[0154] SEQ ID NO: 15 variable region T-20109 beta chain (na)

[0155] SEQ ID NO: 16 variable region T-20332 alpha chain (na)

[0156] SEQ ID NO: 17 variable region T-20332 beta chain (na)

[0157] SEQ ID NO: 18 murine constant region (alpha)

[0158] SEQ ID NO: 19 minimally murine constant region (alpha)

[0159] SEQ ID NO: 20 human constant region (alpha)

[0160] SEQ ID NO: 21 murine constant region (beta)

[0161] SEQ ID NO: 22 minimally murine constant region (beta)

[0162] SEQ ID NO: 23 human constant region (beta)

[0163] SEQ ID NO: 24 reverse primer for TCRA

[0164] SEQ ID NO: 25 reverse primer for TCRB

[0165] SEQ ID NO: 26 sequence from human CD5 (FIG. 1a)

[0166] SEQ ID NO: 27 sequence from mouse CD5 (FIG. 1b)

[0167] SEQ ID NO: 28 crRNA-spacer A2-1

[0168] SEQ ID NO: 29 crRNA-spacer A2-2

[0169] SEQ ID NO: 30 crRNA-spacer A2-3

[0170] SEQ ID NO: 31 crRNA-spacer A2-4

[0171] SEQ ID NO: 32 crRNA-spacer A2-5

[0172] SEQ ID NO: 33 epitope from CD5

[0173] SEQ ID NO: 34 T-7378 alpha chain CDR1

[0174] SEQ ID NO: 35 T-7378 alpha chain CDR2

[0175] SEQ ID NO: 36 T-7378 alpha chain CDR3

[0176] SEQ ID NO: 37 T-7378 beta chain CDR1

[0177] SEQ ID NO: 38 T-7378 beta chain CDR2

[0178] SEQ ID NO: 39 T-7378 beta chain CDR3

[0179] SEQ ID NO: 40 variable region T-7378 alpha chain (aa)

[0180] SEQ ID NO: 41 variable region T-7378 beta chain (aa)

[0181] SEQ ID NO: 42 variable region T-7378 alpha chain (na)

[0182] SEQ ID NO: 43 variable region T-7378 beta chain (na)

[0183] SEQ ID NO: 44 variable region T-7378 alpha chain (na), codon-optimized

[0184] SEQ ID NO: 45 variable region T-7378 beta chain (na), codon-optimized

EXAMPLES

1.1. Selection of Epitopes

[0185] Full length human CD5 protein sequence was obtained from NCBI database. The sequence was submitted to NetMHC V4 for prediction of epitopes binding to HLA-A2 allele. Epitope length was defined as 9-mers. The predicted epitopes with highest binding affinity and minimum homology to mouse CD5 were selected for immunization.

1.2. Immunization of ABabDII Mice

[0186] Predicted peptide (e.g., the peptide of SEQ ID NO: 1, as shown in FIG. 1a, or the peptide of SEQ ID NO: 33) was dissolved in appropriate solvent to a concentration of 2 mg/ml. Mice were primed on day 0 and immunized on day 21 with 100 μg of peptide in a 1:1 solution of incomplete Freund's adjuvant (IFA) and 50 μg CpG1826 by subcutaneous injection. Blood was collected 7 days after each boost and blood cells were cultured with 10.sup.−6 M peptide overnight in the presence of Brefeldin A (BFA). Peripheral response was analyzed by intracellular IFN-γ staining of blood cells after overnight culture (FIG. 1b).

[0187] Mice with IFN-γ-secreting CD8.sup.+ T cells in the periphery were sacrificed. Spleen and inguinal lymph nodes of reactive mice were collected. CD4.sup.+ T cells were depleted by CD4 microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). 1×10.sup.6 splenocytes were seeded per well of a 24-well plate, and expanded for 10 days in RPMI 1640 medium supplemented with 10% FBS gold, HEPES, NEAA, Sodium Pyruvate, 50 μM β-mercaptoethanol, 20 IU/ml human IL-2 and 10.sup.−8 M peptide. Splenocytes were stimulated with 10.sup.−6 M peptide for 4 h before a mouse IFN-γ secretion assay (Miltenyi Biotech, Bergisch Gladbach, Germany). The cells were treated with Fc Block, stained with antibodies against mouse CD3-APC and mouse CD8-PerPC (BD Biosciences, San Jose, Calif., USA). IFN-γ secreting CD8.sup.+ T cells were sorted with BD FACS Aria III (BD Biosciences, San Jose, Calif., USA) (FIG. 1c), and transferred to RTL lysis buffer for RNA isolation with RNeasy Micro Kit (Qiagen, Hilden, Germany).

