scholarly journals Improving TCR Gene Therapy for Treatment of Haematological Malignancies

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Emma Nicholson ◽  
Sara Ghorashian ◽  
Hans Stauss
Keyword(s):  
T Cells ◽  
Gene Transfer ◽  
Helper T Cells ◽  
Cell Populations ◽  
High Avidity ◽  
Effector Cells ◽  
High Affinity ◽  
Tcr Gene Therapy ◽  
Antitumour Response

Adoptive immunotherapy using TCR gene modified T cells may allow separation of beneficial Graft versus tumour responses from harmful GvHD. Improvements to this include methods to generate high avidity or high affinity TCR, improvements in vector design and reduction in mispairing. Following adoptive transfer, TCR transduced T cells must be able to survive and persist in vivo to give most effective antitumour responses. Central memory or naive T cells have both been shown to be more effective than effector cells at expanding and persisting in vivo. Lymphodepletion may enhance persistence of transferred T cell populations. TCR gene transfer can be used to redirect CD4 helper T cells, and these could be used in combination with CD8+ tumour specific T cells to provide help for the antitumour response. Antigen specific T regulatory T cells can also be generated by TCR gene transfer and could be used to suppress unwanted alloresponses.

Gene Therapy With a High-Affinity Single-Chain p53-Specific TCR Mediates Potent Anti-Tumor Response Without Inducing Gvhd In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1657-1657
Author(s):  
Hakim Echchannaoui ◽  
Jutta Petschenka ◽  
Edite Antunes ◽  
Matthias Theobald
Keyword(s):  
T Cells ◽  
Gene Transfer ◽  
Mouse Models ◽  
Adoptive Transfer ◽  
High Avidity ◽  
Single Chain ◽  
High Affinity ◽  
Tumor Associated Antigen ◽  
Human T Cells

Abstract The adoptive transfer of tumor-reactive cells is a promising approach in the treatment of human malignancies, but the challenge of isolating T cells with high-avidity for tumor antigens in each patient has limited its widespread application. Using HLA-A2.1 transgenic mice, we have demonstrated the feasibility of T-cell receptor (TCR) gene transfer into T cells to circumvent self-tolerance to the widely expressed human p53(264-272) tumor-associated antigen and developed approaches to generate high-affinity CD8-independent TCR. However, a safety concern of TCR gene transfer is the risk of pairing between introduced and the naturally expressed endogenous TCR chains, resulting in the generation of self-reactive T cells (off-target autoimmunity). We first genetically modified p53TCR constructs to minimize mispairing and improve correct pairing of the introduced TCR. We and others have shown that, cysteine modifications are able to increase the expression of the introduced TCR but fail to prevent mispairing formation in mouse and human T cells. To further enhance preferential TCR pairing, cell surface expression and TCR function, we introduced additional cysteine residues into the TCR α and β chain constant domains along with codon-optimization of the TCR sequences and cloning of the TCR constructs into one single 2A-based retroviral vector. To overcome TCR mispairing formation, we designed a single chain (sc) TCR by connecting the variable TCRa domain to the TCRb chain via a short peptide linker co-expressed with a truncated constant TCR a domain. Beside off-target toxicity, adoptive transfer of high-avidity T cells may potentially cause severe on-target toxicity for normal cells expressing low level of antigens. In this respect, pre-clinical in vivo studies are still missing. Here, we evaluated the safety issues raised by the risk of p53TCR gene transfer-associated on/off-target toxicities in relevant mouse models of adoptive transfer. In vitro studies showed that, scTCR-modified CD4+ and CD8+ T cells displayed similar high-avidity compared to the full-length TCR, as determined by peptide titration in cytotoxicity assays and were able to mediate specific lysis of p53 mutant A2.1+ tumor cells. Though, genetic modifications preserved the antigen specificity of these TCRs, the full-length version of the TCR could not prevent mispairing-mediated lethal off-target autoimmunity in vivo. In sharp contrast, T cells engrafted with the modified scTCR did not induce graft-versus-host disease (GVHD) following adoptive transfer. We next assessed the potential of scTCR-modified T cells to cause on-target autoimmunity in vivo, using mice which express the human wild type p53 and A2.1Kb (Hupki-A2.1Kb). We found that lymphodepleting preconditioning regimens plus vaccination-induced expansion of transferred TCR-specific T cells did not result in a depletion of hematopoietic cells, as mice recovered normal white blood cell counts, including lymphocytes and monocytes and survived without any sign of toxicity. Importantly, our study demonstrated that high-avidity scTCR-engineered human T cells were able to eradicate established tumors and persist for more than 6 months after infusion without inducing xeno-GVHD in NodScid IL-2R gamma chain-null mice. Taken together, our study provided evidence that an optimized high-affinity scTCR-specific for the broadly expressed tumor-associated antigen p53(264-272) can eradicate p53+A2.1+ tumor cells in vivo without inducing off-target or self-directed toxicities in humanized mouse models of adoptive T-cell transfer. These data strongly support the improved safety and therapeutic efficacy of high-affinity scp53TCR for TCR-based immunotherapy of p53-associated malignancies. Disclosures: No relevant conflicts of interest to declare.

