| Background and Objection:T cell receptor (TCR) is expressed on T cell surface which recognize and bind to antigen-specific receptors. When the body is subjected to the tumor antigen stimulation, TCR will recognize the peptide antigen-MHC complex which presented by APC on the surface of target cells. TCR plays an important role in cell immunity, TCR gene transfer enables instantaneous generation of defined T-cell immunity, and allows the introduction of TCR with specificities not present in the T-cell pool. Therefore in the TCR gene transfer therapy, isolated the tumor antigen-specific TCR gene and transferred into the T cells of patient, can make the patient’s inability T cells to regain the ability of tumor antigen recognition and response. TCR gene modification of T cells as adoptive immunotherapy for cancer patients has progressed significantly in recent years. The group of S. Rosenberg completed the application of TCR gene therapy in patients with melanoma tumors in human clinical trials in 2006. In the first clinical trials using a retroviral MART1-specific TCR modified the T cells isolated from patients, amplified approximately 109-1010 MART-1-specific T cells and then transferred into HLA- A2+ restricted patients with metastatic melanoma. The results shown that in 17 patients there were 2 patients’ tumor cell regression, and the genetically modified TCR expression was still detectable after a year of treatment. Indeed the trial for the first time demonstrated the feasibility of TCR gene therapy in clinical applications, and also provides a practical basis for the transfer TCR gene therapy.TCR genes adoptive therapy is a clinical promise approach for the treatment of malignant tumors and viral diseases. However, several factors currently limit the efficacy and hamper the application of TCR gene immunotherapy, one of critical problem is that the introduced TCR a-and β-chain can potentially assemble with endogenous TCR chains (i.e., TCR mispairing), which not only reduces expression of the desired TCR pair but can create a new TCR with unknown specificity that can potentially cause autoimmunity and off-target toxicity. Recently, Our laboratory have identified a TCR (Vα12 and Vβ7) from TILs of patients (HLA-A2+, AFP+) with hepatocellular carcinoma. We demonstrated that wtTCRal2 and 07 expression in transduced human T cells was lower than the levels of endogenous TCR chains, and mispairing with the endogenous TCR. Since the transferred TCR has to compete for cell surface expression with the endogenous TCR, and mixed TCR dimers composed of the a-chain of one TCR and the β-chain of the other TCR, gene-transferred TCR will be diluted on the cell surface and the activity is also reduced. Furthermore mismatch TCR may also be formed to produce a new unknown specific TCR which will bring this new off-target toxicity autoimmune in TCR gene therapy. Although there is no clinical evidence that the mismatch TCR will cause autoreactive disease right now, the risk of autoimmune TCR brought by heterozygous can’t be ignored. Indeed, T cells expressing mispaired TCRs and expanded under high IL-2 conditions (similar to the current clinical setting) were demonstrated to induce graft-versus-host disease (GvHD) in a preclinical model. Therefore in TCR gene therapy a high affinity of gene-transferred TCR for their specific peptide/HLA complexes is needed, and the efficiency of cell surface expression of the transferred TCR is also considered to be important for the efficacy of the TCR gene-modified T cells.Therefore how to increase preferential TCR pairing and at the same time avoid the generation of mispairing TCR with unknown TCR specificities is crucial for TCR gene therapy in recent years. Several strategies have been explored to prevent the mixed TCR dimers formation. Examples of such strategies are replacement of the C domains of the TCRa and β chains by the corresponding murine domains, introduction of an additional inter-chain disulfide bond between the constant domains of TCRa and β chains, inversion of amino acid residues in the constant region of the TCRa and β chains that form the TCR interface, and use single-chain TCR (scTCR) chimeras including three-domain TCRs that contain other signaling domains such as CD3-ζ or FcεRI-γ (VαVβCβCD3ζ).However, these strategies are still a lot of room for improvement. Such as the human-murine hybrid TCR will introduced the transformation of murine immunogenic fragment, which is likely to induce a host to produce HAMA, and therefore this murined TCR gene is difficult to apply in the clinical treatment. The