MODIFIED IPSCS

Abstract

The present invention provides a modified induced pluripotent stem cell iPSC or haemogenic lineage cell comprising at least one heterologous nucleic acid sequence encoding a heterologous T-cell receptor (TCR) integrated in the cell genome and uses thereof.

Claims

1. A modified induced pluripotent stem cell iPSC or haemogenic lineage cell comprising at least one heterologous nucleic acid sequence encoding a heterologous T-cell receptor (TCR) integrated at or into a locus in the cell genome.

2. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to claim 1, wherein the at least one heterologous nucleic acid sequence encoding a heterologous TCR is an expressible heterologous nucleic acid sequence.

3. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to either claim 1 or claim 2, wherein the modified induced pluripotent stem cell iPSC or haemogenic lineage cell expresses or presents the at least one heterologous TCR encoded by a heterologous TCR encoding nucleic acid sequence, preferably expressed or presented at the cell surface.

4. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any previous claim, wherein the modified haemogenic lineage cell is derived from the modified iPSC.

5. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any previous claim, wherein the haemogenic lineage cell is selected from any one of; (a) a mesoderm cell, optionally which may express any one or more of the mesodermal markers, Brachyury, Goosecoid, MixI1, KDR (also known as FLK1 or VEGFR2), FoxA2, GATA6 or PDGF alpha R, (b) a haemogenic endothelial cell, optionally which may be (a) CD34+ or (b) CD34+CD73 or (c) CD34+CD73CXCR4 (CD184), (c) a haematopoietic progenitor cell, optionally which may be (a) CD34+ or (b) CD34+CD45+ or (c) CD34+ and/or CD45+ in combination with any one or more of CD117+, CD133+, CD45+, FLK+, CD38, (d) CD34+, CD133+, CD45+, FLK1+, CD38 (d) a progenitor T cell, optionally which may be (a) CD5+ and/or CD7+, or (b) CD5+ and/or CD7+ in combination with any one or more of: CD44+, CD25+, CD2+, CD45+, CD3, CD4, CD8, (e) a double positive T cell (DP T cell), optionally which may be (a) CD4+CD8+(b) CD4+CD8+ in combination with any one or more of CD3+, CD28+, CD45+, (f) a single positive T cells (SP T cell) optionally which may be (a) CD4+(b) CD8+(b) CD4+ or CD8+ in combination with any one or more of CD3+, CD28+, CD45+, or (g) a mature T cell, optionally alpha beta T cell, gamma delta T cell, NKT cell, T helper cell (TH) which are CD4+, cytotoxic T cell (TC) which are CD8+.

6. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any previous claim, wherein RAG1 expression is reduced or eliminated.

7. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to claim 6, wherein the rag1 gene is inactivated or knocked-out.

8. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any previous claim, wherein the heterologous nucleic acid sequence encoding a heterologous TCR is integrated at or into one or both alleles of the locus in the cell genome.

9. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to claim 6, wherein the locus is a gene encoding an endogenous protein of the cell.

10. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to claim 7, wherein the heterologous nucleic acid sequence encoding a heterologous TCR is integrated adjacent to or within the gene encoding the endogenous protein

11. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to either of claim 9 or 10, wherein the heterologous nucleic acid sequence encoding a heterologous TCR is integrated within an intron or exon of the gene encoding the endogenous protein, optionally a 3 exon.

12. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to claim 9, wherein the heterologous nucleic acid sequence encoding a heterologous TCR is integrated within the 3 exon before the TAG stop sequence of the gene or nucleic add sequence encoding the endogenous protein.

13. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 7 to 10, wherein the integration of the heterologous nucleic acid sequence encoding a heterologous TCR is non-disruptive to the production of the endogenous protein.

14. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 9 to 13, comprising a fusion sequence between the nucleic acid encoding the heterologous TCR and the nucleic acid encoding the endogenous gene, preferably a fusion gene or sequence or multicistronic fusion gene or sequence between the nucleic acid encoding the heterologous TCR and the nucleic acid encoding the endogenous protein.

15. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to claim 14 wherein the nucleic acid encoding the heterologous TCR is connected to the nucleic acid encoding the endogenous protein by a nucleic acid sequence encoding a peptide comprising an enzymatic cleavage site and/or a nucleic acid sequence which mediates ribosome-skipping.

16. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to either of claim 14 or 15, wherein the nucleic acid encoding the heterologous TCR comprises a coding sequence of a TCR and TCR chain, optionally with an intervening nucleic acid sequence encoding a peptide comprising an enzymatic cleavage site and/or nucleic acid sequence which mediates ribosome-skipping.

17. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to either of claim 15 or 16, wherein the nucleic acid sequence which mediates ribosome-skipping is a T2A or P2A skip sequence.

18. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 15 to 17, wherein the nucleic acid sequence encoding a peptide comprising an enzymatic cleavage site encodes a furin cleavage site, preferably RAKR.

19. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 9 to 18, wherein the transcription and expression of the heterologous TCR and the endogenous protein is from the same promoter.

20. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 9 to 19, wherein the heterologous TCR is expressed as a fusion protein with the endogenous protein, optionally connected by a peptide comprising an enzymatic cleavage site, preferably a furin cleavage site, preferably RAKR and/or ribosome skip sequence, preferably T2A or P2A skip sequence.

21. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 1 to 20, wherein the heterologous TCR is expressed and/or presented in the cell as a nascent heterologous TCR comprising a TCR alpha chain and TCR beta chain, preferably expressed and/or presented at the cell surface.

22. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 9 to 21, wherein the endogenous protein is a protein that is trafficked to the cell surface, optionally via the secretory pathway and/or to the plasma membrane.

23. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 3 to 22, wherein the endogenous protein is a membrane protein or transmembrane protein, optionally a receptor protein, preferably a receptor protein tyrosine phosphatase (PTP), preferably CD45 or protein tyrosine phosphatase receptor type C or PTPRC.

24. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any previous claim, wherein the locus is or is in a gene encoding a membrane protein or transmembrane protein, optionally a receptor protein, preferably a receptor protein tyrosine phosphatase (PTP), preferably CD45 or protein tyrosine phosphatase receptor type C or PTPRC.

25. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any previous claim, wherein the locus is or is in the PTPRC (CD45) gene on chromosome 1.

26. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to claim 25, wherein the integration is at exon 33 of the PTPRC (CD45) gene, optionally before the TAG stop codon or immediately adjacent to and/or before the TAG stop codon.

27. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claim 14 to claim 26, wherein the fusion gene or sequence or multicistronic fusion gene or sequence comprises the nucleic acid encoding the heterologous TCR and nucleic acid encoding PTPRC with an intervening nucleic acid sequence encoding a peptide comprising an enzymatic cleavage site, preferably a furin cleavage site, and nucleic acid sequence which mediates ribosome-skipping, preferably selected from a T2A or P2A skip sequence and wherein the nucleic acid sequence encoding the heterologous TCR comprises the coding sequence of a TCR and TCR chain, with an intervening nucleic acid sequence encoding a peptide comprising an enzymatic cleavage site, preferably a furin cleavage site, and nucleic acid sequence which mediates ribosome-skipping, preferably selected from T2A or P2A skip sequence.

28. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any previous claim, wherein the heterologous TCR binds or specifically binds to an antigen or peptide antigen thereof selected from, (a) a cancer and/or tumour antigen or peptide antigen thereof, or (b) a cancer and/or tumour antigen or peptide antigen thereof associated with a cancerous condition and/or presented by tumour or cancer cell or tissue.

29. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to claim 28 wherein the cancer and/or tumour antigen or peptide antigen thereof is selected from; (a) a cancer-testis antigen, (b) a MAGE antigen, (c) MAGE A4 or peptide antigen thereof, optionally a peptide antigen comprising the sequence GVYDGREHTV (SEQ ID NO: 2), or (d) AFP or peptide antigen thereof, optionally a peptide antigen comprising the sequence FMNKFIYEI (SEQ ID No: 21).

30. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to either one of claim 28 or 29 wherein the cancer and/or tumour antigen or peptide antigen thereof is complexed with a peptide presenting molecule, optionally major histocompatibility complex (MHC) or human leukocyte antigen (HLA), optionally class I or class II, optionally HLA-A2 or HLA-A*02, or HLA-A*0201.

31. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any previous claim, wherein the heterologous TCR comprises a TCR having; (a) an alpha chain variable domain comprising an amino acid sequence that has at least 80%, identity to SEQ ID NO:9 or the sequence of amino acid residues 1-136 of SEQ ID NO:6, and/or the beta chain variable domain comprising an amino acid sequence that has at least 80%, identity to SEQ ID NO:10 or the sequence of amino acid residues 1-133 of SEQ ID NO:7, or (b) an alpha chain variable domain comprising an amino acid sequence that has at least 80%, identity to the sequence of amino acid residues 1-112 of SEQ ID NO:22, and/or the beta chain variable domain comprising an amino acid sequence that has at least 80%, identity to the sequence of amino acid residues 1-112 of SEQ ID NO:23.

32. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any previous claim, wherein the iPSC or haemogenic lineage cell further comprises a nucleic acid encoding and/or expresses or presents a heterologous co-receptor, optionally wherein the co-receptor is a CD8 co-receptor.

33. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to claim 32, wherein the heterologous CD8 co-receptor is heterodimer or homodimer, a CD8b heterodimer or a CD8 homodimer.

34. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to either one of claim 32 or 33, wherein the heterologous CD8 co-receptor comprises; (a) a CDR 1 of at least 80% sequence identity to amino acid sequence VLLSNPTSG, SEQ ID NO:17, CDR 2 of at least 80% sequence identity to amino acid sequence YLSQNKPK SEQ ID NO:18 and CDR 3 of at least 80% sequence identity amino acid sequence LSNSIM SEQ ID NO:19, or (b) an amino acid sequence having at least 80% sequence identity to amino acids number 22 to 235 of SEQ ID NO: 19.

35. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any previous claim, wherein the iPSC or haemogenic lineage cell further comprises a nucleic acid encoding and/or expresses or presents a heterologous co-stimulatory ligand; optionally 4-1BBL or CD80.

36. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 1 to 35, wherein the binding of the iPSC or haemogenic lineage cell and/or heterologous TCR to the cancer and/or tumour antigen or peptide antigen thereof or a cancer and/or tumour cell or tissue expressing or presenting the cancer and/or tumour antigen or peptide antigen thereof according to any of claims 28 to 30 induces activation of the iPSC or haemogenic lineage cell, optionally as determined by any one or more of; (a) up-regulation of T cell activation markers, for example either CD69 and/or CD25 on CD3+ cells, (b) up-regulation of cytokine production, for example any one or more of IFN gamma, IL-2 or Granzyme B, (c) induced cell cytotoxic activity in the presence of the antigen or antigen peptide, or (d) ability to kill tumour cells presenting the antigen or antigen peptide.

37. A nucleic acid construct or vector comprising a nucleic acid region encoding the heterologous TCR according to any previous claim and at least one homology region comprising a nucleic acid region homologous to a nucleic acid region at the locus in the cell genome for integration of the nucleic acid region encoding the heterologous TCR.

38. The nucleic acid construct or vector according to claim 37, wherein the locus is or is in a gene encoding a membrane protein or transmembrane protein, optionally a receptor protein, preferably a receptor protein tyrosine phosphatase (PTP), preferably CD45 or protein tyrosine phosphatase receptor type C or PTPRC.

39. The nucleic acid construct or vector according to either claim 37 or 38, wherein the locus is or is in the PTPRC (CD45) gene on chromosome 1.

40. The nucleic acid construct or vector according to any one of claims 37 to 39, wherein the locus is at exon 33 of the PTPRC (CD45) gene, optionally before the TAG stop codon or immediately adjacent to and/or before the TAG stop codon.

41. The nucleic acid construct or vector according to any one of claims 37 to 40, wherein the nucleic acid encoding the heterologous TCR comprises a coding sequence of a TCR and TCR chain, optionally with an intervening nucleic acid sequence encoding a peptide comprising an enzymatic cleavage site and/or nucleic acid sequence which mediates ribosome-skipping, preferably a furin cleavage site, preferably RAKR and/or ribosome skip sequence, preferably T2A or P2A skip sequence.

42. The nucleic acid construct or vector according to any one of claims 37 to 41, wherein the construct or vector comprises a left hand and a right hand homology region each homologous to a nucleic acid region at the locus in the cell genome for integration of the nucleic acid region encoding the heterologous TCR and which flank opposite sides of the integration site.

43. The nucleic acid construct or vector according to any one of claims 37 to 42, wherein the construct or vector further comprises any one or more of: (a) a recombination target sequence, preferably loxP (locus of X-over P1) sequence, (b) an expressible selection marker sequence, preferably an antibiotic resistance gene, and optionally a neomycin resistance gene, preferably constitutively expressed from a promoter for example from an EF1A promoter.

44. The nucleic acid construct or vector according to any one of claims 37 to 43, wherein the construct or vector comprises a nucleotide sequence encoding the heterologous TCR comprising the sequence SEQ ID No: 45, homology regions comprising the sequences SEQ ID No: 43 and SEQ ID No: 44, a recombination target sequence comprising SEQ ID No: 48 and an expressible selection marker sequence comprising SEQ ID No: 47 and SEQ ID No: 49.

45. A process of producing a modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 1 to 36 comprising introducing the nucleic acid construct or vector according to any one of claims 37 to 44 into an unmodified induced pluripotent stem cell iPSC or haemogenic lineage cell, optionally as defined in claim 5, under conditions to permit integration of the nucleic acid sequence encoding a heterologous T-cell receptor (TCR) at or into a locus in the cell genome and optionally isolating the modified induced pluripotent stem cell iPSC or haemogenic lineage cell.

46. A pharmaceutical composition comprising the modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 1 to 36 and a pharmaceutically acceptable carrier.

47. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 1 to 36, or pharmaceutical composition of claim 46, for use in therapy and/or medicine.

48. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 1 to 36, or pharmaceutical composition of claim 46, for use in treatment, prevention or delaying the progression of cancer and/or tumour in an individual or subject optionally wherein the treatment is cancer immunotherapy therapy and/or adoptive T cell therapy, optionally allogenic adoptive T cell therapy.

49. Use of the modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 1 to 36, or pharmaceutical composition of claim 46, in the manufacture of a medicament for the treatment of cancer and/or tumour in an individual or subject, optionally wherein the treatment is cancer immunotherapy therapy and/or adoptive T cell therapy, optionally allogenic adoptive T cell therapy.

50. A method of treating, preventing or delaying the progression of cancer and/or tumour in an individual or subject comprising administering to the individual the modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 1 to 34, or pharmaceutical composition of claim 44, optionally wherein the treatment is cancer immunotherapy therapy and/or adoptive T cell therapy, optionally allogenic adoptive T cell therapy.

51. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 1 to 36, or pharmaceutical composition of claim 46, for use according to any of claims 47 to 49 or in the method of claim 50, wherein the cancer and/or tumour is a solid tumour.

52. The modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 1 to 36, or pharmaceutical composition of claim 46, for use according to any of claims 47 to 49, or in the method of claim 50, wherein the modified induced pluripotent stem cell iPSC or haemogenic lineage cell, or pharmaceutical composition is for use or used in combination with one or more further therapeutic agent optionally administered or for administration separately, sequentially or simultaneously.

53. A kit comprising, (a) the modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 1 to 36, or pharmaceutical composition of claim 46, and a package insert comprising instructions for use thereof for treating, preventing or delaying the progression of cancer and/or tumour in an individual or subject, or (b) the modified induced pluripotent stem cell iPSC or haemogenic lineage cell according to any one of claims 1 to 36, or pharmaceutical composition of claim 46, and a package insert comprising instructions for use thereof for treating, preventing or delaying the progression of cancer and/or tumour in an individual or subject, in combination with one or more further therapeutic agent optionally administered or for administration separately, sequentially or simultaneously.

Description

FIGURES

[0237] FIG. 1PTPRC splice variants. The PTPRC gene is located on chromosome 1 and contains 33 exons. Multiple splice variants are produced from this gene via alternative splicing. The splice variants are a result of splicing at exons 4-6. Each transcript contains the same 3 exon structure. Figure adapted from Tchilian and Beverley 2006. Trends in Immunology.

[0238] FIG. 2ADP A2M4 knock-in strategyThe targeting strategy was deigned to knock ADP A2M4 SPEAR into PTPRC exon 33. Editing was performed with AAV. The homology arms in the rAAV repair template correspond the 500 bp of genomic sequence upstream and downstream of the stop codon. Editing creates a multicistronic fusion between PTPRC and ADP A2M4 with each polypeptide coding sequence separated by a furin cleavage site (RAKR) and 2A skip peptide (PTPRC_FuT2A_TCR_FuP2A_TCR). This design permits the expression of all 3 polypeptides from a single edited allele. Knock-in into Exon 33 also allows the expression of A2M4 across multiple PTPRC splice variants. The resistance cassette contains a LOXP flanked NEO resistance gene constitutively expressed from the EF1A promoter

[0239] FIG. 3Vector map pAAV MCS_PTPRC_FuT2A_A2M4_LOXP_NEO. The targeting cassette containing the homology arms (LHA and RHA), FuT2A_A2M4 TCR and NEO resistance cassette was cloned between the NotI restriction sites in the pAAV MCS vector (Agilent). With the exception of the vector backbone, each fragment was amplified by PCR and fragments cloned into the vector by Gibson assembly.

[0240] FIG. 4PCR screening strategy. Edited clones were isolated by limiting dilution in 96 well plates. A sample of gDNA was isolated from each clone using QuickExtract. Clones were screened by PCR with primers outside the homology arm regions and within the transgene cassette. The LHA boundary PCR was performed using primers PTPRC-5int-FWD and PTPRC-5int-REV. The RHA boundary PCR was performed with primers PTPRC-3int-FWD and PTPRC-3int-REV. PCR products were sequenced. Three clones were progressed for differentiation. ChiPSC31 PTPRC_A2M4 clone 2, ADAPiJ001_J PTPRC_A2M4 clone 3D10 and ADAPiJ001_J PTPRC_A2M4 clone 3G5.

[0241] FIG. 5Expression of ADP A2M4 in iT cells can be induced via lentiviral transduction of CD7+CD5+ progenitors. The iPSC line ADAPi001_J was differentiated and transduced with lentivirus express containing the ADP A2M4 T cell at Stage 4 (CD7+CD5+ progenitor). Transduced cells were progressed through the differentiation protocol. Cells were phenotyped by FACS and ADP A2M4 expression measured by staining with anti-V24 antibody and A2M4 MAGE-4 dextramer.

[0242] FIG. 6Phenotypes of iT cells differentiated from edited iPSC clones (Stage 5): (A) iT cells differentiated from (A) ChiPSC31 PTPRC_A2M4 clone 2 (single targeted allele), (B) iT cells differentiated from ADAPi001_J PTPRC_A2M4 clone 3D10 (both alleles targeted) and (C) iT cells differentiated from ADAPi001_J PTPRC_A2M4 clone 3G5 (single targeted allele). Differentiation is unaffected by editing. Edited clones express the ADP A2M4 SPEAR. Cells were phenotyped by FACS and A2M4 expression measured by staining with anti-V24 antibodies and ADP A2M4 MAGE-4 dextramer. Knock-in at a single allele is sufficient to promote ADP A2M4 expression.

