T CELL MANUFACTURING PROCESS

Abstract

The present invention relates to improved methods for manufacturing T cells and improved T cell compositions resulting therefrom.

Claims

1. A method of manufacturing modified T cells comprising the steps of: (a) activating an isolated population of T cells, (b) culturing the T cells, (c) modifying the T cells to express at least one heterologous T cell receptor (TCR) or chimeric antigen receptor (CAR), preferably by transducing the T cells with a nucleic acid or vector encoding the at least one heterologous T cell receptor (TCR) or chimeric antigen receptor (CAR), (d) adding an inhibitor of AKT (AKT inhibitor) to the modified T cells, (e) culturing the modified T cell population to expand and/or proliferate the cells or cell population, (f) optionally harvesting and/or cryopreserving the modified T cell population.

2. The method according to claim 1 wherein the T cells are enriched for CD3+ fraction.

3. The method according to claim 1 wherein the activation stimulates the population of T cells to proliferate.

4. The method according to claim 3 wherein the activation is by addition of anti-CD3 antibody or antigen binding fragment thereof and/or anti-CD28 antibody or antigen binding fragment thereof, optionally attached to a removable bead.

5. The method according to claim 1, wherein modification is performed prior to or simultaneously with activation.

6. The method according to claim 1, wherein modification is performed after activation, preferably 18-26 hours after activation.

7. The method according to claim 1, wherein the AKT inhibitor is added after modification, preferably 17-24 hours after modification.

8. The method according to claim 1, wherein the AKT inhibitor is selected from the group consisting of: an allosteric inhibitor, a competitive ATP inhibitor, an inhibitor of interaction between AKT and the phospholipids, an inhibitor of phosphorylation of molecules downstream of AKT preferably of phosphorylation of PRAS 40, Ribosomal S6, TSC2.

9. The method according to claim 8, wherein the allosteric inhibitor is selected from, ARQ092, ARQ751, BAY1125976, or MK-2206.

10. The method according to claim 8, wherein the competitive ATP inhibitor is selected from, Afuresertib (GSK2110183), GSK2141795, GSK690693, Ipatasertib (GDC-0068), LY2780301, Triciribine (TCN-PM; VD-0002), AZD5363, or CCT128930

11. The method according to claim 8, wherein the inhibitor of the interaction between AKT and the phospholipids is Perifosine (D-21266, KRX0401).

12. The method according to claim 1, wherein the AKT inhibitor is added at a concentration of between 0.10 uM and 10 uM, preferably 0.5 uM.

13. The method according to claim 1, wherein the modified T cells or population of T cells produced in the presence of the AKT inhibitor have an increased or higher relative proportion of any one or more of: (a) T cells expressing both CD45RA+, CCR7+, (b) T cells that are CD45RO−, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and IL-7Rα+, (c) T cells that are T.sub.SCM cells (stem memory T cells), (d) T cells with a memory phenotype, preferably CD8 memory phenotype, in comparison to (i) a population of T cells or modified T cells produced in the absence of AKT inhibitor, or (ii) a population of T cells or modified T cells produced the presence of AKT inhibitor, wherein the AKT inhibitor is added prior to modification and/or less than or equal to 24 hours after activation.

14. The method according to claim 1, wherein the modified population of T cells produced in the presence of the AKT inhibitor have improved or increased level of any one or more of: (a) persistence in-vivo, preferably persistence measured over 8 to 14 days post infusion in-vivo, (b) expansion from seeding to harvest, (c) durability of response or durable response rate (DRR), (d) antigen induced interferon gamma production, (e) T cell survival or lifespan or percentage viability, (f) T cell effector function, preferably cytotoxicity, in comparison to (i) a population of T cells or modified T cells produced in the absence of AKT inhibitor, or (ii) a population of T cells or modified T cells produced the presence of AKT inhibitor, wherein the AKT inhibitor is added prior to transduction and/or less than or equal to 24 hours after stimulation.

