Patent ID: 12215313

EXAMPLES

1.1 Generation of an MHC Vector Library

Genes for common MHC I alleles were cloned into the γ-retroviral vector MP71 (68-70) to first generate an MHC vector library, to generate single-MHC-expressing K562 cells (23), which were used as artificial APCs comprising the MHC cell library. Allelic versions of HLA-A, -B or -C genes are highly polymorphic. Sequences are open access at the IMGT/HLA database. However, the 5′ and 3′ ends of different types have high sequence similarities, making it challenging to PCR amplify one specific HLA gene from a cells' cDNA. To overcome this problem, cDNA was generated from lymphoblastoid cells (LCL) obtained from the International Histocompatibility Workshop, which were homozygous for the desired HLA-A, -B and -C alleles to enable efficient gene amplification by PCR. Amplified HLA fragments were fused to an IRES-GFP or IRES-CFP expression marker and cloned into the retroviral expression vector MP71 (FIG.1).

1.2 Generation of MHC Cell Library

The erythroleukemic cell line K562 (20) was used as artificial APC scaffold for the generation of the MHC cell library. K562 cells lack endogenous expression of MHC class I molecules though expressing β-2 microglobulin, one ubiquitous component of functional MHC complexes. However, upon transfection with an MHC class I α-chain allele, the cells can be shown to possess a functional antigen processing machinery with MHC surface expression, thereby making K562 an attractive scaffold for the generation of artificial APCs (19, 23, 24). Stable transduction of K562 cells with single HLA alleles was conducted using the MP71 retroviral vector-based HLA library. Production of retroviral supernatant in 293T packaging cells and transduction was performed as described (72) and resulted in GFP- or CFP-expressing populations, as was confirmed by flow cytometric analysis. Functional assembly and surface expression of MHC complexes was indicated by MHC class I antibody staining of GFP- or CFP-positive cell populations (FIG.2). All HLA alleles transduced in K562 cells were expressed at the cell surface. For later analysis of isolated TCRs, panels of K562 cells were generated covering all of the six MHC class I alleles of the original T cell donor.

1.3 Antigen Expression in the MHC Cell Library

To use K562 cells of the MHC cell library as artificial APCs, single-MHC-expressing K562 cells were transduced with the retroviral vector MP71 to stably express antigenic constructs in the context of a single MHC allele. Retroviral transduction was performed as described (71). Antigen expression in K562 cells allowed for endogenous processing and presentation of epitopes in the context of single MHC alleles. Many antigens (HPV16 E5, E6 and L1, CMV pp65 and IE-1) could not be detected readily by intracellular FACS staining. Furthermore, antibodies were not available for truncated antigens of HPV16 E5 (minigene constructs), which were used for epitope mapping, as well as mutated nucleotide sequences. Thus, all antigens were fused to an IRES-mCherry marker to indirectly confirm expression by flow cytometry (FIG.3).

A second strategy to express antigenic sequences in target cells was to transfect ivtRNA via electroporation. Therefore, antigen sequences were cloned into expression vectors to enable T7 promoter-dependent generation of ivtRNA and subsequent polyadenylation using mMessage mMachine and poly(A) kits from Ambion (Life Technologies). Electroporation of ivtRNA into K562 cells was performed with a BioRad GenePulser using an exponential electroporation protocol. Generally, ivtRNA encoding GFP was used as a control for electroporation efficiency.FIG.4shows that HLA-transduced K562 cells expressed GFP after ivtRNA electroporation.

1.4 Induction of Antigen-Specific T Cell Response by Target Cells of MHC Cell Library

In the previous experiments, it was shown that HLA-transduced K562 cells of the MHC cell library express a defined antigen after retroviral transduction or after transfection with antigen ivtRNA. The next step was to test the capacity of the MHC cell library to endogenously process and present epitopes to induce antigen-specific T cell responses. It has been described that HLA antigen-specific stimulation of T cells via the TCR leads to the upregulation of the early activation marker CD137 (32-34).