1.3. Identification and Cloning of TCRs

[0188] 5′RACE-ready cDNA was synthesized with SMARTer RACE kit (Clontech, Calif., USA) according to instructions of the manufacturer. cDNA was diluted 1:3 prior to use. TCRA and TCRB variable chains were amplified by 5′RACE-PCR in a 50 μL reaction mix of 5 μL diluted cDNA, 2X Q5 Hot Start High-Fidelity master mix (New England Biosciences, Ipswich, Mass., USA), 5 μL forward primer from the SMARTer RACE kit (10X Universal Primer A Mix (UPM)) and 0.5 μM reverse primers for TCRA: 5′-CGGCCACTTTCAGGAGGAGGATTCGGACC-3′ (SEQ ID NO: 24) or TCRB:5′-CCGTAGAACTGGACTTGACAGCGGAAGTGG-3′ (SEQ ID NO: 25). Initial denaturation was done at 98° C. for 2 min seconds followed by 30 cycles of denaturation at 98° C. for 30 s, annealing at 72° C. for 30 s and elongation at 72° C. for 45 s. Annealing temperature was decreased by 2° C. at every 5 cycles for the first 10 cycles. Reaction was carried out for total 35 cycles. Final elongation was done at 72° C. for 5 min.

[0189] PCR products were separated on 2% gel. Bands corresponding to the correct size were eluted from the gel and cloned using Zero Blunt TOPO PCR Cloning Kit (Invitrogen) and sequenced with SP6 primer. Dominant TCR-α/(3 chains were selected and paired. The TCR constant regions were replaced with mouse counterparts. Paired TCR-α/(3 chains were linked with a p2A element (FIG. 1d). TCR cassette was codon optimized, synthesized by GeneArt (Thermo Fisher Scientific, Waltham, Mass., USA) and cloned into μMP71 by restriction site cloning.

1.4. Generation of RNAi Vectors

[0190] Three different miRNA sequences 100% complementary to the HLA-A2 allele, i.e., crRNA sequences suitable for knocking out HLA-A2 by CRISPR/Cas9, were designed in silico (SEQ ID NO: 28, 29 and 30) and produced by overlap polymerase chain reaction (PCR) to introduce into the MP71-GFP vector as previously described (Bunse et al., 2014. Molecular Therapy 22(11):1983-1991). RNAi-TCR vectors were generated by swapping the TCR cassette with GFP by restriction enzyme cloning using cut sites Notl and EcoRl.

1.5. Formation of RNP Complexes

[0191] crRNAs targeting HLA-A2 allele were predicted in silico with CRISPRGold (ttps://crisprgold.mdcberlin.de) and top five sequences with minimum off-target risks were selected (A2-1, A2-2, A2-.3, A2-3, A2-4 and A2-5), comprising, in this order, SEQ ID NO: 28-32. crRNAs and tracrRNAs were chemically synthesized (Dharmacon, IDT) and recombinant SpCas9 was obtained from the protein facility of MDC in in 20 mM HEPES-KOH pH 7.5, 150 mM KCl, 10% glycerol, 1 mM DTT. Lyophilized RNA was resuspended in the provided resuspension buffer to reach 100 μM concentration, aliquoted and stored at −20° C. crRNA and tracrRNA aliquots were thawed, mixed 1:1 by volume, annealed by incubation at 95° C. for 5 min and let cool down to RT on benchtop for 10 min. SpCas9, stored at 40 μM, was then mixed at 1:1 molar ratio with the gRNA at RT for 15 min to form an RNP at 20 μM. RNPs were electroporated immediately after complexing.

1.6. Electroporation of Human T Cells

[0192] PBMCs were isolated from fresh blood of HLA-A2*01 positive blood donors by Ficoll separation. T cells were MACS sorted from the PBMCs using a pan T cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany). 1×10.sup.6 isolated T cells were stimulated either on anti-CD3/antiCD28 coated plates or with human T-activator CD2/CD28 Dynabeads (Thermo Fisher Scientific) in RPMI 1640 medium supplemented with 10% FBS, HEPES, 100 IU/ml IL-2 in a 24-well plate. Cells were collected 48 hours after stimulation, resuspended in 20 μL Lonza P3 buffer per 1×10.sup.6 cells and electroporated with 54 of RNP complex in the Amaxa 4D Nucleofector using the program EH110. Cells were incubated in RPMI 1640 medium supplemented with 10% FBS, HEPES, 100 IU/ml IL-2 in a 48-well plate for 24 hours before transduction.