introduction of an additional inter-chain disulfide bond between the TCR a and P chain constant domains reduces the neoreactivity of TCR-transduced T cells, but mismatch TCRs still produced. The stategues that inversion of amino acid residues in the constant region of the TCR a and P chains solve the TCR mismatch to a certain extent, however the ability of exogenous TCR priority paired limited and the pMHC binding and T cell functions are redirected not ideal. For single-chain TCR, whether the conformational can be maintained stably, and combined with CD3 molecules effectively still remains to be studied. All in a word it is still necessary to find some other way to modified the TCR molecules to prevent or reduce the formation of mixed TCR dimers and also able to maintain the antigen recognition specificity.This study will modify the specific TCR (Val2 and Vβ7) which was identified from TILs of patients (HLA-A2+) with hepatocellular carcinoma by our laboratory, we designed four modified TCR variants, named Chimeric TCR (chimTCR). The first modification plan come from the previous studies suggest that the γδ TCR which is homologous with αβTCR is unable to form mixed TCR dimers with αβTCR chains and the formation of mixed dimers can be completely prevented when transfer of αβ TCR chains into γδ T cells. Therefore, we designed three chimTCR variants which were made using a domain-exchange strategies. The first chimeric TCR (chimlTCR) was replacement of the C domains of the αβTCR by the corresponding γδTCR domains, but retained the extracellular connecting region, transmembrane and intracellular region form a sandwich structure. The second chimeric TCR (chim2 TCR) was swapped the extracellular connecting region, transmembrane and intracellular region with the corresponding γδTCR region. The third one (chim3 TCR) was replacement of the entire C domains of the αβTCR by the corresponding y8TCR domains. The second modification plan was based on the intracellular region of the TCR is too short to transduce the intracellular signal, and needs to bind CD3 subunits to perform the signal transduction. So we generated a fourth chimeric TCR (chim4TCR) which modified by fusing the original constant domains downstream of the extracellular cysteine of TCRa and β chains to complete human CD3ζ. We than investigated these four chimTCR variants. On one hand we investigated whether these chimeric TCR molecules can prevent mismatch with the endogenous TCR, and on the other hand through the study of the domain replacement chimTCRs to explore the function of αβTCR or γδTCR domains. The whole study is divided into four parts. First part, we cloned the γ9δ2TCR and CD3ζ gene from peripheral blood mononuclear cells of healthy people, and then use bioinformatics methods to analyze primary structure characteristics of α12p7TCR, y982TCR and CD3ζ sequences, and use the secondary structure prediction online services identified the possible replacement sites, than use tertiary structure prediction analysis to confirm and optimize the domains integration sites. The second part, then we design of the four sets of overlapping PCR primers base on the bioinformatics prediction domain substitution sites, cloned and identified four recombination vectors pIRES-chimTRBYFP-chimTRACFP which were fused a pair of fluorescent proteins for the fluorescence resonance energy transfer (FRET) experiment, and also cloned and identified four adenovirus shuttle plasmid pDC315-chimTRB-IRES-chimTRA which were removed the fluorescent proteins for packaging adenovirus. The third part, we transfected the four vectors pIRES-chimTRBYFP-chimTRACFP into cells and analyze the expression and assembly capacity of these chimTCRs using FRET method, and detected the expression of chimTCRs by flow cytometry. The fourth part we first investigated the interaction of chimTCRs with CD3ζ by FRET method, than we studied the CTL effects of chimTCRs modified T cells and the ability of cytokine secretion, finally we analyzed the initial signal state of chim4TCR in T cells by colocalization. In summary, the study was aimed to investigate the pairing, assembly of chimTCRs, the ability of binding CD3 molecules as well as antigens recognition and signal transduction of chimTCRs. And may provide an adequate strategy to enhance efficacy and safety of TCR gene transfer and pave the way to potential immunologic studies dealing with lymphocytes function and differentiation.Chapter 1 Acquisition of γδTCR gene and bioinformatics analysis of chimeric Objective To determine the al2p7TCR, y982TCR and CD3ζ domain replacement