For this, two well-characterized TCRs (B23, S51) were used, which were isolated from antigen-specific T cell clones, recognizing endogenously processed and presented epitope on HLA-B*27:05. PBMCs engineered to express the TCRs (FIG.5a, b) were used to analyze the capacity of antigen-expressing K562 cells of the MHC cell library to activate antigen-specific T cells. T cell activation was measured by CD137 expression via flow cytometry. As shown inFIG.5c, all TCR-engineered PBMCs expressed CD137 activation marker after 20 h of coculture with antigen ivtRNA-transfected K562-B*27:05 target cells. The amount of CD8+/CD137+T cells was around 6% for TCR-B23-engineered T cells and 8% for TCR-S51-engineered T cells, which reflects the total amount of TCR-engineered/CD8+T cells (FIG.5a, b) used for the coculture. In conclusion, K562 cells endogenously processed the antigenic epitope and presented it on the transgenic HLA-B*27:05, which led to the stimulation of all antigen-specific T cells in the sample as measured by CD137 expression. Additionally, CD137 expression correlated with antigen-specific IFNγ release of TCR-transduced T cells as measured by ELISA.

2.1 Antigen-Specific Expansion of T Cells

In the following, the setup of a screening approach to detect and isolate TCRs with desired antigen specificity is described. It can be transferred to different antigens, e.g., from different viruses, or different tumor-specific antigens.

Therefore, DCs were generated and matured from plate adherent monocytes (72,73) using endotoxin-free medium. Maturation state of mature DCs (mDC) was confirmed by staining for T cell activation markers CD80, CD83 and CD86 as well as MHC II expression followed by flow cytometry (FIG.6). Antigen ivtRNA was generated from six viral antigens (CMV pp65 and IE1, HPV16 L1, E5, E6 and E7), which represented full-length reference HPV16 and CMV wild type gene sequences as indicated in the open access UniProt database. mDCs were transfected with ivtRNA of antigens to ensure the presentation of naturally processed and presented epitopes at the cell surface (74). ivtRNA encoding GFP was used as transfection control. Expression was measured 4-6 h after transfection (FIG.6). Antigen-expressing mDCs were used at a PBMC to DC ratio of 10:1. PBMC stimulation with DCs was performed using medium containing 10% human serum (74,75). IL-2 (20 U/ml) and IL-7 (5 ng/ml) were provided with the medium from day 2 of stimulation on to favor T cell proliferation. Three times a week IL-2 (20 U/ml) and IL-7 (5 ng/ml) was provided to the culture. Proliferating PBMCs were splitted at ratios of 1:2 to 3:4.

2.2 Screening for Virus-Specific T Cells

A second stimulation was performed 14 days after the first round of stimulation using autologous mDCs expressing one of the six viral antigens. After 28 days, the 12 T cell cultures were screened for reactivity to specific antigen-MHC combinations employing the MHC cell library (FIG.2). Cells of the MHC cell library were transfected with antigen ivtRNA via electroporation and each T cell culture raised against one antigen was screened for reactivity to the antigen in combination with one MHC type. Contacting of the library with T cells was performed preferably 18-22 h to achieve optimal activation of antigen-specific T cells within the T cell sample. The addition of cytokines was avoided during contacting to prevent T cells from unspecific activation. IFNγ release of antigen-specific T cells was measured by ELISA. Further, the T cells in the coculture were analyzed for expression of CD137 by flow cytometry (33). T cells showed specific reactivity to pp65 and E5 in combination with HLA-B*07:02 and HLA-B*15:01, respectively. Antigen-MHC-specific T cell reactivity could be measured by release of IFNγ and upregulation of CD137 at the T cell surface. Thus, combining cytokine release ELISA and flow cytometric analysis of T cell activation marker represents a robust two-method read out system for detecting antigen-MHC-specific T cell responses.