15. A population of modified T cells produced according to the method of claim 1, or a composition comprising the population and a physiologically acceptable excipient.

16. (canceled)

17. A method of adoptive therapy or for treating cancer or tumor, comprising administering to a patient in need thereof of population of modified T cells or composition according to claim 15.

18. (canceled)

19. A kit comprising a population of modified T cells produced according to the method of claim 1 and a package insert comprising instructions for using the T cells to treat or delay the progression of cancer and/or tumour in a subject.

Description

FIGURES

[0240] FIG. 1. Timepoints for addition of AKTi. Schematic diagram showing the timepoints at which MK-2206 was added to T cell expansion culture, comparison was made between cultures with a day 0+day 2 addition vs day 2 alone. Day 0 addition occurs post bead addition, T-Cell activation, but pre-transduction/expansion. Day 2 addition occurs post transduction but pre-expansion. Added to media used in G-Rex top up, does not disrupt process.

[0241] FIG. 2. T Cell Total Nucleated Cell (TNC) counts in Response to MK-2206 for healthy donor materials (HDM) HDM were expanded in 10 M G-Rex for 10 days. Cultures were treated with MK-2206 at various concentrations added at 3 different timepoints; day 0(+D2), Day 2. The total nucleated cell (TNC) counts were taken on day 10 post-harvest on the auto cell counter ViCELL.

[0242] FIG. 3 Multiple Healthy Donor expansion on day 10 harvest following MK-2206 compound addition. Six healthy donor materials were expanded for 10 days in 10 M G-Rex following the described process. Cultures were treated with MK-2206 at concentrations 10 uM titrated down to 0.05 uM. Graph A detail compounds added on day 0 (+day 2 media addition), Graph B details compounds added on day 2 alone. Harvested cells were counted using the ViCELL. Results displayed as fold change in expansion to untreated control. Where replicate cultures were expanded, an average of 2 replicates was shown for datapoint+/−SEM.

[0243] FIG. 4. Healthy Donor Memory Phenotype Distribution in transduced CD8+ compartments. Healthy donor material was expanded for 10 days in 10 M G-Rex following the described process. Cultures were treated with AKTi MK-2206 at multiple concentrations in uM shown on X-axis. The compounds were added either on day 0+day 2 or day 2 alone. Harvested cells were analysed by flow cytometry gated CD3+/Dextramer+/CD8+ compartment for Memory Phenotype Markers (CCR7+/−, CD45RA+/−).

[0244] FIG. 5. Antigen-stimulated IFNgamma secretion by T-cells expanded in the presence of MK-2206 Expanded T-Cells were thawed from frozen, rested for 2 hours, and then co-cultured with Antigen-positive cell line (A375, seeded a day before) for 48 hours. Supernatants from stimulated T-Cell cultures were analysed for cytokine release using IFNγ ELISA. Each datapoint shows an average of 3 replicates+/−SEM. MK-2206 was added on day 0 +day 2, Day 2.

EXAMPLES

Example 1

[0245] 1.1. Objectives

[0246] The objective of the following study was to investigate new processes of producing T cells in culture which could lead to an improved T cell function without detriment to T cell expansion, such that the T cell population produced would have improved functional effectiveness in adoptive therapy and in the treatment of cancer. It was particularly desirable for the process to increase the proportion of the less differentiated T cell population within functional CD8+ compartment, particularly with minimal or no negative impact on T cell expansion, T cell viability, transduction, CD8 frequency in final population and also having an improved cytokine secretion in response to antigen specific activation. It was expected that such a cell population would have improved anti-tumour activity as gauged by effector function in response to antigen (cytotoxic cell killing activity and cytokine production), and demonstrate improved persistence as gauged by prolonged survival in response to antigen stimulated survival, additionally the T cells would have improved persistence and memory formation, survival, and antigen stimulated survival. The process incorporates the use of an AKT inhibitor, MK-2206 is exampled here which is a small molecule inhibitor of Protein Kinase B (AKT), which acts upstream of the glycolysis pathway. Improved T cell expansion was achieved when MK-2206 was added on Day 2 after activation of the T cells on day 0 and transduction on day 1, analysis showed improved functionality of the resultant T cell population as explained below.