[0243] FIG. 7iT cells differentiated from edited iPSC clones exhibit specific and potent cytolytic effector function: iT cell cytolytic function was assessed with the KILR assay. T2 cells were pulsed with decreasing concentrations (10.sup.5 M-10.sup.11 M) of the MAGE-A4 derived peptide GVYDGREHTV and incubated with iT cells differentiated from ADAPi001_J (ADAP001_J WT NTD (non-transduced)), ADAPi001_J derived, iT cells transduced with ADP A2M4 lentivirus (ADAPi001_J WT A2M4 TD) and iT cells differentiated from edited ADAPi001_J PTPRC_A2M4 clone 3G10. Cytolytic function was compared to ADP A2M4 transduced and non-transduced PBL isolated from healthy donors.

[0244] FIG. 8iT cells up-regulate the activation markers CD69 and CD25 when incubated with antigen positive tumour cells lines: iT cells differentiated from the ADAPi001_J iPSC line (NTD non-transduced), ADAPi001_J derived iT cells transduced with ADP A2M4 lentivirus (TD) and iT cells differentiated from edited ADAPi001_J PTPRC_A2M4 clone 3G10 were co-cultured (24 hrs) with the tumour lines A375 (HLA-A*02 positive and MAGE-A4 positive) and COLO205 (HLA-A*02 positive and MAGE-A4 negative) in the presence or absence of the MAGE-A4 peptide GVYDGREHTV (0.5 g). Following co-culture, up-regulation in expression of the T cell activation markers CD69 and CD25 was determined by flow cytometry. The expression of activation markers was compared to ADP A2M4 transduced and non-transduced PBL isolated from healthy donors.

[0245] FIG. 9Sequence validation of the INDELs present within the two RAG1 alleles (Exon2) in ADAPi001_J_7D10_RAG1.sup./. One allele (top) contains an 11 bp deletion and the second (bottom) contains a 16 bp deletion. Both INDELs are predicted to inactivate RAG1. Clone 7D10 ADAPi001_J_7 D10_RAG1.sup./ was used to knock-in the A2M4 TCR into PTPRC (Exon33)

[0246] FIG. 10RAG1.sup./ iT cells do not express CD3 and TCR. The expression of cell surface markers CD45, CD4, CD8, CD8, CD56, CD3, TCR chain V-24 (binds to A2M4-TCR) and TCR were measured by FACS. Phenotypes of differentiated iT-cells (A) Wild Type ADAPi001_J (B) A2M4 lentivirus transduced wild type ADAPi001_J (C) ADAPi001_J_7 D10_RAG1.sup./ (D) A2M4 lentivirus transduced ADAPi001_J_7 D10_RAG1.sup./.

[0247] FIG. 11Monoallelic knock-in of the A2M4 TCR into PTPRC rescues CD3 and TCR expression RAG1.sup./ iT-cells. A single copy of the A2M4 TCR was knocked into PTPRC (Exon33) in the RAG1.sup./ iPSC clone ADAPi001_J_7 D10. Three clones were isolated and differentiated into iT cells The expression of cell surface markers CD45, CD4, CD8, CD8, CD56, CD3, TCR chain V-24 (binds to A2M4-TCR) and TCR were measured by FACS. (A) ADAPi001_J_1B8_RAG1.sup./_PTPRC.sup.A2M4/WT (B) ADAPi001_J_2D4_RAG1.sup./_PTPRC.sup.A2M4/WT (C) ADAPi001_J_2E7_RAG1.sup./_ PTPRC.sup.A2M4/WT.

[0248] FIG. 12iT-cells differentiated from a RAG1.sup./ iPSC clone can kill antigen positive tumour cell lines following transduction with a lentivirus encoding the A2M4 TCR. MAGE-A4 peptide (GVYDGREHTV) peptide and non-pulsed GFP.sup.+ve A375 (MAGE-A4.sup.+ve, HLA-A2*02.sup.+ve) cells were co-cultured with non-transduced or A2M4 transduced iT-cells differentiated from wild type ADPAi001_J or ADPAi001_J_c7D10_RAG1.sup./. A decrease in Green object counts (GFP) represents death of GFP.sup.+ve A375 tumour cell lines.

[0249] FIG. 13iT-cells differentiated from RAG1.sup./ PTPRC.sup.A2M4/WT iPSC clones can kill antigen positive tumour cell. MAGE-A4 peptide (GVYDGREHTV) pulsed and non-pulsed GFP.sup.+ve A375 (MAGE-A4.sup.+ve, HLA-A2*02.sup.+ve) cells were co-cultured iT-cells differentiated from the iPSC clones ADAPi001_J_c1B8_RAG1.sup./_ PTPRC.sup.A2M4/WT, ADPAi001_J_c2D7_RAG PTPRC.sup.A2M4/WT or ADPAi001_J_c2E4_RAG1.sup./ PTPRC.sup.A2M4/WT. A decrease in Green object counts (GFP) represents death of GFP.sup.+ve A375 tumour cell lines.

EXAMPLES

Introduction

[0250] We describe here the generation of iT-cells expressing a defined heterologous recombinant TCR. In a first set of experiments, starting with a GMP compliant hiPSC source, we knocked-in the recombinant heterologous T-cell receptor ADP A2M4 that recognises the MAGE-A4 peptide (GVYDGREHTV) presented by HLA-A*02. We show that ADP A2M4 iT-cells derived from engineered iPSCs specifically express the ADP A2M4 TCR as measured by anti-TCRV24 and dextramer staining. Edited ADP A2M4 iT-cells up regulate activation markers including CD25 and CD69 when incubated with HLA-A*02 expressing tumour lines that express the cognate antigen and exhibit potent antigen dependent killing of these lines. In a second set of experiments, we knocked-in the ADP A2M4 TCR into a RAG1 null iPSC line ADAPi001_J. The following examples represents the development of an allogeneic hiPSC derived platform, with limited genome editing, that permits the production of ADP A2M4 TCR iT-cells of therapeutic value and activity.

[0251] ADP A2M4 TCR is stably introduced into the iT-cells by knock-in at the PTPRC locus. PTPRC encodes a transmembrane protein tyrosine phosphatase (CD45) that is an important regulator of TCR signalling. The PTPRC gene contains 33 exons and encodes multiple isoforms (CD45RABC, CD45R0, CD45RAB, CD45RAC, CD45RBC, CD45RA, CD45RB and CD45RC) which are generated by alternative splicing of exons 4-6 (FIG. 1). The expression of CD45 isoforms is altered during T cell development, activation and differentiation. For example, CD45RA is primarily expressed on nave T cell subsets whereas CD45RO is primarily expressed on memory T cell subsets.

[0252] The PTPRC isoforms all share the same 3 exon structure and thus modification of this region will be shared between all of the PTRPC isoforms. An rAAV targeting plasmid was generated to enable the knock-in of the ADP A2M4 TCR into PTPRC exon 33 immediately before the TAG stop codon. The construct was deigned to generate a multicistronic fusion gene between PTPRC and ADP A2M4 SPEAR. This permits the regulated transcription and expression of PTPRC isoforms and ADP A2M4 from the endogenous PTPRC promoter. A furin cleavage site combined with T2A or P2A skip sequence peptide separates the PTPRC and ADP A2M4 TCR and A2M4 TCR coding sequences (FIGS. 2 and 3). This allows the production of multiple polypeptides from a single mRNA. Cleavage by the furin protease in the endoplasmic reticulum will remove the skip sequence peptides allowing the expression of nascent protein sequences.

Production of iPSC Cell Lines

[0253] The iPSC lines (ADAPi001) were created via re-programming of CD34+ progenitors isolated from umbilical cord blood using the pEB-C5 and pEB-TG episomal plasmids [Chou, B. K., et al., Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures. Cell Res, 2011. 21(3): p. 518-29]. All ADAPi001 clones are traceable and produced under GMP conditions. Nine ADAPi001 hiPSC clones were characterised with a small working cell bank of early passage number (p10) produced for each clone. Genomic stability was assessed via cyto-SNP analysis, karyotyping and WGS (whole genome sequencing). Pluripotency was determined with IHC (immunohistochemistry), flow cytometry and Pluritest [Muller, F. J., et al., A bioinformatic assay for pluripotency in human cells. Nat Methods, 2011. 8(4): p. 315-7]. Differentiation of iT-cells from ADAPi001 iPSC clones was also confirmed.

iPSC Cell Culture

[0254] The iPSC lines ChiPSC31 (Takara) and ADAPi001_J were used for editing experiments. The ChiPSC31 iPSC line was cultured on the COATI matrix with DEF-CS 500 culture media (Takara). The ADAPi001_J iPSC line was cultured on the Synthemax II-SC Substrate (Corning) with mTeSR Plus culture media (STEMCELL Technologies). Both iPSC lines were adapted for enzymatic dissociation with TrypLE Select (ThermoFisher) and passaged every 4-5 days. iPSC cultures were maintained in a humidified 37 C., 5% O.sub.2, 5% CO.sub.2 incubator. The expression of pluripotency markers (POU5F1, NANOG, TRA-160 and SOX2) and absence of the differentiation marker SSEA-1 was routinely monitored by FACS analysis.

Generation of the AAV Targeting Vector.

[0255] The pAAV-MCS (Agilent) plasmid was digested with NotI (NEB) to remove the sequences between the left and right inverted terminal repeat (ITR) regions. The vector backbone was purified by agarose gel extraction. The left and right homology arms (LHA, SEQ ID No: 43 and RHA, SEQ ID No: 44), FuTA_A2M4 transgene cassette, SEQ ID No: 45, and LOPX, SEQ ID No: 48, flanked EF1A_Neo selection cassette SEQ ID No: 47 and SEQ ID No: 49 were amplified by PCR. Each primer contained overlapping sequence to allow generation of the targeting vector by Gibson assembly. The left and right homology arm sequences (500 bp) were amplified from genomic DNA (gDNA) isolated from the iPSC line ADAPi001_J. The LHA arm corresponds to nucleotides 198755679 to 198756178 on and RHA corresponds to nucleotides 198756182 to 198756681 Chromosome 1 (Genome build GRCh38 Ensembl release 99January 2020). The ADP A2M4 TCR transgene contains the coding sequence of the TCR and TCR chains with a furin cleavage site (RAKR) and P2A skip sequence between two TCR chains. The LOXP flanked selection cassette was synthetically synthesised (GeneArt). The pAAV_MCS_PTPRC_FuT2A_A2M4_LOXPNEO plasmid map is shown in FIG. 3.