2.3 Sorting of Virus-Specific T Cells to Analyze the TCR Repertoire

T cells, which showed specific responses to one antigen-MHC combination in both assays, were selected for FACS sorting to analyze the TCR repertoire. T cells, which were expanded with CMV pp65, were cocultured with pp65-transfected K562-B*07:02 target cells, and CD137+T cells were sorted from the culture. Nearly half of the CD8+T cells were CD137+in this setting. Thirteen percent of CD8+T cells expressed CD137 upon coculture with HPV16 E5-transfected K562-B*15:01 target cells.

After sorting of antigen-MHC-specific T cells, RNA was isolated and cDNA was generated using the SMARTer RACE cDNA amplification kit (Clontech) for 5′-RACE PCR of TCR alpha and beta genes. The PCR amplification generated TCR alpha and beta gene fragments, which quantitatively represented the amount of each T cell clonotype in the FACS-sorted T cell sample. PCR products of TCR alpha and beta gene fragments were ligated into sequencing vectors using the TOPO® cloning system (Invitrogen, Life Technologies), transformed into bacteria and grown on plates containing selective medium. Each bacterial colony was regarded as containing one sequencing vector with one PCR TCR alpha or beta gene fragment. Vector DNA preparations of numerous bacterial colonies were followed by sequencing of vector inserts (TCR alpha or beta gene fragments). Sequencing results of each bacterial colony were analyzed by using the web-based IMGTN-Quest. Frequencies of identical TCR alpha or beta chains reflected the proportion of identical T cell clonotypes within the FACS-sorted T cell sample. Next, frequency matching of TCR alpha and beta chains was performed to reconstitute functional TCRs, which had accounted for antigen-MHC-specific IFNγ release and CD137 upregulation.

TCR analysis revealed TRAV17 and TRAV38-2 chains each to be present in nearly 40% of all T cells sorted upon response to HPV16 ES and HLA-B*15:01. Three TCR beta variable chains were found to be present in 21-32% of T cells (Table 1). It was assumed that each of the two TCR alpha chains could assemble a functional ES-specific TCR with one of the three TCR beta chains. Thus, there were six possible combinations of candidate TCR alpha and TCR beta chains to assemble a functional HPV16 E5-specific TCR.

T cells sorted for reactivity to CMV pp65 and HLA-B*07:02 had one predominant TCR with a TRAV17 and a TRBV7-9 chain, both being present in about 70% of TCR alpha and TCR beta colonies, respectively (Table 1).

In sum, TCR analysis showed that CD137 sorting of antigen-MHC-specific T cells is accompanied by a strong enrichment for few predominant TCR alpha and beta chains, which appear at high frequency and which may reconstitute functional antigen-MHC-specific TCRs.

TABLE 1TCR analysisMHC class IV segmentCDR-3SEQ ID NO:Frequency% of totalAntigen HPV16E5HLA-B*15:01Homsap TRAV17*01 FCAESEYGNKLVF21038.5Homsap TRAV38-2/DV8*01 FCAYRSWNYGQNFVF191038.5Homsap TRAV8-6*02 FCAVSEPAAGNKLTF2027.7Homsap TRAV26-2*01 FCILRGAGGTSYGKLTF2113.8Homsap TRAV8-6*02 FCAVITNAGKSTF2213.8Homsap TRAV25*01 FCAGPPSGTYKYIF2313.8Homsap TRAV6*02 (F)CALPMEYGNKLVF243.8Homsap TRBV5-1*01 FCASSSRGHQNTGELFF25621.4Homsap TRBV12-3*01 FCASSPEGEGVTGELFF26828.6Homsap TRBV6-5*01 FCASSYRQQETQYF6932.1Homsap TRBV5-1*01 FCASTLRGYTEAFF2713.6Homsap TRBV5-5*02 (F)CASSPWADSNQPQHF2813.6Homsap TRBV20-1*01 FCSAGTSGGPAYEQYF2913.6Homsap TRBV27*01 FCASSSPLADDYNEQFF3013.6Homsap TRBV6-2*01 F . . .CASSHRRAHRAREQYF3113.6Antigen CMV pp65HLA-B*07:02Homsap TRAV14/DV4*01 FCAMREGKDSSYKLIF3228.7Homsap TRAV17*01 FCATVIRMDSSYKLIF111773.9Homsap TRAV8-1*01 FCAVNRGGSNYKLTF3314.3Homsap TRAV3*01 FCAVRDIGGFKTIF3414.3Homsap TRAV1-2*01 FCALDGQKLLF3514.3Homsap TRAV4*01 FCLVGGLRGNVLHC3614.3Homsap TRBV7-9*03 FCASSLIGVSSYNEQFF151368.4Homsap TRBV27*01 FCASRLGGGNYNEQFF37421.1Homsap TRBV20-1*01 FCSASPRDRKFSGNTIYF3815.3Homsap TRBV7-9*03 FCASSSHDNQGAKSPLHF3915.3TCR alpha and TCR beta chains were amplified with TRAC- and TRBC-specific reverse primers from 5'-RACE cDNA of T cells, which had shown antigen-MHC-specific T cell responses. TCR analysis was performed with IMGT/V-Quest. V(D)J gene usage and CDR3 sequences specify identical TCR alpha or TCR beta chains. Frequencies of colonies carrying one TCR alpha or TCR beta chain are indicated. Percentage of total indicates the proportion of colonies with identical TCR alpha or TCR beta chains.
2.4 Functional Analysis of TCR Alpha and TCR Beta Chain Combinations