[0247] 1.2. T Cell Isolation Expansion and Culture

[0248] All cells were expanded and transduced, using a static expansion system, (scalable G-Rex®). Cryopreserved leukapheresis starting materials from healthy donors or cancer patients were thawed, washed, activated with CD3/CD28 magnetic Dynabeads, and CD3+ positive fraction was magnetically separated. The starting leukapheresis material was similar across the healthy donors. CD3+ cells ranged from 44-64%, all with an increased purity of 79-87% following positive isolation using anti CD3/CD28 Dynabeads. CD3+ phenotyping identified a range of stem cell memory (SCM) markers (CCR7+/CD45RA+) ranging from 11.6-57% in the CD8+ compartment between individuals. Enriched CD3+ T cells were seeded based on total nucleated cell (TNC) counts at 1.5×10.sup.6 TNC/cm.sup.2 density in the G-Rex® static cell expansion system device, in 10% of final culture volume using TexMACS+5% human AB serum (HABS) supplemented with 100 IU/ml IL-2. Transduction with vector expressing a heterologous TCR (recognising MAGE-A4) was performed 18-26 hours post addition of CD3/CD28 Dynabeads to the cells, using lentivirus (LV) vector at a MOI of 0.45. Media was then added to top up to the final culture volume, this was performed 17-24 hours post transduction with TexMACS+5% HABS+500 IU/ml IL-2. Cells were then incubated at 37° C., 5% CO.sub.2 for a further 8 days. MK-2206 was added to the media at defined intervals; in day 0 seeding media and supplemented in media used for day 2 media addition, or solely supplemented in media for day 2 media addition. The concentration of the compound varied for each experiment, 10 uM, 7.5 uM, 5 uM, 2.5 uM, 2 uM, 1.5 uM, 1 uM, 0.5 uM, 0.25 uM, 0.1 uM, 0.05 uM as dictated in the following examples. T cells were harvested 10 days post seeding. After bead removal, T-cells were counted by automated cell analysis (with ViCELL). Harvested cells were then frozen for later phenotypic and functional analysis as described. An overview of the process is given in FIG. 1.

[0249] 1.3 Antigen-Stimulation and Cytokine Release Analysis (IFNγ)

[0250] Function of expanded T-cells was evaluated based on the levels of cytokine release by expanded T-cells following antigen-stimulation with the MAGEA4-expressing cell line, A375. A375 MAGEA4 positive target cells were counted using automated cell counter and seeded in a 96 well U-bottom plate at 30,000 viable nucleated cells (VNC) per well, in 100 uL volume per well, one day prior to T-cell stimulation and incubated overnight at 37° C., 5% CO.sub.2. Wells that were used as “T cells alone” control, received 100 uL R10 media (RPMI medium with 10% FBS and 1% Penicillin/Streptomycin) only. Following overnight incubation of target cells, harvested and frozen T-cells were thawed at 37° C. in the water bath and washed with R10 media. Cells were rested in R10 media at a density of 2×10.sup.6 VNC/mL for 2 hours at 37° C., 5% CO.sub.2. After 2-hour rest, T-cells were counted using automated cell counter, and cultures were normalised to 35% transduction efficiency for all donors combining calculated amount of transduced and non-transduced VNC for each condition. The normalised T-cells were seeded at 150,000 VNC/well in 100 uL volume to the plate containing A375 target cells, seeded a day before. Plates were returned to the incubator at 37° C., 5% CO.sub.2 for 48 hours to allow for T-cell stimulation. Following 48-hour stimulation, plates were centrifuged for 5 minutes at 400 G and supernatants transferred to a new 96 well plate. Supernatants were stored at −20° C.