AAV Production

[0256] The packaging plasmids pHelper (Agilent 240071) and AAV-3 Rep-Cap Plasmid (Cell Biolabs VPK-423) used for rAAV production were grown in NEB 5-alpha Competent E. coli. The AAV targeting plasmid containing the homology arms, FuT2A_A2M4 and NEO selection cassette were grown in NEB Stable Competent E. coli. All plasmid DNA was purified using Plasmid Plus Giga Kit (Qiagen) or EndoFree Plasmid Giga Kit (Qiagen). The ITR integrity in the AAV transgene plasmid was confirmed with AhdI and BglI single restriction enzyme digest.

[0257] The 293AAV Cell Line (Cell Biolabs) used to produce rAAV was cultured with 10% (v/v) Heat Inactivated FBS (Life Technologies), DMEM, high glucose, GlutaMAX Supplement, pyruvate (Life technologies) Pen/Strep (Life Technologies) and Non Essential Amino Acids (Life Technologies). 293AAV were seeded in a 5-cell stack (Corning) at a density of 21610.sup.6/5CS 24 hrs before transfection. 293AAV were transfected with the rAAV transgene plasmid, pHelper and AAV-3 Rep-Cap at a molar ratio of 2:1:1 using Turbofect transfection reagent (Thermofisher). A total 2.6 mg plasmid DNA was used for each transfection. Cells were cultured for 72 hrs before harvesting and virus purification. To harvest cells, EDTA (Thermofisher) was added to a final concentration of 6.25 mM to promote cell detachment from the tissue culture plastic. rAAV virus was purified via iodixanol gradient ultracentrifugation or the AAVpro Purification Kit (Takara) according to the manufacturers instructions.

[0258] For iodixanol gradient purification cell pellets were collected by centrifugation (300 g, 5 min), resuspended in 5 ml cell lysis buffer (150 mM NaCl, 5 mM Tris-HCl pH8.5) and subjected to three rounds of freeze thaw using a dry-ice/ethanol bath and a 37 C. water bath. Following the final thaw, the lysate was treated with Benzonase (200 U/ml) (Sigma) for 1 hr at 37 C. Remaining debris was removed by centrifugation (1000 g, 29 min, 4 C.) before loading onto the iodixanol gradient. The iodixanol gradient (9 ml 15% iodixanol, 6 ml 25% iodixanol, 5 ml 40% iodixanol and 5 ml, 60% iodixanol) was prepared by diluting 60% iodixanol (OptiPrep), (STEMCELL Technologies). The 25% and 40% iodixanol phases were prepared by diluting 60% iodixanol in PBS-MK buffer (1PBS, 2.76 mM MgCl2, 2 mM KCl). The 15% iodixanol phase was prepared by diluting 60% iodixanol in PBS-MK buffer (1PBS 1 M NaCl, 2.76 mM MgCl.sub.2, 2 mM KCl). Phenol red was added to the 25% and 60% phases into order to distinguish these layers. Gradient phases are added step wise into OptiSeal Polypropylene Centrifuge tubes (Beckman Coulter). The cell lysate is loaded on top of the 15% phase and tubes centrifuged in a 70Ti ultracentrifuge rotor at 67,000 rpm for 90 min at 18 C. rAAV is extracted from the 40% phase following centrifugation. rAAV virus preps were concentrated further using centrifugal filter units (MWCO 100 kDa) (Millipore) and the buffer was exchanged for PBS 0.001% (v/v) Pluronic Acid (Sigma). Virus preps were stored at 80 C.

[0259] Viral titres were confirmed by qPCR using the QuantiTect Probe PCR kit (Qiagen). Primer and probe sequences are designed against the ITR (Aurnhamer et al., 2012. Human Gene Ther Methods). Primer sequences ITR_F GGAACCCCTAGTGATGGAGTT, SEQ ID NO: 54, ITR_R CGGCCTCAGTGAGCGA SEQ ID NO: 55, Probe 56-FAM/CACTCCCTC/ZEN/TCTGCGCGCTCG/3IABkFQ. Viral quantification was determined against a standard curve prepared from pAAVS derived plasmid DNA of known concentration and copy number. The PCR program consisted of one cycle of denaturation at C. for 15 min followed by 40 cycles of denaturation at 95 C. for 15 s and primer annealing/extension at 60 C. for 60 s. qPCR reactions were performed on the QuantStudio 7 and analysed with the QuantStudio Real Time PCR software.

Gene EditingKnock-In of A2M4 SPEAR into Exon 33 at the PTPRC Locus iPSC lines CHiPSC31 and ADAPi001-J were passaged enzymatically using TrypLE Select (ThermoFisher) as a single cell suspension. Following dissociation, iPSCs were counted (NucleoCounter NC-250). CHiPSC31 (200-30010.sup.3) were seeded in a well of a coated (Coati) 6-well plate containing complete DEF-CS 500 supplemented with GF3. Media was exchanged for complete DEF-CS 500 the following day. ADAPi001-J (2-310.sup.5) were seeded into a well of a Synthemax (Corning) coated 6 well plate containing mTeSR Plus (STEMCELL Technologies) supplemented with 1 CloneR (STEMCELL Technologies), Media was exchanged for mTeSR Plus the following day. iPSC s (CHiPSC31 or ADAPi001-J) were cultured for 48 hours prior to AAV transduction. CHiPSC31 or ADAPi001-J were transduced with AAV3 serotyped virus (500-10000 Vg/cell). Antibiotic selection (Geneticin, 30-100 g/ml) was commenced 48 hrs post transduction and was continued throughout the remaining culture. 3-6 days after viral transduction, iPSC s were seeded into coated 96 well plates for screening and clonal isolation. Clones were expanded in 96 well plates for 10-16 days before PCR screening. In some instances, positive clones were subjected to additional clonal isolation by limiting dilution. All clones were screed by PCR in order to confirm integration. All PCR products were sequence verified.

Clone Isolation and Screening

[0260] iPSC clones were isolated following selection of the edited iPSC cell pools with G418. Edited ChiPSC31 (3000 cells) were seeded into 10 cm dishes with complete DEF-CS 500 supplemented with GF3 and G418 (50 g/ml). Media was exchanged every 48 hrs for complete DEF-CS 500 supplemented with G418 (50 g/ml). Clones were manually picked following 11 days of expansion. A sample was taken for genomic DNA extraction using QuickExtract (Lucigen). Clones were screened for transgene integration by PCR (FIG. 4).

[0261] Edited clones from the ADAPi001_J line were isolated by limiting dilution in 96 well plates coated with Synthemax. Cells were seeded at an average of 100 cells/well. 9600 iPSC were seeded in well A1 before 2 serial dilution in column 1 (A1-H1). Cells were then diluted 2 across all rows of the plate. Edited ADAPiJ001_J were seed into mTeSR Plus supplemented with 1 CloneR. Media was exchanged every 2 days for complete mTeSR Plus. Cells were cultured for 12-14 days before screening wells for the presence of single colonies. Clones were expanded and genomic DNA isolated using QuickExtract (Lucigen) for screening by PCR in order to confirm targeted transgene insertion.

[0262] PCR reactions were performed across homology arm boundaries. The 5 LHA boundary was amplified with the primers PTPRC-5int-FWD CAGTGATTCCTGCCCTGATTCTTA (SEQ ID No: 50) and PTPRC-5int-REV TACAGCCACAGGATCACGAGAAAG (SEQ ID No: 51). The primer PTPRC-5int-FWD corresponds to nucleotides 198755562 to 198755585 (+) on chromosome 1 (Genome build GRCh38 Ensembl release 99January 2020) and lies 93 nucleotides upstream of the LHA sequence. The PTPRC-5int-REV primer is in the A2M4 transgene sequence. The 3 RHA boundary was amplified with the primers PTPRC-3int-FWD CCTGCCGAGAAAGTATCCATCAT (SEQ ID No: 52) and PTPRC-3int-REV TAGCATACACACACATACCACCTT (SEQ ID No: 53). The primer PTPRC-3int-FWD1 is in the NEO cassette and PTPRC-3int-REV1 corresponds to nucleotides 198756729 to 198756752 () on chromosome 1 and lies 47 nucleotides downstream from the RHA. PCR reactions were performed with Q5 Hot Start High-Fidelity 2 Master Mix (NEB). PCR program denaturation 98 C., 30 s, 1 cycle, 40 cycles denaturation 98 C., 5s, annealing 66 C., 10s, Extension 72 C., 25 s, 1 cycle extension 72 C., 5 min. PCR products were gel purified and sanger sequenced to confirm integration. Positive clones were expanded, and small cell banks produced. Maintenance of pluripotency marker expression was confirmed by flow cytometry. Three clones were progressed for differentiation. ChiPSC31 PTPRC_A2M4 clone 2, ADAPiJ001_J PTPRC_A2M4 clone 3D10 and ADAPiJ001_J PTPRC_A2M4 clone 3G5. ChiPSC31 PTPRC_A2M4 clone 2 and ADAPiJ001_J PTPRC_A2M4 clone 3G5 had targeted knock-in of ADP A2M4 at one allele whereas ADAPiJ001_J PTPRC_A2M4 clone 3D10 had ADP A2M4 knock-in at both alleles.