For transgenic expression of TCRs, each TCR alpha and TCR beta chain gene was cloned into the γ-retroviral vector MP71. Cell surface expression and functional analysis of TCRs was performed after stable transduction of PBMCs with different TCR alpha and TCR beta gene combinations.

Variable regions of predominant TCR alpha and TCR beta chain genes (TRAV and TRBV), which are responsible for peptide-MHC (pMHC) I binding, were fused to codon-optimized murine constant TCR alpha and TCR beta gene segments (mTRAC and mTRBC) to enable preferential pairing of transgenic TCR chains after retroviral transduction of T cells (39, 40, 71). Staining of transgenic TCRs with an antibody specific for the mTRBC was followed by flow cytometric analysis and showed that all TRA/TRB chain combinations were expressed in PBMCs (FIG.9a). However, transduction rates of different combinations varied between 6-25%. For functional analysis, TCR-transduced T cells were tested for reactivity to K562-B*15:01 or K562-B*07:02 target cells, which were transfected via electroporation with HPV16 E5 or CMV pp65 antigen ivtRNA, respectively. Strikingly, T cells expressing TRAV17 (SEQ ID NO: 3) in combination with TRBV6-5 (SEQ ID NO: 7) recognized E5-transfected target cells, whereas none of the other five TRA/TRB combinations showed antigen-specific reactivity to E5 (FIG.9b). Therefore, TRAV17 in combination with TRBV6-5 reconstituted a functional antigen-specific TCR. The only candidate TRA/TRB combination of CMV-reactive T cells (TRAV17 (SEQ ID NO: 12) and TRBV7-9 (SEQ ID NO: 16)) showed pp65-specific recognition of K562-B*07:02 target cells. TRA/TRB-transduced T cells showed no background reactivity to H2O-transfected target cells (FIG.9b), thus allowing clear identification of functional antigen-MHC-specific TCRs.

In conclusion, reconstitutions of TCRs from antigen-specific T cell clonotypes were achieved through the combination of TRA and TRB chains, which were found at high frequencies in FACS-sorted T cell samples. Screening, detection and isolation of TCRs with desired antigen specificity could be achieved by this approach, which was based on the use of the MHC cell library.