Memory Effector Cells but Not Effector Cells Derived from Naive T Cells Can Utilize a Non-Perforin and Non-FasL Pathway To Inhibit Allogeneic Progenitor Cell Function Ex-Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3029-3029
Author(s):  
Zachary F. Zimmerman ◽  
Monica V. Jones ◽  
Stephen A. Stohlman ◽  
Robert B. Levy
Keyword(s):  
T Cells ◽  
Progenitor Cell ◽  
Ex Vivo ◽  
Cell Populations ◽  
Naive T Cells ◽  
Effector Cells ◽  
Naïve T Cells ◽  
Effector Pathways ◽  
Dependent Pathway

Abstract We are interested in understanding the effector pathways used by host T cells to inhibit engraftment following allogeneic HCT. Previous work in MHC matched models found that mice deficient in either perforin or fasl continue to mediate resistance, however, the numbers of BMCs necessary to override the host barrier is less than in cytotoxically normal mice. These findings suggest both cytotoxic pathways are likely to contribute to the barrier. Notably, in the combined absence of both pathways, resistance is virtually absent. In contrast, animals which contain memory CD8 T cells can resist large numbers of BMCs regardless of the absence of both perforin and fasl pathways. The present studies examined the use of these pathways by T cell populations with respect to the inhibition of progenitor cell (PC)activity (CFU-IL3) from BMC ex-vivo to determine the relative contributions of perforin and/or fasL in inhibition mediated by effector cells derived from naive vs. memory cell populations. Effector cells were generated from unsensitized BALB/c and BALB/c-perf−/− mice as well as mice immunized against H2b alloantigens. Primary effectors from unsensitized mice and memory effectors were generated following 5 and 2–3 day MLC cultures, respectively. Effector cells were co-cultured up to 48 hrs. with BMC and then plated with rmIL-3 to elicit CFU formation. Effector cells from unsensitized BALB/c-wt mice inhibited allogeneic B6-wt CFU (>80%) as well as B6-lpr CFU (>70%) formation. Syngeneic BM was not inhibited. These data demonstrate that the perforin dependent pathway is sufficient for inhibition, consistent with in vivo resistance observed in FasL defective recipients. Effector cells generated from unsensitized BALB/c-perf−/− also effectively inhibited B6-wt CFU (>80%), consistent with a fas mediated pathway. However, in contrast to effectors generated from unsensitized BALB/C-wt mice, effector cells generated from BALB/c-perf−/−did not inhibit CFU from B6-lpr BM, suggesting that: 1) both perforin and fasl effector pathways can be utilized to inhibit CFU formation ex-vivo and, 2) in the absence of both pathways, these primary effector cells could not mediate such inhibition. As anticipated, memory effector cells from BALB/c-wt mice exhibited the same pattern as the "naive" effectors (i.e.>90% inhibition) against both B6-wt and B6-lpr BM. This finding is consistent with inhibition by perforin and/or additional effector pathways. However, memory effector cells from cytotoxically deficient BALB/c-perf−/− mice failed to mediate the same pattern of inhibition as "naive" effectors from this strain. In contrast to the inability of primary effectors to inhibit B6-lpr CFU activity, the memory effectors efficiently (i.e.>90% inhibition) inhibited CFU generation from B6-lpr BM. These findings demonstrate that unlike effector cells generated from naive T cells, memory effectors can use a non-perforin/fasL dependent pathway to inhibit allogeneic PC activity. The results are consistent with the inhibition of engraftment in vivo following conditioning and transplant into cytotoxically double (perforin + fasL) defective mice and support the notion that memory - but not primary effectors utilize a non-perforin/fasl pathway to inhibit allogeneic CFU PC function. Current experiments are examining the kinetics of CFU inhibition and precise characterization of the effector T cells.

scholarly journals Functional Avidity: A Measure to Predict the Efficacy of Effector T Cells?