1.7. TCR Re-Expression in Human PBLs

[0193] HEKT-GALV-g/p cells were transfected with 18 μg μMP71 vector carrying the TCR cassette with or without HLA-A2-targeting miRNA sequence. The virus supernatant was collected 48 h after transfection.

[0194] For RNAi mediated HLA-A2 knock down, the cells were collected and activated as described for electroporation and transduced 48h and 72h after activation with the TCR vectors carrying the miRNA sequences.

[0195] CRISPR/Cas9 mediated HLA-A2 edited cells were transduced with the vector carrying TCR cassette 48h and 72h after electroporation

[0196] Transduction efficiency was determined by FACS staining for human HLA-A2-PE (BD Biosciences, San Jose, Calif., USA), human CD8-APC (BD Biosciences, San Jose, Calif., USA) and mouse TRBC-PerCP (Biolegend, San Diego, Calif., USA).

[0197] TCR-transduced-T cells were expanded in T cell medium supplemented with 100 IU/ml IL-2 for 15 days and analysed per FACS every other day to measure HLA-A2, CD8, TCR expression and cell viability. A fraction of cells were frozen on day 8 to be used as effector cells for functional assays.

1.8. Functional Assays

[0198] 1.8.1. Detection of Cytokine Release

[0199] For detection of cytokine release, 2×10.sup.4 target cells and 2×10.sup.4 TCR-transduced cells were seeded in 200 uL final volume in a 96-well format to reach 1:1 effector to target ratio. Cell-free supernatant was collected after overnight incubation to detect IFN-γ secretion by ELISA.

[0200] 1.8.2 Detection of T Cell Activation

[0201] Cells were collected for further analysis and stained with antibodies against human CD137-PE (BD Biosciences, San Jose, Calif., USA), human CD8-APC-H7 (BD Biosciences, San Jose, Calif., USA), mouse TRBC-APC (Biolegend, San Diego, Calif., USA) and run on BD FACSCanto II Flow cytometer. Data was analyzed with FlowJo version 10.0.8.

2. Results

[0202] a) TCR recognizing the CD5.sub.51-59 epitope (SEQ ID NO: 1) were isolated, T-20109 and T 20332. Both TCR share the CDRs of the alpha chain, but, interestingly, differ in the CDR3 region of the beta chain. FIG. 2a shows FACS analysis of HLA-A2.sup.− human peripheral blood lymphocytes (hPBLs) after transduction with T-20109 and T-20332 TCRs. The transduction rate varied between 30-80% depending on the virus titer.

[0203] T2 cells were loaded with serial dilutions of peptide at 10.sup.−5 M to 10.sup.−12 M for peptide titration experiments (FIG. 2b). T-20109 and T 20332 both have a high peptide sensitivity, with T20109 reacting at slightly lower peptide concentrations.

[0204] For experiments with FACS analysis, target cells were selected based on their CD5 expression and labeled with 1 μM CFSE (ab113853, Abcam, Cambridge, UK) prior to seeding to differentiate them from effector cells.

[0205] H9 cells and CCRF-CEM cells express CD5. The TCR-transduced cells were tested for cytokine release after overnight incubation with these cells lines which had either been engineered to express HLA-A2 or not (FIG. 2c), showing HLA restriction of both TCR constructs. A corresponding analysis with other CD5.sup.+ HLA-A2-cells as target cells confirmed HLA restriction (FIG. 2d).

[0206] For co-culture with primary human cells as targets, PBMCs were isolated from HLA-A2 positive and negative blood donors. To obtain a CD5 positive fraction, cells were stained with CD5-APC antibody, labeled with anti-APC magnetic beads and MACS sorted. MACS-sorted CD19 positive cells served as the CD5 negative fraction. T cell activation was tested after overnight incubation with or without peptide (1 μM/L).

[0207] FIG. 2e shows, for T-20109 and a control TCR not reactive with CD5, that T-cells expressing said TCR are activated by CD5-positive cells if these cells also express HLA-A2. Activation, in particular by HLA-A2-positive cells, is also induced—or increased—by addition of peptide.

[0208] b) A TCR recognizing the CD5283-291 epitope (SEQ ID NO: 33) were isolated, T-7378. FIG. 6a shows FACS analysis of HLA-A2.sup.− human peripheral blood lymphocytes (hPBLs) after transduction with T-7378 TCR. The transduction rate varied between 20-80% depending on the virus titer.