2.5 Optimization of HPV- and CMV-Specific TCRs for Transgenic Expression in PBMCs

To increase efficiency of transgenic TCR expression, several optimizations of TCR transgene sequences were applied (71). TRA and TRB chain sequences were codon-optimized and human TRAC and TRBC gene segments were replaced by their murine counterparts to increase preferential binding of transgenic TCR chains and to reduce pairing with endogenous TCR chains expressed by recipient T cells (SEQ ID NO: 5, 9, 14, 18). The optimized TRB gene was then linked via a P2A element to the TRA genes and resulting single TCR transgene cassettes (E5-specific: SEQ ID NO: 40, pp65-specific: SEQ ID NO: 41) were molecularly cloned into the γ-retroviral vector MP71. Retroviral particles carrying the optimized TCR transgene cassettes were generated via a three-plasmid transfection of 293T cells and donor PBMCs were stably transduced with retroviral particles encoding the TCRs (70). TCR gene-modified T cells within the PBMC sample were analyzed by flow cytometry after antibody staining of the transgenic murine TRBC. TCR transduction rates of 45% for the ES-specific TCR (TRAV17+TRBV6-5, SEQ ID NO: 40) and 37% for pp65-specific TCR (TRAV17+TRBV7-9, SEQ ID NO: 41) could be achieved in PBMCs, whereby 27% and 22%, respectively, were positive for CD8 and the transgenic TCR. In conclusion, transgenic TCR expression could be improved markedly from 6-7% when using the non-optimized TRA and TRB single chain transgene cassettes, which were used to reconstitute functional TCRs, to approximately 40% when using the optimized TCR transgene cassettes.

2.6 Epitope Mapping of the HPV16 E5-Specific TCR

After detection of T cell clonotypes recognizing immunogenic antigen-MHC combination without prior knowledge of immunogenic epitopes, epitope mapping was performed to reveal the exact peptide sequence within the antigenic HPV16 ES protein, which is recognized by the ES-specific TCR. The HPV16 E5-specific TCR composed of TRAV17 and TRBV6-5 sequences was an unique TCR, which has not been described before. To map the antigenic sequence recognized by the TCR, 3′-truncated minigene versions of HPV16 E5 generated by PCR with primers amplifying the respective gene region of interest. E5 minigenes were cloned into the retroviral vector MP71 and stably transduced into K562-B*15:01 target cells (FIG.11a). T cells transduced with the optimized E5-specific TCR gene cassette recognized target cells carrying the full-length E5 and the 189 nt E5 minigene sequence but not 126 and 63 nt E5 minigenes as indicated by IFNγ release (FIG.11b). Furthermore, ES TCR-transduced T cells released IFNγ irrespective of whether the target cells harbored E5 wt or E5co gene sequences (FIG.11b). In conclusion, the HPV16 E5 TCR was specific for an epitope within the E5 protein region between amino acid 42-63.

To narrow down candidate epitopes that may be the target of ES-specific TCR recognition, in silico epitope prediction was performed using the web-based IEDB T cell epitope combined predictor, which integrates predictions of proteasomal cleavage, TAP transport, ER processing and MHC class I binding. Integrated epitope prediction was performed including 8-14-mer peptides in one analysis. Epitope prediction results were calculated as total score, which can be interpreted as the probability of a given peptide to be processed and presented on an MHC molecule at the cell surface. Table 2 includes all epitopes (p1-p17) of the prediction with a positive total score using constitutive proteasome prediction. In contrast to DCs, K562 cells express a constitutive proteasome, which resembles proteasomes expressed in tumor cells (76). All epitopes, which were not expressed from amino acid sequence 42-63 of HPV16 E5 were excluded from further analysis. Surprisingly, the top three predicted epitopes (p1-p3) had to be discarded.