[0251] ELISAs were performed using Human IFNγ by ELISA. Standard controls were prepared for the plate to a maximum concentration of 10000 pg/ml for IFNy and serially diluted 1 in 2 to generate a standard curve for each assay. Colorimetric read-out was analysed (BMG LABTECH FLUOstar Omega plate reader) at OD 450.

Example 2

[0252] 2.1. T Cell Expansion Analysis

[0253] Healthy donor material (leukapheresis material comprising white blood cells separated from a donor sample of blood) was processed for culture in static expansion (G-Rex device) following the aforementioned process for a 10 day duration. The cells were cultured with MK-2206 (1.25, 2.5, 5.0, 10 uM) added to cultures on day 0+day 2 or day 2 alone (with the day 2 media addition). On harvest, Total Nucleated Cell (TNC) counts were performed using an automated cell counter (ViCELL) to provide expansion data, and flow cytometry performed to determine phenotype. The harvested cultures were frozen and later thawed for functional assessment, an antigen stimulated cytokine release assay (1.3 described above).

[0254] The data (FIG. 2) showed increased expansion in the presence of the lower concentrations of MK-2206 when compared to the untreated control. All timepoints for addition of MK-2206 showed reduced expansion at 10 uM. With exception of 10 uM, day 2 alone addition provided improved TNC yield. Day 0(+2) was consistently providing low yield.

Example 3

[0255] 3.1. Extended Titration and Expansion Analysis

[0256] The concentration of MK-2206 was tested in further titratration from 0.05 to 10 uM to fully determine optimal dosing and day 0(+2) and day 2 alone are both assessed to determine the optimal time of addition. Again, expansion, phenotype and cytokine production data has been assessed. An additional four donors were tested at the varying concentrations, to provide a full titration range in this study, 10 uM to 0.05 uM. All donors were tested in 10 M G-Rex following the described process and were harvested on Day 10. Expansion data considered the timepoint of addition of MK-2206; Day 0+2 and Day 2 alone.

[0257] The addition timepoint of MK-2206 has an impact on expansion. Day 0+2 did not show a consistent response. There was donor variation across all concentrations tested, with limits of expansion increase of 1.5-fold and expansion decrease of 0.15 fold. Peak expansion was at the lowest concentrations (0.05 and 0.1 uM), where all donors responded similar or 0.5 fold better than the untreated control (FIG. 3A). Day 2 addition had no negative impact on expansion. Donor variation did exist; however, all conditions provided a similar or better response to the control (≥1-2fold). Day 2 addition provided peak expansion for 3 donors at 0.5 uM, before expansion began to decrease in a dose dependent response (0.05-0.25 uM). Expansion response appears to plateau >0.5 uM, with some donor variation (FIG. 3B). Overall, Day 2 addition provided a measurably better response to expansion that of Day 0+2.

Example 4

[0258] 3.4. T Cell Phenotype Analysis

[0259] Flow cytometry was performed on healthy donor material on day 10 harvest from the described process to determine Memory Phenotype markers of cultured cells using CCR7 and CD45RA staining

[0260] FIG. 4 data shows that cells cultured with MK-2206 showed increased CCR7+CD45RA+ populations at harvest when added on day 0+2 and day 2 alone when compared to the untreated control. The concentration of additive did not have a significant effect on increasing CCR7+CD45RA+. The increase in these stem cell like markers were more reflective of the day 0 starting leukapheresis material (FIG. 4 D0), suggesting perhaps that the additive maintains this population by slowing differentiation.

[0261] MK-2206 has an impact on memory phenotype markers (CCR7/CD45RA), as shown (FIG. 4), there is also an observed increase in CD62L expression, increasing the population of these double positive SCM-like cells which are less terminally differentiated less subject to functional exhaustion and more able to persist with longer survival time in-vivo and provide a longer lasting and more durable immune response. This increase in CCR7+ populations indicates an increase in Stem Cell-like and central Memory markers, and a consequent decrease in CCR7−CD45RA−, i.e. the more terminally differentiated effector memory (EM) populations, this cell subpopulation has potential for exhaustion and decreased T-Cell survival and proliferation in-vivo. The EMRA population generally remains similar to the untreated control across all concentrations tested. The increase in CCR7+ population signifies that cells cultures with MK-2206 are generating ‘fitter’ T-Cells which have the ability to survive and persist, this is of functional benefit for the T- Cell population particularly in the context of adoptive therapy and treatment of cancer.