T Cell Differentiation

[0263] HiPSC maintenance medium was removed, the cells washed twice with DMEM/F12 (Invitrogen). 2 mL of StemPro34 PLUS (StemPro34 from Invitrogen; StemPro34 basal media, with supplement added and Penicillin Streptomycin (1% v/v: Invitrogen) and Glutamine (2 mM: Invitrogen), Ascorbic Acid (50 mg/ml: Sigma Aldrich) and monothioglycerol (100 mM: Sigma Aldrich), further supplemented with 50 ng/mL of Activin A (Miltenyi Biotec) was added and incubated for 4 hours. Volumes are dependent of culture flask size, typically at least 2 mls/9 cm.sup.2, and 20 mls/150 cm.sup.2.

[0264] After 4 hours, the medium was removed, and the cells washed twice with DMEM/F12 to remove residual high concentration Activin A. The medium was replaced with 2 mL of StemPro34 PLUS supplemented with 5 ng/mL of Activin A, 10 ng/ml of BMP4 (Miltenyi Biotec) and 5 ng/ml of bFGF (Miltenyi Biotec) and incubated for 44 hours (Stage 1 media). The medium was then replaced with fresh Stage 1 media and supplemented with 10 M CHIR-99021 (Selleckchem) and further cultured for 48 hours.

[0265] On Day 4, the medium was removed, and the cells washed twice with DMEM/F12 to remove residual stage 1 cytokines. The medium was then replaced with StemPro34 PLUS supplemented with 100 ng/mL of SCF (Miltenyi Biotec) and 15 ng/ml of VEGF (Miltenyi Biotec) and incubated for 48 hours (Stage 2 media). The medium was then replenished with fresh Stage 2 media and the cells cultured for a further 48 hours.

[0266] The medium was then replaced by the Stage 3 medium which requires the minimum of SCF and VEGF and the cells cultured for between 16-18 days, with demi depletion feeding every 48h. Typically this involved harvest of media and collection of cells in suspension by centrifugation (300 g, 10 min), and returning suspension cells to culture with fresh media (i.e 20 ml for a T150 flask).

[0267] On approximately day 16 CD34+ cells were isolated from resulting monolayers for onward culture. CD34+ cells were harvested by sequential incubation with Accutase (STEMCELL Technologies) for 30 mins at 37 C. and then Collagense II (Invitrogen: 2 mg/ml) for 30 mins at 37 C. Cell suspensions were collected and washed (2 centrifugation at 300 g for 12 min in DMEM/F12), prior to CD34+ cell isolation via Magnetic activated beads (MACS) isolation (Miltenyi: according to manufacturer's instructions).

[0268] For continued lymphoid proliferation and differentiation, STEMCELL Technologies proprietary 2 stage (Lymphoid Proliferation/T cell Maturation) media was employed.

[0269] During culture in STEMCELL Technologies Lymphoid Proliferation media T-cell progenitors non-edited control lines were lentivirally transduced with the ADP A2M4 TCR in the presence of poloxymer F108 (Sigma).

T Cell Phenotyping

[0270] iPSC derived T Cells were phenotyped using flow cytometry. The cells were stained with CD3 (clone SK7); A2M4 dextramer and live/dead stain EF506 to show expression of ADP A2M4 TCR in iPSC derived T Cells.

[0271] FIG. 5 shows phenotyping data for transduced clones. Expression of ADP A2M4 in iT cells can be induced via lentiviral transduction of CD7+CD5+ progenitors as described above. The iPSC line ADAPi001_J was differentiated and transduced with lentivirus express containing the ADP A2M4 T cell at Stage 4 (CD7+CD5+ progenitor) and the transduced cells were progressed through the differentiation protocol. Cells were phenotyped by FACS and ADP A2M4 expression measured by staining with anti-TCR antibody anti-V24 and MAGE-A4 GVYDGREHTV/HLA-A*0201 dextramer.

[0272] FIG. 6 shows phenotyping data for iT cells differentiated from edited iPSC clones (Stage Panel (A) shows iT cells differentiated from an alternative cell line ChiPSC31 PTPRC_A2M4 clone 2 (single targeted allele), panel (B) iT cells differentiated from ADAPi001_J PTPRC_A2M4 clone 3D10 (both alleles targeted) and panel (C) iT cells differentiated from ADAPi001_J PTPRC_A2M4 clone 3G5 (single targeted allele). Differentiation is unaffected by editing. iT-cells were phenotyped by flow cytometry and expression of CD45, CD7, CD5, CD4, CD8, CD56 and CD3 were detected. Edited clones express the ADP A2M4 TCR. Cells were phenotyped by FACS and A2M4 expression measured by staining with anti-TCR antibody anti-V24 and MAGE-A4 GVYDGREHTV/HLA-A*0201 dextramer. Knock-in at a single allele is sufficient to promote ADP A2M4 TCR expression, where both alleles are targeted the expression is improved.

Flow Cytometry Staining

[0273] The following antibodies were used for flow cytometry: CD8-BV650 (clone 2ST8.5H7), TCR-APC (done B1), TCR V24-PE (Clone IP26), CD3-APC-R700 (clone UCHT1), CD4-BV605 (clone RPA-T4), CD8-PE-CY7 (clone RPA-T8), CD69-BUV395 (clone FN50), CD25-BV421 (Clone BC96), HLA-DR-AF488 (clone L243), and EF506-BV510. Samples were acquired on the BD Fortessa.

KILR Cytotoxicity Assay

[0274] 10,000 KILR T2s (generated by transduction with KILR retroparticles (DiscoverX)) were added per well of a 384 well white LUMITRAC 600 microplate (Greiner). T cells derived from iPSC lines at the end of stage 6 were added at 20,000 cells per well. MAGE-A4 peptide (GVYDGREHTV) was added to cells in a titration from 10.sup.5 M to 10.sup.11 M. Cells and peptide were co-cultured for 24 hours at 37 C. under normoxia before addition of KILR detection solution (KILR Detection Kit (DiscoverX)) for 1 hr at room temperature. Luminescence from samples was detected using the FLUOstar Omega Microplate Reader. The cytolytic activity of iT cells was compared to non-transduced and ADP A2M4 lentivirus transduced PBL controls from healthy donors. FIG. 7 shows data for iT cells differentiated from ADAPi001_J (ADAP001_J WT NTD (non-transduced)), ADAPi001_J derived, iT cells transduced with ADP A2M4 lentivirus (ADAPi001_J WT A2M4 TD) and iT cells differentiated from edited ADAPi001_J PTPRC_A2M4 clone 3G10. Increased signal indicates increased cell death and the data demonstrate potent cytolytic effector function of the knock-in edited ADAPi001_J PTPRC_A2M4 clone 3G10.

[0275] Edited ADAPi001-J ADP A2M4 3D10 iT-cells, ADP A2M4 transduced and non-transduced ADAPi001-J (WT) derived iT-cells were also co-cultured with A375 (MAGE-A4+HLA-A*02+) or COLO205 (MAGE-A4 HLA-A*02+) in the presence or absence of MAGE-A4 peptide (GVYDGREHTV) for 24 hrs. Cytokines. (IFN and IL-2) and Granzyme B secretion was measured by ELISA. The edited ADP A2M4 iT-cells were seen to release high levels of each cytokine in an antigen dependent manner.

Antigen-Specific Activation Assay

[0276] A375 (HLA-A*02 positive and MAGE-A4 positive) and Colo205 (HLA-A*02 positive and MAGE-A4 antigen negative) tumour cell lines were used as target cells. 200,000 targets were co-cultured in a 96 well round bottom plate, with either 110{circumflex over ()}6 iPSC derived T cells, or PBL controls (rested over 2 hours). Target cell lines and T cells were then cultured with or without 0.5 g of ADP-A2A4 peptide (GVYDGREHTV). Additionally, iPSC derived T cells were cultured without peptide and target cells. All cells were then incubated at 37 C. for 24 hours prior to cell staining. Following co-culture, up-regulation in expression of the T cell activation markers CD69 and CD25 was determined by flow cytometry. iT cells were compared to non-transduced and ADP A2M4 lentivirus transduced PBL controls (FIG. 8). The edited ADP A2M4 iT-cells were seen to up-regulate the activation markers CD69 and CD25 when incubated with antigen positive tumour cells lines.

RAG1 Null Cell Knock-In

[0277] iPSC Cell Culture

[0278] Sequential RAG1 editing and knock-in of the A2MR TCR into the PTPRC locus were performed the iPSC line ADAPi001_J. The ADAPi001_J iPSC line was cultured on the Synthemax II-SC Substrate (Corning) with mTeSR Plus culture media (STEMCELL Technologies). Both iPSC lines were adapted for enzymatic dissociation with TrypLE Select (ThermoFisher) and passaged every 4-5 days. iPSC cultures were maintained in a humidified 37 C., 5% 02, 5% CO.sub.2 incubator. The expression of pluripotency markers (POU5F1, NANOG, TRA-160 and SOX2) and absence of the differentiation marker SSEA-1 was routinely monitored by FACS analysis. Editing experiments were performed sequentially. Clone 7D10 with RAG1 inactivation was derived from wild type ADAPi001_J. Clones 167, 2D7 and 2E7 (RAG1.sup./ PTPRC.sup.A2M4/WT) were generated via knock-in of the A2M4 TCR into PTPRC Exon 33 into the RAG1.sup./ clone 7D10.

Guide RNA Sequences

[0279] RAG1 Exon 2 was targeted with the guide RNA TCTTTTCAAAGGATCTCACC. This sequence corresponds to nucleotides 36,573,405-36,573,425, chromosome 11 (Human GRCh38 Ensembl release 103February 2021). Exon 33 PTPRC was targeted with the guide RNA GCAAGTCCAGCTTTAAATCA (nucleotides 198756151-198756171, Chromosome 1. Human GRCh38, Human GRCh38 Ensembl release 103February 2021). Guide RNA sequences were synthesised by IDT.