sites using bioinformatics analysis and make the structure of chimeric TCR molecules designed stably and reasonably.Methods and materialsThere is a kind of y8 T cells (accounted for 5% CD3+ T cells) besides αβT cells (accounted for 95% CD3+ T cells) in healthy human peripheral blood. Since the TCRVy9 and TCRV82 chains always pair together in y8T cells in healthy human peripheral blood (Vγ9Vδ2 T cells accounted for 50%～95% of peripheral blood y8T cells in most healthy human). We first cloned TCRVy9, TCRV82 and CD3ζ sequences from healthy human PBMC by RT-PCR and confirmed by sequencing, than the primary structure characteristics of TCRVy9, TCRV82, TCRVα12 and TCRVβ7 were identified by sequence alignment in IMGT database. The primary structure characteristics of CD3ζ was analyzed in Uniprot database. The secondary structure of TCRVy9, TCRV82, TCRVal2, TCRVβ7 and CD3ζ were predicted by Jpred and Prediction Protein online servers. The transmembrane region of the TCRVy9, TCRV82, TCRVal2, TCRVβ7 and CD3ζ were predicted by TMHMM and the other four programs.3D structure of the TCRVy9, TCRVS2, TCRVα12, TCRVβ7 were constructed by homology method in Swiss-Model server. Finally the 3D structure of the chimTCRs were predicted by Modeller9V7 and the models were evaluated the antigen recognition region by spatial structure comparison.ResultsThe results of IMGT database analysis showed that the CDR3 region were encoded by the V-D-J or V-J joining region genes of TCRa12, β7, y9 and 82 chains. Secondary structure prediction indicated that there is α β-sheet displayed after CDR3 region in the V domain, then a section of loop structure connected with the C domain of the TCR. Combined with the results of several transmembrane prediction, the transmembrane regions of TCRa12,07, y9,82 and CD3ζ were determined. The 3D structure prediction showed that the TCRa12 was homologous with TCR82 and the TCRβ7 was homologous with TCRy9. Finally the replacement sites of TCR were confirmed by combination of all the above results. The 3D modeling results of chimeric TCR showed that the structure of chimeric TCR molecules can maintain the V domain stability.ConclusionIn conclusion, we designed 4 chimTCR variants by replacement of the domains of the αβTCR by the corresponding y8TCR domains and CD3ζ protein. Chapter 2 Cloning of chimTCR variants and construction of adenovirus containing chimTCR genesObjectiveTo construct a set of chimeric TCR genes containing a pair of fluorescent proteins used to detect the expression and pairing of chimTCR by FRET method, and a set of shuttle vectors containing chimTCR genes for packaging of adenovirus.Methods and materialsWe designed four sets of overlapping PCR primers according to the predicted domain fusion site using primer premier5.0 and Oligo6.0 softwares. The fusion genes chimTRA-CFP and chimTRB-YFP were obtained by splice overlapping extension (SOE) PCR. To construct four bicistronic recombinant plasmids pIRES2-chimTRB/YFP-chimTRA/CFP, the fusing genes chimTRA-CFP were inserted into the BstXI/NotI restriction site of pIRES2-EGFP vector, located downstream of an internal ribosome entry site (IRES) sequence of the plasmid to replace the EGFP gene, then the fusion genes chimTRB-YFP were inserted into the NheI/SalI restriction site, located upstream of IRES. The other bicistronic vector expressing both non-modified wtTCR-CFP and wtTCR-YFP constructed similar to the above procedure. The recombinant plasmid pIRES2-chimTRB-chimTRA were constructed in the same manner, then the expression cassette chimTRB-IRES-chimTRA were cloned into shuttle plasmid pDC315 respectively. All recombinant plasmids were confirmed by DNA sequencing. Adenoviral particles Ad5F35 were produced by co-transfection of the packaging cells HEK-293 with pBHGIoxdeElCre vector and recombinant pDC315 vectors, containing the wtTCR or four chimTCR genes. Recombinant adenovirus particle titers were determind by TCID50 method and the recombinant adenoviruses were confirmed by PCR. The supernatants were directly used for infection of the target Jurkat T cells to identify and explore the best multiplicity of infection (MOI) and time of infection.Statistical analysisThe experimental data are expressed as mean (x)±standard deviation (s). Statistical analysis was carried out by SPSS v19.0 software (SPSS Inc, Chicago, USA). After checking for normal distribution and homogeneity of variances, Factorial design variance analysis was used to detect the main effects and interaction of factors. Analysis of one fact was evaluated using Independent samples T test or One way