[0262] SCM T cells are “stem memory cells” (T.sub.SCM cells) CCR7+/CD45RA+ and have similarity to naive T cells, they also express large amounts of CD95, IL-2R beta, CXCR3, and LFA-1, and show numerous functional attributes distinctive of memory cells. SCM T cells are less terminally differentiated T cells with long term response and high capability for self renewal and survival. CM T cells are “central memory T cells” (T.sub.CM cells) express CCR7+/CD45RA− and also have intermediate to high expression of CD44. CM T cells are a memory subpopulation commonly found in the lymph nodes and in the peripheral circulation. EM T cells are “effector memory T cells” (T.sub.EM cells) which are CCR7-/CD45RA− i.e. but lack expression of CCR7 and CD45RA. They also have intermediate to high expression of CD44. These memory T cells lack lymph node-homing receptors and are thus found in the peripheral circulation and tissues, they are immediate response effector cells and subject to exhaustion of function due to their terminally differentiated state. EMRA cells (T.sub.EMRA) are terminally differentiated effector memory cells re-expressing CD45RA (CCR7−/CD45RA+), which is a marker usually found on naive T cells.

Example 5

[0263] 5.1. Cytokine Production

[0264] Expanded T-cells derived from the T cell culture process described above were thawed and seeded in the presence of MAGEA4-positive cell line, A375. For the cytokine release assays, the target and effector cells were co-cultured for 48 hours then the supernatants were collected and analysed for IFN gamma levels by ELISA. Non-transduced controls were also included in the assay as an additional control measure. All samples were normalised to a 35% transduction rate.

[0265] The levels of IFN gamma produced by antigen stimulated T-Cells was investigated as an indication of T cell population functionality. MK-2206 was assessed against the untreated control for each donor. The response in IFN gamma production following antigen stimulation was to a degree donor dependent. The increase in MK-2206 concentration also appeared to decrease the cytokine production of the cells (>5 uM). The highest increase was seen with day 2 addition of MK-2206 (FIG. 5).

Sequences

[0266]