Preparation of Ribonucleoprotein (RNP) Complexes

[0280] crRNA and tracrRNA were annealed by initial denaturation 95 C. for 5 min before cooling to room temperature. Equimolar quantities of annealed crRNA/tracrRNA duplexes and Cas9 protein were incubated at room temperature for 15 min to generate 10 M Ribonucleoprotein (RNP) complexes.

Inactivation of RAG1

[0281] RNP complexes were introduced into iPSC cells via nucleofection with the 4D-Nucleofector using the 16-well Nucleocuvette strips (Lonza). 10010.sup.3 ADAPi-001_J (Wildtype) were resuspended in buffer P3 (Lonza) (510.sup.6/ml). 3 l of RNP complex (10 M) was added to 20 l cell suspension. Nucelofection was performed with program CA-137. Following nucleofection cells were immediately seeded into mTeSR Plus culture media (STEMCELL Technologies) supplemented with 1 CloneR (STEMCELL Technologies). Edited cell pools were expanded for two additional passages before seeding for clonal isolation. Cells were seeded at an average of 100 cells/well. 9600 iPSC were seeded in well A1 before 2 serial dilution in column 1 (A1-H1). Cells were then diluted 2 across all rows of the plate. Edited ADAPiJ001_J were seed into mTeSR Plus supplemented with 1 CloneR. Media was exchanged every 2 days for complete mTeSR Plus. Cells were cultured for 12-14 days before screening wells for the presence of single colonies. Clones were expanded and genomic DNA isolated using QuickExtract (Lucigen) for screening by PCR in order to sequence the INDELS in RAG1 (Exon2). PCR reactions (NEB Q5 HotStart) were performed using FWD primer CTTGGGACTCAGTTCTGCCC (Chromosome 11 nucleotides 36573327-36573346 (+) Human GRCh38 Ensembl release 103February 2021) and REV primer GAACTCAGTGGGGTGGATCG. Chromosome 11 nucleotides 36573809-36573829 () Human GRCh38 Ensembl release 103February 2021). PCR reactions were performed with 05 Hot Start High-Fidelity 2 Master Mix (NEB). PCR program denaturation 98 C., 30 s, 1 cycle, 40 cycles denaturation 98 C., 5s, annealing 69 C., 10s, Extension 72 C., 30 s, 1 cycle extension 72 C., 2 min. PCR products from each clone were cloned into TOPO Blunt (Thermofisher) and plasmid clones were sanger sequenced to characterise the INDELS present in each edited iPSC clone. This analysis revealed that clone ADAPi001_J_c7D10_RAG1.sup./ had a 11 bp deletion in 1 allele and a 16 bp deletion in the second allele. Both mutations are predicted to introduce inactivating frameshift mutations into RAG1 (FIG. 1).

Knock-In of A2M4 TCR into PTPRC (Exon 33)ADAPi001_J_c7D10_RAG1.sup./

[0282] Knock-in of A2M4 into the PTPRC locus (Exon 33) was performed using CRISPR Cas9 and rAAV supplied repair templates. The editing experiment was performed with the gRNA sequence gcaagtccagctttaaatca (nucleotides 198756152 to 198756171 on Chromosome 1. Human GRCh38, Ensembl release 103February 2021). crRNA was synthesised by Integrated DNA Technologies.

[0283] 110.sup.5 iPSC were seeded in a well of a twelve well plate 24 hrs before transfection. Cells were seeded in complete mTeSR-Plus (STEMCELL Technologies) supplemented with 1 ClonerR (STEMCELL Technologies). The transfection mix was prepared according to manufacturer's instructions. 500 ng of annealed crRNA/tracrRNA duplexes and 2.5 g Cas9 protein were added to 50 l Optimem (Thermofisher) and 5 l Cas9 and incubated at room temperature for 15 min to generate Ribonucleoprotein (RNP) complexes. 3 l Lipofectamine CRISPR-MAX (Thermofisher) in 50 l Optimem was added following RNP complex formation and then the complete transfection mix added to cells. 4 hrs post transfection iPSC were transduced with rAAV (Serotype3) encoding PTPRC_FuT2A_A2M4_LOXPNEO (13 Vg/cell). Media was exchanged for complete mTeSR-Plus (STEMCELL Technologies) supplemented with 100 g/ml G418 (Thermofisher) 24 hrs post rAAV transduction. Edited cell pools were expanded for 1 passage before clonal isolation with limiting dilution in 96 well plates coated with Synthemax. 1.510.sup.4 iPSC were seeded in well A1 before 2 serial dilution in column 1 (A1-H1). Cells were then diluted 2 across all rows of the plate. Edited iPSC were seeded into mTeSR Plus supplemented with 1 CloneR. Media was exchanged every 2 days for complete mTeSR Plus supplemented with 100 g/ml G418 (Thermofisher). Cells were cultured for 12-14 days before screening wells for the presence of single colonies. Clones were expanded and genomic DNA isolated using QuickExtract (Lucigen) for screening by PCR in order to confirm targeted transgene insertion.

[0284] PCR reactions were performed across homology arm boundaries. The 5 LHA boundary was amplified with the primers PTPRC-5int-FWD CAGTGATTCCTGCCCTGATTCTTA and PTPRC-5int-REV TACAGCCACAGGATCACGAGAAAG. The primer PTPRC-5int-FWD corresponds to nucleotides 198755562 to 198755585 (+) on chromosome 1 (Human GRCh38, Ensembl release 103February 2021) and lies 93 nucleotides upstream of the LHA sequence. The PTPRC-5int-REV primer is in the A2M4 transgene sequence. The 3 RHA boundary was amplified with the primers PTPRC-3int-FWD CCTGCCGAGAAAGTATCCATCAT and PTPRC-3int-REV TAGCATACACACACATACCACCTT. The primer PTPRC-3int-FWD1 is in the NEO cassette and PTPRC-3int-REV1 corresponds to nucleotides 198756729 to 198756752 () on chromosome 1 and lies 47 nucleotides downstream from the RHA. (Human GRCh38, Ensembl release 103February 2021). PCR reactions were performed with Q5 Hot Start High-Fidelity 2 Master Mix (NEB). PCR program denaturation 98 C., 30 s, 1 cycle, 40 cycles denaturation 98 C., 5s, annealing 66 C., 10s, Extension 72 C., 25 s, 1 cycle extension 72 C., 5 min. PCR products were gel purified and sanger sequenced to confirm integration. Positive clones were expanded, and small cell banks produced. Maintenance of pluripotency marker expression was confirmed by flow cytometry

IncuCyte Cytotoxicity Assay

[0285] GFP.sup.+ve A375 (MAGE-A4.sup.+ve, HLA-A2*02.sup.+ve) targets cells were seeded in 384 well plates (Greiner Bio-One) at a density of 1500/well in 200 of complete RPMI 1640 (RPMI 1640, 10% (v/v) FCS, 100 U/ml penicillin 100 g/ml streptomycin, 2 mM L-Glutamine). Plates were added to the Incucyte Zoom, and incubated at 37 C., with 5% CO.sub.2 in a humidified incubator.

[0286] 24 hours later, targets were pulsed with MAGE-A4 peptide (GVYDGREHTV) at a final concentration of 10 M. 6000 iT cells were added in triplicate in 10 l media. Images were taken every 2 hours, for six days in the Incucyte. Data was then analysed using ZOOM2018A software using Top-Hat process definitions.

CONCLUSION

[0287] We have presented a novel editing strategy that has enabled the differentiation of ADP A2M4 TCR expressing iT-cells. Edited ADP A2M4 TCR iT-cell activation and cytolytic effector function has been shown to be antigen dependent. The phenotype of edited ADP A2M4 iT-cells including CD56 and CD8 expression, potent cytokine and cytotoxic activity suggests that edited ADP A2M4 iT-cells possess an innate and adaptive like phenotype.

[0288] Although dextramer staining suggests that edited ADP A2M4 iT-cells only express the ADP-A2M4 TCR the technology is applicable to a full TCR repertoire. The edited A2M4 TCR iT-cells exhibit potent cytolytic and effector function, which is comparable or increased compared to A2M4 TCR transduced PBL from healthy donors. This suggests that, like autologous A2M4 TCR T-cells, ADP-A2M4 iT-cells will be effective in cell therapy and bring clinical benefit to patients.

[0289] The generation ADP-A2M4 iT-cells is an important milestone in producing an iPSC derived allogeneic platform. The ability to promote TCR expression in iT-cells via genetic knock-in at a single, defined locus offers an opportunity to produce multiple clonal iPSC banks encoding specific TCRs against a range of tumour antigens. In the future, this off-the-shelf platform could deliver a range of defined and consistent T-cell therapies to patients specific to their tumour antigen expression profile in a timely manner.