Anova. Probabilities (P)<0.05 was considered to be statistical difference and P<0.01 was considered to be significantly statistical difference.ResultsWe obtained four pairs of fusion genes successfully, and constructed four bicistronic recombinant plasmids pIRES2-chimTRB/YFP-chimTRA/CFP and four adenovirus shuttle plasmids pDC315-chimTRB-IRES-chimTRA successfully. Recombinant adenoviruses were packaged correctly and the titers were all greater than 109IU/ml, the best MOI for the recombinant adenoviruses infected Jurkat cells was MOI=100 (PFU) and the best infection time was 48h.ConclusionIn conclusion, we constructed four bicistronic recombinant plasmids expressed chimTRA-CFP and chimTRB-YFP successfully. And also constructed four capacity of recombinant adenoviruses successfully.Chapter 3 Analysis of the expression and assembly of chimeric TCRs ObjectiveTo investigated the expression level and pairing ability of the chimeric TCRs in recipient cells, as well as their degree of mismatch with the endogenous TCR.Methods and materialspIRES-TRB/YFP-TRA/CFP and four recombinant plasmids pIRES-chimTRBYFP-chimTRACFP were transfected into Jurkat and BEL-7402 cells with lipofectamine LTX/PLUS according to the manufacturer’s instructions respectively. Cells transfected with pIRES-TRA/CFP and pIRES-TRB/YFP as a control to remove the SBT contamination in FRET analysis. After 24h of transfection, Jurkat cells grown on glass-bottom dishs were washed twice with PBS and immobilized with 0.05% Low Metling Agarose for 15 min and 7402 cells washed twice with PBS solution. The confocal images of cells were acquired using Olympus FluoView1000 CLSM with FV10-ASW 1.7 software. The FRET donor (TRA-ECFP or chimTRA/CFP) was excited with a 458 nm Ar- laser and the acceptor (TRB-YFP or chimTRB/YFP) was excited with a 515 nm Ar- laser. Cells were scanned from 475 to 585nm with 10nm step-size and 20nm band-width to obtain original images. Than FRET efficiency was analyzed using the sensitized acceptor emission (SE) method. Mean FRET efficiency from multipale (n=4) cell images in each group and five random regions of interest (ROI) in each cell image. Statistical analysis was performed with Two-way ANOVA with a Bonferroni’s multiple comparisons test using SPSS 19 software. Differences with P values<0.05 were considered significant. Data are expressed as mean±SD. chimeric TCRs viral supernatant were transduced into Jurkat cells with MOI100 PFU, TCR-transduced Jurkat T cells (1×106) were analyzed for transgene expression by flow cytometry using FITC-conjugated anti-TCRVα12 mAb and PE-conjugated anti-TCRVβ7 on a Epics-XL flow cytometer. Samples were analyzed using EXPO32 software and dispalyd as dotplots.Statistical analysisThe experimental data are expressed as mean (x) ± standard deviation (s). Statistical analysis was carried out by SPSS v19.0 software (SPSS Inc, Chicago, USA). After checking for normal distribution and homogeneity of variances, Factorial design variance analysis was used to detect the main effects and interaction of factors. Analysis of one fact was evaluated using Independent samples T test or One way Anova. Probabilities (P)<0.05 was considered to be statistical difference and P<0.01 was considered to be significantly statistical difference.ResultsThe fluorescence observation with CLSM was shown that the introduced fusion genes chimTRA/CFP and chimTRB/YFP could located at the plasma membrane of cells and expressed in T cells and non-T cells, especially the chim4TCR which carried the CD3ζ molecule expressed strongly in T cells and non-T cells, which indicated that chim4TCR can expressed independent CD3 components. The SE-FRET method and statistical analysis showed that the FRET efficiency of chim1TCR and chim2TCRwas higher than wtTCR in both BEL-7402 and Jurkat but they still paired with the endogenous TCR and produced a small amount of mismathed TCRs. Chim1TCR and chim2TCR can’t prevent mismatches in Jurkat cells, but reduced mismatches to some extent compared to wtTCR. The FRET efficiency of chim3TCR and chim4TCR in BEL-7402 cells and Jurkat cells have no significant difference which means they can’t paired with the endogenous TCR and can prevent mismatches. The results of Flow cytometry showed that the chim1TCR, chim2TCR and chim4TCR expressed higher than the wtTCR on the surface of jurkat cells, while chim3TCR displayed reduced surface expression comparing to wtTCR. Specially, chim4TCR displayed increased surface expression comparing to the wtTCR and the other three chimeric TCRs.ConclusionTaken together, this part of studies confirmed that