[0209] T2 cells were loaded with serial dilutions of peptide of SEQ ID NO: 33 at 10.sup.−5 M to 10.sup.−12 M for peptide titration experiments (FIG. 6b).

[0210] For experiments with FACS analysis, target cells were selected based on their CD5 expression and labeled with 1 μM CFSE (ab113853, Abcam, Cambridge, UK) prior to seeding to differentiate them from effector cells.

[0211] H9 cells and CCRF-CEM cells express CD5. The TCR-transduced cells were tested for cytokine release after overnight incubation with these cells lines which had either been engineered to express HLA-A2 or not (FIG. 6c), showing HLA restriction of both TCR constructs. A corresponding analysis with other CD5.sup.+ HLA-A2-cells as target cells confirmed HLA restriction (FIG. 6d).

[0212] Further, to test for a potential alloreactivity of T-7378, T-7378 transduced effector cells were co-cultured with 14 different LCL lines with known HLA genotype (Table 3). The LCL lines do not express any CD5. No IFN-γ release by effector cells against any of the LCLs was detected, showing T-7378 does not have allo-reactive response to any of the HLA alleles covered by the LCL lines. T-7378 can thus be safely used in patients having a large variety of HLA-genotypes, e.g., those tested.

TABLE-US-00001 TABLE 3 List of the LCLs and their MHC Class I alleles. HLA-A HLA-B HLA-C LCL1 A*02 A*26 B*13 B*27 LCL2 A*32 A*68 B*44 LCL3 A*01 A*31 B*08 B*40:02 LCL4 A*02 A*24 B*15 LCL5 A*24 B*08 B*51 LCL6 A*01 B*08 GOELK A*11 A*24 B*13 B*38 C*05:01:01 C*12:03:01 MDB1 A*01 A*11 B*08 B*15 FSB1 A*24 A*26 B*07 B*38 JNB3 A*01 A*02:01 B*07 B*40:01 STA01 A*02:01 A*02:01 B*07:02 B*15:01 RZB2/22 A*02:01 A*29:01 B*44:02 B*45:01 C*06:02 KOEB2 A*01 A*29 B*44 B*51 LSKB1 A*01 A*02 B*07 B*08 C*07 C*07 AMB13 A*01 A*26 B*35:01 B*57:01 KH1 A*01 A*03 B*07 B*08 ML A*02 A*23 B40:01 B*44

2.1 RNAi Downregulation of CD5

[0213] hPBLs were isolated from HLA-A2.sup.+ blood donors and transduced with vectors carrying CD5 TCRs with or without the HLA-A2 targeting miRNA sequences (FIGS. 3a and 8a). The percentage of living T cells transduced with CD5 TCR decreased in time due to fratricide. Introduction of HLA-A2-targeting miRNA sequence (RNAi-T-20109 and RNAi-T-20332) to the vector rescued the viability of the after day 8 (FIGS. 3b and 8b), and the percentage of HLA-A2-negative cells increased due to the selective pressure (FIGS. 3c and 8c). No significant decrease in peptide sensitivity or affinity of the TCRs was seen due to introduction of the miRNA (FIG. 3d).

2.2 CRISPR/Cas-Mediated Downregulation of CD5

[0214] FIG. 4a shows that some of the crRNA constructs selected, in particular, A2-2, A2-4 and A2-5, were able to reduce HLA-A2 expression on the surface of hPBLs. crRNA A2-5 (comprising SEQ ID NO: 32) yielded highest KO efficiency, therefore, it is preferred and was selected for downstream experiments.

[0215] hPBLs were isolated from HLA-A2.sup.+ blood donors, electroporated with RNP complexes targeting HLA-A2 genomic sequence or Cas9 only and transduced with T-20109 and T-20332 TCRs. The viability of T cells transduced with aCD5 TCR together with A2-5 after d8 was higher than the viability of T cells transduced with aCD5 TCR and Cas9 only, in particular for T-20109 (FIG. 4b). The percentage of HLA-A2-negative cells increased due to the selective pressure (FIG. 4c). No significant decrease in peptide sensitivity or affinity of the TCRs was seen due to reduced expression of HLA-A2 (FIG. 4d).

[0216] FIGS. 5a and b compares T cell activation of T cells expressing T-20109 (a) and T-20332 (b) which either were from an HLAA2-negative donor or wherein HLA-A2 had been downregulated by miRNA or the CRISPR-based approach. All TCR-transgenic T cells only recognized the HLA-A2 positive target cells.