TABLE-US-00001 Sequences SEQIDNO:1,MAGEA4 MSSEQKSQHC KPEEGVEAQE EALGLVGAQA PTTEEQEAAV SSSSPLVPGT LEEVPAAESA GPPQSPQGAS ALPTTISFTC WRQPNEGSSS QEEEGPSTSP DAESLFREAL SNKVDELAHF LLRKYRAKEL VTKAEMLERV IKNYKRCFPV IFGKASESLK MIFGIDVKEV DPASNTYTLV TCLGLSYDGL LGNNQIFPKT GLLIIVLGTI AMEGDSASEE EIWEELGVMG VYDGREHTVY GEPRELLTQD WVQENYLEYR QVPGSNPARY EFLWGPRALA ETSYVKVLEH VVRVNARVRI AYPSLREAAL LEEEEGV SEQIDNO:2,MAGEA4peptide GVYDGREHTV SEQIDNO:3;(CD8)CDRsboldunderlined,signalsequenceitalic underlined MALPVTALLLPLALLLHAARPSQFRVSPLDTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPT FLLYLSQNKPKAAGLDTQRFSCKRLGDTFVLTLSDFRRENECYYFCSALSNSIMYFSHFVPVFLPAK PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL YCNHRNRRRVCKCPRPVVKSGDKPSLSARYV SEQIDNO:4;(CD8) ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGAGCCA GTTCCGGGTGTCGCCGCTGGATCGGACCTGGAACCTGGGCGAGACAGTGGAGCTGAAGTGCCAGGTGC TGCTGTCCAACCCGACGTCGGGCTGCTCGTGGCTCTTCCAGCCGCGCGGCGCCGCCGCCAGTCCCACC TTCCTCCTATACCTCTCCCAAAACAAGCCCAAGGCGGCCGAGGGGCTGGACACCCAGCGGTTCTCGGG CAAGAGGTTGGGGGACACCTTCGTCCTCACCCTGAGCGACTTCCGCCGAGAGAACGAGGGCTACTATT TCTGCTCGGCCCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAG CCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCT GCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTG ATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTT TACTGCAACCACAGGAACCGAAGACGTGTTTGCAAATGTCCCCGGCCTGTGGTCAAATCGGGAGACAA GCCCAGCCTTTCGGCGAGATACGTCGGTTCAAGAGCTAAAAGAAGTGGTAGTGGTGCCCCTGTGA SEQIDNO:5;(MAGEA4TCRchain)CDRsboldunderlined MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSL TILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQFGAGTQVVVTPD IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKS DFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDINLNFQNLSVIGFRILLLKVAGENLLMTLR LWSSGSRAKR SEQIDNO:6;(MAGEA4TCRchaincodingsequence) ATGAAGAAGCACCTGACCACCTTTCTCGTGATCCTGTGGCTGTACTTCTACCGGGGCAACGGCAAGAA CCAGGTGGAACAGAGCCCCCAGAGCCTGATCATCCTGGAAGGCAAGAACTGCACCCTGCAGTGCAACT ACACCGTGTCCCCCTTCAGCAACCTGCGGTGGTACAAGCAGGACACCGGCAGAGGCCCTGTGTCCCTG ACCATCCTGACCTTCAGCGAGAACACCAAGAGCAACGGCCGGTACACCGCCACCCTGGACGCCGATAC AAAGCAGAGCAGCCTGCACATCACCGCCAGCCAGCTGAGCGATAGCGCCAGCTACATCTGCGTGGTGT CCGGCGGCACAGACAGCTGGGGCAAGCTGCAGTTTGGCGCCGGAACACAGGTGGTCGTGACCCCCGAC ATCCAGAACCCTGACCCTGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCT GTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGACA AGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAATAGCGCCGTGGCCTGGTCCAACAAGAGC GACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGA GAGCAGCTGCGACGTCAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACC TGAGCGTGATCGGCTTCAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGAGA CTGTGGTCCAGCGGCAGCCGGGCCAAGAGA SEQIDNO:7;(MAGEA4TCRchain)CDRsboldunderlined MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYS FDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYEEQFFGPGTRLTVLEDLK NVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDS RYCLSSRLRVSATFWQNPRNHERCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQ GVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQIDNO:8;(MAGEA4TCRchaincodingsequence) ATGGCCAGCCTGCTGTTCTTCTGCGGCGCCTTCTACCTGCTGGGCACCGGCTCTATGGATGCCGACGT GACCCAGACCCCCCGGAACAGAATCACCAAGACCGGCAAGCGGATCATGCTGGAATGCTCCCAGACCA AGGGCCACGACCGGATGTACTGGTACAGACAGGACCCTGGCCTGGGCCTGCGGCTGATCTACTACAGC TTCGACGTGAAGGACATCAACAAGGGCGAGATCAGCGACGGCTACAGCGTGTCCAGACAGGCTCAGGC CAAGITCAGCCTGTCCCTGGAAAGCGCCATCCCCAACCAGACCGCCCTGTACTTTTGTGCCACAAGCG GCCAGGGCGCCTACGAGGAGCAGTTCTTTGGCCCTGGCACCCGGCTGACAGTGCTGGAAGATCTGAAG AACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAAATCAGCCACACCCAGAAAGC CACACTCGTGTGTCTGGCCACCGGCTTCTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCA AAGAGGTGCACAGCGGCGTGTCCACCGATCCCCAGCCTCTGAAAGAACAGCCCGCCCTGAACGACAGC CGGTACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCAGAAACCACTTCAG ATGCCAGGTGCAGTTTTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACAGAGCCAAGCCCGTGA CACAGATCGTGTCTGCCGAAGCTTGGGGGCGCGCCGATTGTGGCTTTACCAGCGAGAGCTACCAGCAG GGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGAAAGGCCACACTGTACGCCGTGCTGGT GTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC SEQIDNO:9;(MAGEA4TCRachainvariableregion)136AA-CDRsbold underlined MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSL TILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQFGAGTQVVVTPD SEQIDNO:10;(MAGEA4TCRchainvariableregion)133AA-CDRs boldunderlined MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYS FDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGOGAYEEQFFGPGTRLTVLE SEQIDNO:11;CDR1MAGEA4TCRchain,(residues48-53) VSPFSN SEQIDNO:12;CDR2MAGEA4TCRchain,(residues71-76) LTFSEN SEQIDNO:13;CDR3MAGEA4TCRchain,(residues111-125) CVVSGGTDSWGKLQF SEQIDNO:14;CDR1MAGEA4TCRchain,(residues46-50) KGHDR SEQIDNO:15;CDR2MAGEA4TCRchain,(residues68-73) SFDVKD SEQIDNO:16;CDR3MAGEA4TCRchain,(residues110-123) CATSGQGAYEEQFF SEQIDNO:17;CDR1CD8(residues45-53) VLLSNPTSG SEQIDNO:18;CDR2CD8(residues72-79) YLSQNKPK SEQIDNO:19;CDR3CD8(residues118-123) LSNSIM SEQIDNO:20HumanAlpha-fetoprotein MKWVESIFLIFLLNFTESRTLHRNEYGIASILDSYQCTAEISLADLATIF FAQFVQEATYKEVSKMVKDALTAIEKPTGDEQSSGCLENQLPAFLEELCH EKEILEKYGHSDCCSQSEEGRHNCFLAHKKPTPASIPLFQVPEPVTSCEA YEEDRETFMNKFIYEIARRHPFLYAPTILLWAARYDKIIPSCCKAENAVE CFQTKAATVTKELRESSLLNQHACAVMKNFGTRIFQAITVTKLSQKFTKV NFTEIQKLVLDVAHVHEHCCRGDVLDCLODGEKIMSYICSQQDTLSNKIT ECCKLTTLERGQCIIHAENDEKPEGLSPNLNRFLGDRDENQFSSGEKNIE LASFVHEYSRRHPQLAVSVILRVAKGYQELLEKCFQTENPLECQDKGEEE LOKYIQESQALAKRSCGLFQKLGEYYLQNAFLVAYTKKAPQLTSSELMAI TRKMAATAATCCQLSEDKLLACGEGAADIIIGHLCIRHEMTPVNPGVGQC CTSSYANRRPCFSSLVVDETYVPPAFSDDKFIFHKDLCQAQGVALQTMKQ EFLINLVKQKPqITEEQLEAVIADFSGLLEKCCOGQEQEVCFAEEGQKLI SKTRAALGV SEQIDNO:21HumanAlpha-fetoproteinpeptide FMNKFIYEI ParentalAFPTCRTRAV12-2*02/TRAJ41*01/TRACalphachainaminoacid extracellularsequence(SEQIDNo:22) 1020 ** QKEVEQNSGPLSVPEGAIASLNCTYSD 304050 *** RGSQSFFWYRQYSGKSPELIMSIYSNG 607080 *** DKEDGRETAQLNKASQYVSLLIRDSQP 90100 ** SDSATYLCAVNSDSGYALNFGKGTSLL 110120130 *** VIPHIQNPDPAVYQLRDSKSSDKSVCL 140150160 *** FTDFDSQTNVSQSKDSDVYITDKTVLD 170180 ** MRSMDFKSNSAVAWSNKSDFACANAFN 190200 ** NSIIPEDTFFPSPESS ParentalAFPTCRTRBV9*01/TRBD2/TRBJ2-7*01/TRBC2betachainamino acidextracellularsequence(SEQIDNo:23) 1020 ** DSGVTQTPKHLITATGQRVtLRCSPRS 304050 *** GDLSVYWYQQSLDQGLQFLIQYYNGEE 607080 *** RAKGNILERFSAQQFPDLHSELNLSSL 90100 ** ELGDSALYFCASSLGGESEQYFGPGTR 110120130 *** LTVTEDLKNVEPPEVAVFEPSEAEISH 140150160 *** TQKATLVCLATGFYPDHVELSWWVNGK 170180 ** EVHSGVSTDPQPLKEQPALNDSRYCLS 190200210 *** SRLRVSATFWQNPRNHFRCQVQFYGLS 220230240 *** ENDEWTQDRAKPVTQIVSAEAWGRAD ReferenceTCRalphachainDNAsequence(SEQIDNo:24) caaaaagaagttgagcagaattctggacccctcagtgttccagagggagccattgcctctctcaactg cacttacagtgaccgaggttcccagtccttcttctggtacagacaatattctgggaaaagccctgagt tgataatgtccatatactccaatggtgacaaagaagatggaaggtttacagcacagctcaataaagcc agccagtatgtttctctgctcatcagagactcccagcccagtgattcagccacctacctctgtgccgt