TABLE 2Epitope prediction of HPV16 E5Integrated MHC class I epitope prediction (IEDB) was used to rankthe likelihood of candidate target epitopes (p1-17) for the HPV16E5-specific TCR. Depicted is the position of the first amino acid ofpredicted peptides within the full-length HPV16 E5 protein (Pos.),peptide length (-mer), amino acid sequence, predicted bindingaffinity to HLA-B*15:01 and the SEQ ID NO. The algorithm usesa combined total score (arbitrary units), which integrates predictionsfor proteasomal cleavage, TAP transport and MHC (HLA-B*15:01)binding affinity. The table shows only peptides with a total scorehigher than zero. The higher the total score, the higher the efficiencyof a peptide to be processed and presented at the cell surface.Epitopes in bold print were translated from 126-189 ntsequence of E5. Epitopes in italics were recognized by the TCR.Affinity toTotalSEQ#Pos.-merSequenceMHC [nM]scoreID NOp12812LIRPLLLSVSTY42,980,7042p22713LLIRPLLLSVSTY39,490.6743p3729FLIHTHARF32,600,6044p4559SAFRCFIVY97,770,601p55410ASAFRCFIVY102,300,5845p64810LLWITAASAF39,050,5846p75113ITAASAFRCFIVY79,800,5147p8328LLLSVSTY63,700,3848p95311AASAFRCFIVY143,790,3649p105514SAFRCFIVYIIFVY138,790,3650p115014WITAASAFRCFIVY144,350,3251p125212TAASAFRCFIVY149,570,2852p134711LLLWITAASAF70,870,2553p14119LLACFLLCF119,990,1054p15508WITAASAF108,630,0855p16738LIHTHARF116,820,0556p174813LLWITAASAFRCF169,110,0057

The result of the integrated epitope prediction of HPV16 E5 is listed in Table 2. The best predicted epitope was ranked first. Peptides encoded within amino acid sequence 42-63 are shown in bold print. These ten candidate epitopes were clustered according to sequence similarities (FIG.12a) and peptides were exogenously loaded on K562-B*15:01 target cells. E5 TCR-transduced PBMCs specifically recognized HPV16 E5 epitope p4 (SAFRCFIVY, SEQ ID NO: 1). Additionally, the TCR clearly recognized all N-terminally elongated derivatives of SAFRCFIVY (SEQ ID NO: 1) including 12-, 13- and 14-mers conferring unconventional peptide lengths for MHC I (FIG.12b). HLA-B*15:01 may tolerate binding of peptides which protrude at the N-terminus. In contrast, none of the other peptides was recognized, although peptide p6 and p13 were predicted to have higher binding affinities to FILA-B*15:01 than p4.

In sum, integrated epitope prediction facilitated mapping of the exact epitope recognized by the E5-specific TCR. However, the algorithm could not predict the immunogenic epitope, and mapping of antigenic sequence with truncated minigenes of E5 was necessary prior to epitope prediction.

The isolated TRAV17 and TRBV7-9 sequences resembled a CMV-specific TCR. TCR sequences specific for CMV pp65 and restricted to HLA-B*07:02 have been published with one to five amino acids difference in the CDR3 regions (66,67) (Table 1). These TCRs had been reported to recognize the TPRVTGGGAM (SEQ ID NO: 10) 10-mer epitope of CMV pp65 when presented on HLA-B*07:02. Here, pp65 TCR-transduced PBMCs were cocultured with K562-B*07:02 target cells loaded with CMV pp65-derived epitope p1 (TPRVTGGGAM, SEQ ID NO: 10) (FIG.12a). Indeed, TCR-transduced T cells recognized p1-pulsed target cells and released IFNγ (FIG.12b). pp65 TCR-transduced PBMCs were specific for p1, despite sequence differences in the CDR3 region compared to previously published pp65-B*07:02-specific TCRs.

In summary, this approach enabled the identification of a novel immunogenic HPV16 E5 epitope and its corresponding TCR and of an immunodominant CMV pp65 epitope and its corresponding TCR. Both TCRs were specific for endogenously processed epitopes and reflected the T cell response caused by only one T cell clonotype in the initial MHC cell library-based screening. Thus, an unbiased screening of natural T cell responses and the identification of TCRs rapidly after antigen-specific in vitro stimulation is possible with the method of the invention, without prior knowledge of the epitope, thereby avoiding limitations of epitope prediction programs to predict functional T cell responses to a defined antigen and avoiding resource-intensive and unfavorable T cell clone culture. This approach can also be applied to TILs or tissue-resident T cells and screenings can be extended to further pathogen-derived and tumor-specific antigens as well as any antigen to be targeted by a TCR.

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