TABLE-US-00001 GVYDGREHTV, (SEQ ID NO: 1), MAGE A4 peptide FMNKFIYEI (SEQ ID No: 2) alpha fetoprotein (AFP) peptide or residues 158-166 derived from SEQ ID NO: 3 Human Alpha-fetoprotein SEQ ID NO: 3 MKWVESIFLI FLLNFTESRT LHRNEYGIAS ILDSYQCTAE ISLADLATIF FAQFVQEATY KEVSKMVKDA LTAIEKPTGD EQSSGCLENQ LPAFLEELCH EKEILEKYGH SDCCSQSEEG RHNCFLAHKK PTPASIPLFQ VPEPVTSCEA YEEDRETFMN KFIYEIARRH PFLYAPTILL WAARYDKIIP SCCKAENAVE CFQTKAATVT KELRESSLLN QHACAVMKNF GTRTFQAITV TKLSQKFTKV NFTEIQKLVL DVAHVHEHCC RGDVLDCLQD GEKIMSYICS QQDTLSNKIT ECCKLTTLER GQCIIHAEND EKPEGLSPNL NRFLGDRDFN QFSSGEKNIF LASFVHEYSR RHPQLAVSVI LRVAKGYQEL LEKCFQTENP LECQDKGEEE LQKYIQESQA LAKRSCGLFQ KLGEYYLQNA FLVAYTKKAP QLTSSELMAI TRKMAATAAT CCQLSEDKLL ACGEGAADII IGHLCIRHEM TPVNPGVGQC CTSSYANRRP CFSSLVVDET YVPPAFSDDK FIFHKDLCQA QGVALQTMKQ EFLINLVKQK PQITEEQLEA VIADFSGLLE KCCQGQEQEV CFAEEGQKLI SKTRAALGV SEQ ID NO: 4; MAGE A4 TCR α chain, CDRs bold underlined MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTG RGPVSLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQ FGAGTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLD MRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQN LSVIGFRILLLKVAGFNLLMTLRLWSSGSRAKR SEQ ID NO: 5; MAGE A4 TCR α chain coding sequence ATGAAGAAGCACCTGACCACCTTTCTCGTGATCCTGTGGCTGTACTTCTACCGGGGCAAC GGCAAGAACCAGGTGGAACAGAGCCCCCAGAGCCTGATCATCCTGGAAGGCAAGAACT GCACCCTGCAGTGCAACTACACCGTGTCCCCCTTCAGCAACCTGCGGTGGTACAAGCAGG ACACCGGCAGAGGCCCTGTGTCCCTGACCATCCTGACCTTCAGCGAGAACACCAAGAGC AACGGCCGGTACACCGCCACCCTGGACGCCGATACAAAGCAGAGCAGCCTGCACATCAC CGCCAGCCAGCTGAGCGATAGCGCCAGCTACATCTGCGTGGTGTCCGGCGGCACAGACA GCTGGGGCAAGCTGCAGTTTGGCGCCGGAACACAGGTGGTCGTGACCCCCGACATCCAG AACCCTGACCCTGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTG CCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGT ACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAATAGCGCC GTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATC CCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTCAAGCTGGTGGAAAA GAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCAGAA TCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGAGACTGTGGTCCAGCG GCAGCCGGGCCAAGAGA SEQ ID NO: 6; (MAGE A4 TCR β chain) CDRs bold underlined MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGL GLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYEEQFF GPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVH SGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRA KPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG SEQ ID NO: 7; (MAGE A4 TCR β chain coding sequence) ATGGCCAGCCTGCTGTTCTTCTGCGGCGCCTTCTACCTGCTGGGCACCGGCTCTATGGAT GCCGACGTGACCCAGACCCCCCGGAACAGAATCACCAAGACCGGCAAGCGGATCATGCT GGAATGCTCCCAGACCAAGGGCCACGACCGGATGTACTGGTACAGACAGGACCCTGGCC TGGGCCTGCGGCTGATCTACTACAGCTTCGACGTGAAGGACATCAACAAGGGCGAGATC AGCGACGGCTACAGCGTGTCCAGACAGGCTCAGGCCAAGTTCAGCCTGTCCCTGGAAAG CGCCATCCCCAACCAGACCGCCCTGTACTTTTGTGCCACAAGCGGCCAGGGCGCCTACGA GGAGCAGTTCTTTGGCCCTGGCACCCGGCTGACAGTGCTGGAAGATCTGAAGAACGTGTT CCCCCCAGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAAATCAGCCACACCCAGAAAG CCACACTCGTGTGTCTGGCCACCGGCTTCTACCCCGACCACGTGGAACTGTCTTGGTGGG