gaatagtgattccgggtatgcactcaacttcggcaaaggcacctcgctgttggtcacaccccatatcc agaaccctgaccctgccgtgtaccagctgagagactctaagtcgagtgacaagtctgtctgcctattc accgattttgattctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaatg tgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgact ttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagt tcc ReferenceTCRbetachainDNAsequence(SEQIDNo:25) gattctggagtcacacaaaccccaaagcacctgatcacagcaactggacagcgagtgacgctgagatg ctcccctaggtctggagacctctctgtgtactggtaccaacagagcctggaccagggcctccagttcc tcattcagtattataatggagaagagagagcaaaaggaaacattcttgaacgattctccgcacaacag ttccctgacttgcactctgaactaaacctgagctctctggagctgggggactcagctttgtatttctg tgccagcagcctcgggggggaatctgagcagtacttcgggccgggcaccaggctcacggtcacagagg acctgaaaaacgtgttcccacccgaggtcgctgtgtttgagccatcagaagcagagatctcccacacc caaaaggccacactggtgtgcctggccaccggtttctaccccgaccacgtggagctgagctggtgggt gaatgggaaggaggtgcacagtggggtctgcacagacccgcagcccctcaaggagcagcccgccctca atgactccagatacgctctgagcagccgcctgagggtctcggccaccttctggcaggacccccgcaac cacttccgctgtcaagtccagttctacgggctctcggagaatgacgagtggacccaggatagggccaa acccgtcacccagatcgtcagcgccgaggcctggggtagagcagac DRGSQS(CDR1),SEQIDNO:26 IYSNGD(CDR2),SEQIDNO:27 AVNSDSGYALNF(CDR3),SEQIDNO:28 SGDLS(CDR1),SEQIDNO:29 YYNGEE(CDR2),SEQIDNO:30 ASSLGGESEQY(CDR3),SEQIDNO:31 DRGSQA(CDR1),SEQIDNO:32 AVNSDSSYALNF(DR2),SEQIDNO:33 AVNSDSGVALNE(CDR2),SEQIDNO:34 AVNSQSGYALNF(CDR2),SEQIDNO:35 AVNSQSGYSLNE(CDR2),SEQIDNO:36 AVNSQNGYALNF(CDR2),SEQIDNO:37 DRGSFS(CDR1),SEQIDNO:38 DRGSYS(CDR1),SEQIDNO:39 AVNSQSSYALNF(xCDR2),SEQIDNO:40 VariantAFPTCR(AFPTRAV12-2*02/TRAJ41*01/TRACalphachainamino acidextracellularsequence,CDRsunderlined(SEQIDNo:41) QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQAFFWYRQYSGKSPELIMSIYSNGDKEDGRETAQLNKA SQYVSLLIRDSQPSDSATYLCAVNSQSGYALNFGKGTSLLVTPHIQNPDPAVYQLRDSKSSDKSVCLF TDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPES S VariantAFPTCRTRBV9*01/TRBD2/TRBJ2-7*01/TRBC2betachainamino acidextracellularsequence,CDRsunderlined(SEQIDNo:42) DSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLOFLIQYYNGEERAKGNILERFSAQQ FPDLHSELNLSSLELGDSALYFCASSLGGESEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHT QKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWONPRN HFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD LeftHomologyArm(LHA),SEQIDNO:43 TTTGGGGTTGCTCCAAGGTAAAGTTCAAAAAGTATCCTGCAGTCAACCCTTTAGCACCATAAAGAAAC TAAATTATTTAGATGTTTTTATGAGAACATATCAAAAAGTACTTTTCTGTCATCCAATACTTCCACAA ATAAATCATTAGTTCTTGCTAATCTTCATCTGGCATAAAAATAATGACATCAACTTTCTTCATGTAAT TTCCCACTTAATTCCTTTACTAGGAGCAATATCAATTCCTATATGACGTCATTGCCAGCACCIACCCT GCTCAGAATGGACAAGTAAAGAAAAACAACCATCAAGAAGATAAAATTGAATTTGATAATGAAGTGGA CAAAGTAAAGCAGGATGCTAATTGTGTTAATCCACTTGGTGCCCCAGAAAAGCTCCCTGAAGCAAAGG AACAGGCTGAAGGTTCTGAACCCACGAGTGGCACTGAGGGGCCAGAACATTCTGTCAATGGTCCTGCA AGTCCAGCTTTAAATCAAGGTTCA RightHomologyArm(RHA),SEQIDNO:44 GAAAAGACATAAATGAGGAAACTCCAAACCTCCTGTTAGCTGTTATTTCTATTTTTGTAGAAGTAGGA AGTGAAAATAGGTATACAGTGGATTAATTAAATGCAGCGAACCAATATTTGTAGAAGGGTTATATTTT ACTACTGTGGAAAAATATTTAAGATAGTTTTGCCAGAACAGTTTGTACAGACGTATGCTTATTTTAAA ATTTTATCTCTTATTCAGTAAAAAACAACTTCTTTGTAATCGTTATGTGTGTATATGTATGTGTGTAT GGGTGTGTGTTTGTGTGAGAGACAGAGAAAGAGAGAGAATTCTTTCAAGTGAATCTAAAAGCTTTTGC TTTTCCTTTGTTTTTATGAAGAAAAAATACATTTTATATTAGAAGTGTTAACTTAGCTTGAAGGATCT GTTTTTAAAAATCATAAACTGTGTGCAGACTCAATAAAATCATGTACATTTCTGAAATGACCTCAAGA TGTCCTCCTTGTTCTACTCATATA FuT2A_A2M4,SEQIDNO:45 TCCAGCGGCAGCCGGGCCAAGAGATCTGGATCAGGTGAGGGCAGAGGCAGCCTGCTGACATGTGGCGA CGTGGAAGAAAACCCTGGCCCTATGAAGAAGCACCTGACCACCTTTCTCGTGATCCTGTGGCTGTACT TCTACCGGGGCAACGGCAAGAACCAGGTGGAACAGAGCCCCCAGAGCCTGATCATCCTGGAAGGCAAG AACTGCACCCTGCAGTGCAACTACACCGTGTCCCCCTTCAGCAACCTGCGGTGGTACAAGCAGGACAC CGGCAGAGGCCCTGTGTCCCTGACCATCCTGACCTTCAGCGAGAACACCAAGAGCAACGGCCGGTACA CCGCCACCCTGGACGCCGATACAAAGCAGAGCAGCCTGCACATCACCGCCAGCCAGCTGAGCGATAGC GCCAGCTACATCTGCGTGGTGTCCGGCGGCACAGACAGCTGGGGCAAGCTGCAGTTTGGCGCCGGAAC ACAGGTGGTCGTGACCCCCGACATCCAGAACCCTGACCCTGCCGTGTACCAGCTGCGGGACAGCAAGA GCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGAC AGCGACGTGTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAATAGCGC CGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGG ACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTCAAGCTGGTGGAAAAGAGCTTCGAGACAGAC ACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCAGAATCCTGCTGCTGAAGGTGGCCGGCTT CAACCTGCTGATGACCCTGAGACTGTGGTCCAGCGGCAGCCGGGCCAAGAGATCTGGATCCGGCGCTA CCAACTTTAGCCTGCTGAAGCAGGCCGGGGACGTGGAAGAAAACCCTGGCCCTAGGATGGCCAGCCTG CTGTTCTTCTGCGGCGCCTTCTACCTGCTGGGCACCGGCTCTATGGATGCCGACGTGACCCAGACCCC CCGGAACAGAATCACCAAGACCGGCAAGCGGATCATGCTGGAATGCTCCCAGACCAAGGGCCACGACC GGATGTACTGGTACAGACAGGACCCTGGCCTGGGCCTGCGGCTGATCTACTACAGCTTCGACGTGAAG GACATCAACAAGGGCGAGATCAGCGACGGCTACAGCGTGTCCAGACAGGCTCAGGCCAAGTTCAGCCT GTCCCTGGAAAGCGCCATCCCCAACCAGACCGCCCTGTACTTTTGTGCCACAAGCGGCCAGGGCGCCT ACGAGGAGCAGTTCTTTGGCCCTGGCACCCGGCTGACAGTGCTGGAAGATCTGAAGAACGTGTTCCCC CCAGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAAATCAGCCACACCCAGAAAGCCACACTCGTGTG TCTGGCCACCGGCTTCTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACA GCGGCGTGTCCACCGATCCCCAGCCTCTGAAAGAACAGCCCGCCCTGAACGACAGCCGGTACTGCCTG AGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCAGAAACCACTTCAGATGCCAGGTGCA GTTTTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACAGAGCCAAGCCCGTGACACAGATCGTGT CTGCCGAAGCTTGGGGGCGCGCCGATTGTGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGC GCCACCATCCTGTACGAGATCCTGCTGGGAAAGGCCACACTGTACGCCGTGCTGGTGTCTGCCCTGGT GCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGCTAA SyntheticPolyAsequence,SEQIDNO:46 AATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTG EF1Apromoter,SEQIDNO:47 GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTC GGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCT CCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTC GCAACGGGTTTGCCGCCAGAACACAG LOXP,SEQIDNO:48 ATAACTTCGTATAATGTATGCTATACGAAGTTAT NEOResistancegene,SEQIDNO:49 ATGGGATCGGCCATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATT CGGCTATGACTGGGCACAACAGACGATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGG GGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCG CGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGG AAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCG AGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTC GACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGA TGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGC CCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGC CGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGC TACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCG CCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGA PrimerPTPRC-5int-FWD(SEQIDNO:50) CAGTGATTCCTGCCCTGATTCTTA PrimerPTPRC-5int-REV(SEQIDNO:51) TACAGCCACAGGATCACGAGAAAG PrimerPTPRC-3int-FWD(SEQIDNO:52) CCTGCCGAGAAAGTATCCATCAT PrimerPTPRC-3int-REV(SEQIDNO:53). TAGCATACACACACATACCACCTT PrimerITR_F(SEQIDNO:54) GGAACCCCTAGTGATGGAGTT PrimerITR_R(SEQIDNO:55) CGGCCTCAGTGAGCGA