chim4TCR showed enhanced surface expression and highly preferred pairing between chim4TCRa and chim4TCRβ in Jurkat cells. Chim3TCR can prevent mismatches, but the expression level was compromised. Chim1TCR and chim2TCR expressed enhanced but can’t prevent mismatches.Chapter 4 Detection of the function of the chimeric TCRs in T cellsObjectiveTo investigate interaction between chimeric TCRs and CD3ζ, and the antigen recognition capability of the chimeric TCRs when modified T cells, and the initial signal state of chim4TCR in cells.Methods and materialsWe constructed a pair of plasmids employed to analyze the interaction between chimeric TCRs and CD3ζ by FRET method. The recombinant plasmids pIRES-chimTRB-chimTRACFP as donors and pIRES-CD3ζTMYFP and pIRES-CD3ζFP as accepters. These recombinant vectors were co-transfected into BEL-7402 cells and scanned by CLSM and analyzed using SE-FRET method which according to the chapter two. PBMC of HLA-A2+ were isolated from healthy donors, Transduction was performed 2 days after initial stimulation using Ad5F35-TRBV-TRAV and Ad5F35-chimTRBV-chimTRAV adenoviruses supernatant. Cytotoxicity of chimTCR genes transferred or untransferred PBMC against target cells (HepG-2, Huh-1 and BEL-7402) at different E/T ranging from 30:1 to 1:1 was evaluated by the calcein release assay. chimTCR genes transferred or untransferred PBMC were cocultured overnight with target cells (HepG-2, Huh-1 and BEL-7402) at 30:1(E/T). IFN-y contents in the supernatants were determined by ELISA assays. Constructed a recombinant plasmids pIRES-chim4TRBYFP-chim4TRA. The chim4TCRYFP or CD3ζYFP genes were transfected into BEL-7402 and jurkat cells, then the membrane of BEL-7402 and jurkat cells were stained by DiD, the cells were excited with an excitation wavelength of 515nm and 635nm and original images of the excited cells scanned from 500nm to 700nm by CLSM. These original images were then applied to analyze the colocalization of chim4TCRYFP and cell membrane.Statistical analysisThe experimental data are expressed as mean (x)± standard deviation (s). Statistical analysis was carried out by SPSS v19.0 software (SPSS Inc, Chicago, USA). After checking for normal distribution and homogeneity of variances, Factorial design variance analysis was used to detect the main effects and interaction of factors. Analysis of one fact was evaluated using Independent samples T test or One way Anova. Probabilities (P)<0.05 was considered to be statistical difference and P<0.01 was considered to be significantly statistical difference.ResultsThe results of the FRET between chimTCR and CD3ζ showed that the wtTCR and the three chimTCRs which replaced y8TCR domains had weak interaction with CD3ζTM, while the FRET between the chim4TCR and CD3ζ can be detected, indicated that the chim4TCR and CD3ζ still can interact with each other. chim4TCR, chimlTCR and wtTCR transfected PBMC enhanced the ability of T cells to lyse HLA-A2+ target cells. Furthermore, chim4TCR transfected PBMC showed higher ability to lyse HLA-A2+ target cells compare to chimlTCR and wtTCR. But chim2TCR and chim3TCR transferred PBMC did not show enhanced CTL effect of HLA-A2+target cells compare to control. There are no statistical differences in level of specific lysis between TCR gene transferred T cells and untransferred control groups in the CTL assays using HLA-A2- BEL-7402 as target cells, which indicates antigen specificity of TCR gene modification. After coculture with HLA-A2+target cells, chim4TCR, wtTCR and chimlTCR gene transferred PBMC can produced IFN-y than untransferred PBMC group. Furthermore, Content of IFN-y secretion in chim4TCR gene transferred PBMC was higher than the wtTCR and chimlTCR gene transferred PBMC. There are no significant difference difference between chim2TCR,chim3TCR and TCR-untransferred controls. There are no statistical differences in level of IFN-y secretion between TCR gene transferred T cells and untransferred control groups after coculture with HLA-A2- BEL-7402 cells. Finally, the colocalization analysis showed that the chim4TCR was colocalized to the cell membrane.ConclusionTaken together, this part of studies confirmed that chim4TCR but not the other three chimTCRs can still interacted with CD3ζ in non-T cells. Chim4TCR, chimlTCR still maintained the original specificity of antigen recognition region, and recognized target tumor cells in an HLA-A2-restricted manner.while chim2TCR and chim3TCR lost antigen recognition capability. Besides signal status of chim4TCR is controlled in T cells. |
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