TCAACGGCAAAGAGGTGCACAGCGGCGTGTCCACCGATCCCCAGCCTCTGAAAGAACAG CCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTT CTGGCAGAACCCCAGAAACCACTTCAGATGCCAGGTGCAGTTTTACGGCCTGAGCGAGA ACGACGAGTGGACCCAGGACAGAGCCAAGCCCGTGACACAGATCGTGTCTGCCGAAGCT TGGGGGCGCGCCGATTGTGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGC CACCATCCTGTACGAGATCCTGCTGGGAAAGGCCACACTGTACGCCGTGCTGGTGTCTGC CCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC SEQ ID NO: 8; (MAGE A4 TCR α chain variable region)136AA - CDRs bold underlined MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTG RGPVSLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQ FGAGTQVVVTPD SEQ ID NO: 9; (MAGE A4 TCR β chain variable region)133AA - CDRs bold underlined MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGL GLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYEEQFF GPGTRLTVLE SEQ ID NO: 10; CDRI MAGE A4 TCR α chain VSPFSN SEQ ID NO: 11; CDR2 MAGE A4 TCR α chain LTFSEN SEQ ID NO: 12; CDR3 MAGE A4 TCR α chain CVVSGGTDSWGKLQF SEQ ID NO: 13; CDRI MAGE A4 TCR β chain KGHDR SEQ ID NO: 14; CDR2 MAGE A4 TCR β chain SFDVKD SEQ ID NO: 15; CDR3 MAGE A4 TCR β chain, CATSGQGAYEEQFF Parental AFP TCR TRAV12-2*02/TRAJ41*01/TRAC alpha chain amino acid extracellular sequence (SEQ ID No: 16) Q K E V E Q N S G P L S V P E G A I A S L N C T Y S D R G S Q S F F W Y R Q Y S G K S P E L I M S I Y S N G D K E D G R F T A Q L N K A S Q Y V S L L I R D S Q P S D S A T Y L C A V N S D S G Y A L N F G K G T S L L V T P H I Q N P D P A V Y Q L R D S K S S D K S V C L F T D F D S Q T N V S Q S K D S D V Y I T D K T V L D M R S M D F K S N S A V A W S N K S D F A C A N A F N N S I I P E D T F F P S P E S S Parental AFP TCR alpha chain DNA sequence (SEQ ID No: 17) caaaaagaagttgagcagaattctggacccctcagtgttccagagggagccattgcctctctcaactgcacttacagtgaccgaggttcccagtcctt cttctggtacagacaatattctgggaaaagccctgagttgataatgtccatatactccaatggtgacaaagaagatggaaggtttacagcacagctca ataaagccagccagtatgtttctctgctcatcagagactcccagcccagtgattcagccacctacctctgtgccgtgaatagtgattccgggtatgcac tcaacttcggcaaaggcacctcgctgttggtcacaccccatatccagaaccctgaccctgccgtgtaccagctgagagactctaagtcgagtgaca agtctgtctgcctattcaccgattttgattctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaatgtgtgctagacatgagg tctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacac cttcttccccagcccagaaagttcc Parental AFP TCR TRBV9*01/TRBD2/TRBJ2-7*01/TRBC2 beta chain amino acid extracellular sequence (SEQ ID No: 18) D S G V T Q T P K H L I T A T G Q R V T L R C S P R S G D L S V Y W Y Q Q S L D Q G L Q F L I Q Y Y N G E E R A K G N I L E R F S A Q Q F P D L H S E L N L S S L E L G D S A L Y F C A S S L G G E S E Q Y F G P G T R L T V T E D L K N V F P P E V A V F E P S E A E I S H T Q K A T L V C L A T G F Y P D H V E L S W W V N G K E V H S G V S T D P Q P L K E Q P A L N D S R Y C L S S R L R V S A T F W Q N P R N H F R C Q V Q F Y G L S E N D E W T Q D R A K P V T Q I V S A E A W G R A D Parental AFP TCR beta chain DNA sequence (SEQ ID No: 19) gattctggagtcacacaaaccccaaagcacctgatcacagcaactggacagcgagtgacgctgagatgctcccctaggtctggagacctctctgtg tactggtaccaacagagcctggaccagggcctccagttcctcattcagtattataatggagaagagagagcaaaaggaaacattcttgaacgattct ccgcacaacagttccctgacttgcactctgaactaaacctgagctctctggagctgggggactcagctttgtatttctgtgccagcagcctcggggg ggaatctgagcagtacttcgggccgggcaccaggctcacggtcacagaggacctgaaaaacgtgttcccacccgaggtcgctgtgtttgagccat cagaagcagagatctcccacacccaaaaggccacactggtgtgcctggccaccggtttctaccccgaccacgtggagctgagctggtgggtgaat gggaaggaggtgcacagtggggtctgcacagacccgcagcccctcaaggagcagcccgccctcaatgactccagatacgctctgagcagccg cctgagggtctcggccaccttctggcaggacccccgcaaccacttccgctgtcaagtccagttctacgggctctcggagaatgacgagtggaccc aggatagggccaaacccgtcacccagatcgtcagcgccgaggcctggggtagagcagac Variant AFP TCR (AFP TRAV12-2*02/TRAJ41*01/TRAC alpha chain amino acid extracellular sequence (SEQ ID No: 20) QEVEQNSGPLSVPEGAIASLNCTYSDRGSQAFFWYRQYSGKSPELIMSIYSNGDKEDGRFT AQLNKASQYVSLLIRDSQPSDSATYLCAVNSQSGYALNFGKGTSLLVTPHIQNPDPAVYQLR DSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACA NAFNNSIIPEDTFFPSPESS Variant AFP TCR TRBV9*01/TRBD2/TRBJ2-7*01/TRBC2 beta chain amino acid extracellular sequence (SEQ ID No: 21) DSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILE RFSAQQFPDLHSELNLSSLELGDSALYFCASSLGGESEQYFGPGTRLTVTEDLKNVFPPEVAV FEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD DRGSQS (αCDR1), AFP TCR, SEQ ID NO: 22 IYSNGD (αCDR2), AFP TCR, SEQ ID NO: 23 AVNSDSGYALNF (αCDR3), AFP TCR, SEQ ID NO: 24 SGDLS (βCDR1), AFP TCR, SEQ ID NO: 25 YYNGEE (βCDR2), AFP TCR, SEQ ID NO: 26 ASSLGGESEQY (PCDR3), AFP TCR, SEQ ID NO: 27 DRGSQA (αCDR1), AFP TCR, SEQ ID NO: 28 AVNSDSSYALNF (αCDR2), AFP TCR, SEQ ID NO: 29 AVNSDSGVALNF (αCDR2), AFP TCR, SEQ ID NO: 30 AVNSQSGYALNF (αCDR2), AFP TCR, SEQ ID NO: 31 AVNSQSGYSLNF (αCDR2), AFP TCR, SEQ ID NO: 32 AVNSQNGYALNF (αCDR2), AFP TCR, SEQ ID NO: 33 DRGSFS (αCDR1), AFP TCR, SEQ ID NO: 34 DRGSYS (αCDR1), AFP TCR, SEQ ID NO: 35 AVNSQSSYALNF (αCDR2), AFP TCR, SEQ ID NO: 36 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGA AASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYF SHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPL AGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV, (CD8α), CDRs bold underlined, signal sequence italic underlined, SEQ ID NO: 37 ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGG CCGAGCCAGTTCCGGGTGTCGCCGCTGGATCGGACCTGGAACCTGGGCGAGACAGTGGA GCTGAAGTGCCAGGTGCTGCTGTCCAACCCGACGTCGGGCTGCTCGTGGCTCTTCCAGCC GCGCGGCGCCGCCGCCAGTCCCACCTTCCTCCTATACCTCTCCCAAAACAAGCCCAAGGC GGCCGAGGGGCTGGACACCCAGCGGTTCTCGGGCAAGAGGTTGGGGGACACCTTCGTCC TCACCCTGAGCGACTTCCGCCGAGAGAACGAGGGCTACTATTTCTGCTCGGCCCTGAGCA ACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGA CGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGC GCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTT CGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCA CTGGTTATCACCCTTTACTGCAACCACAGGAACCGAAGACGTGTTTGCAAATGTCCCCGG CCTGTGGTCAAATCGGGAGACAAGCCCAGCCTTTCGGCGAGATACGTCGGTTCAAGAGC TAAAAGAAGTGGTAGTGGTGCCCCTGTGA, SEQ ID NO: 38; (CD8α) nucleic acid sequence VLLSNPTSG, CD8α CDR1, SEQ ID NO: 39 YLSQNKPK, CD8α CDR2, SEQ ID NO: 40 LSNSIM, CD8α CDR3, SEQ ID NO: 41