2012 ◽  
Vol 2012 ◽  
pp. 1-14 ◽  
Author(s):  
Selena Viganò ◽  
Daniel T. Utzschneider ◽  
Matthieu Perreau ◽  
Giuseppe Pantaleo ◽  
Dietmar Zehn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Response ◽  
Cell Populations ◽  
High Avidity ◽  
Chronic Infections ◽  
Functional Avidity ◽  
Cognate Antigen

The functional avidity is determined by exposing T-cell populationsin vitroto different amounts of cognate antigen. T-cells with high functional avidity respond to low antigen doses. Thisin vitromeasure is thought to correlate well with thein vivoeffector capacity of T-cells. We here present the multifaceted factors determining and influencing the functional avidity of T-cells. We outline how changes in the functional avidity can occur over the course of an infection. This process, known as avidity maturation, can occur despite the fact that T-cells express a fixed TCR. Furthermore, examples are provided illustrating the importance of generating T-cell populations that exhibit a high functional avidity when responding to an infection or tumors. Furthermore, we discuss whether criteria based on which we evaluate an effective T-cell response to acute infections can also be applied to chronic infections such as HIV. Finally, we also focus on observations that high-avidity T-cells show higher signs of exhaustion and facilitate the emergence of virus escape variants. The review summarizes our current understanding of how this may occur as well as how T-cells of different functional avidity contribute to antiviral and anti-tumor immunity. Enhancing our knowledge in this field is relevant for tumor immunotherapy and vaccines design.

MATHEMATICAL MODELING OF IN-VIVO TUMOR-IMMUNE INTERACTIONS FOR THE CANCER IMMUNOTHERAPY USING MATURED DENDRITIC CELLS

2018 ◽  
Vol 26 (01) ◽  
pp. 167-188 ◽  
Author(s):  
ABAZAR ARABAMERI ◽  
DAVUD ASEMANI ◽  
JAMSHID HAJATI
Keyword(s):  
Mathematical Modeling ◽  
Experimental Data ◽  
Genetic Algorithm ◽  
T Cells ◽  
Compartment Model ◽  
Helper T Cells ◽  
Effector Cells ◽  
Proposed Model ◽  
Immunological Mechanisms

To develop an anticancer drug, the mathematical models are nowadays indispensable because of complex immunological mechanisms defying with high experimentation costs as well as a large number of parameters. Based on immunological theories and vision of experimentation data, a simple and sufficient compartment model is designed that can accurately interpret and predict the effects of dendritic cell (DC)-based immunotherapy in accordance with experimentation data. The model includes effector cells, regulatory T cells, helper T cells, and DCs. A new key feature is the inclusion of immunotherapy with DCs matured with different materials. All the parameters of the model have been optimally obtained by fitting the experimental data using genetic algorithm. The proposed model has been used to predict a near-optimal pattern that minimizes tumor size after vaccination. This pattern has been validated by carrying out the associated in-vivo experimentation. The model recommends maturation materials and doses that activate a small amount of Treg in the early days and a large Th1/Treg ratio in the next days. The performance of the model compared with the previous study was shown to be superior, both qualitatively and quantitatively.

scholarly journals The First Functional HLA-a*01:01-Restricted EBV-LMP2-Specific T-Cell Receptors for TCR Gene Therapy of Patients with EBV-Associated Type II/III Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4448-4448
Author(s):  
Wesley Huisman ◽  
Ilse Gille ◽  
Lieve E. van der Maarel ◽  
Lois Hageman ◽  
Rob C.M. de Jong ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Nuclear Antigen ◽  
Cell Populations ◽  
High Avidity ◽  
Type Ii ◽  
Low Frequencies ◽  
Increased Risk ◽  
Tcr Gene Therapy

Epstein Barr virus (EBV) is associated with the development of a broad range of malignancies, including Burkitt's lymphoma, Hodgkin and non-Hodgkin lymphomas, post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma and gastric carcinoma. Differential expression of immunogenic antigens (e.g. EBV Nuclear Antigen (EBNA2-6) and Latent membrane proteins (LMPs)) is seen at the different latent phases of the viral infection. Although many EBV-associated lymphomas only express weakly immunogenic EBV antigens (e.g. EBNA1 and BARF1), lymphomas with type II or III latency express LMP1 and LMP2. Growth of such lymphomas can be curbed using adoptively transferred EBV-LMP1/2-specific T cells. Surprisingly, T cells recognizing EBV-derived peptides in the common HLA allele A*01:01 have not been found. In addition, an HLA-A*01:01-associated increased risk for EBV+ Hodgkin lymphomas and infectious mononucleosis has been reported, suggesting that HLA-A*01:01-restricted EBV-specific T cells may be absent or present at very low frequencies in these patients. A need thus exists for HLA-A*01:01-restricted EBV-specific T-cell products, especially directed against EBV-LMP1/2. Based on MHC class I peptide predictions, HLA-A*01:01-binding peptides derived from different immunogenic EBV antigens were identified and tetramer complexes were synthesized (EBNA3A-YTDHQTTPT, EBNA3A-FLQRTDLSY, BZLF1-FTPDPYQVPF, LMP2-ESEERPPTPY, LMP2-LTEWGSGNRTY). HLA-A*01:01-restricted EBV-specific T cells were present at very low frequencies in total PBMCs from all 6 donors. After sorting using flow cytometry, only EBV-LMP2-ESE specific T cells could be expanded (for 5/6 donors), yielding pure tetramer+ CD8 T-cell populations. Four out of 5 isolated T-cell populations exhibited intermediate to high avidity recognition of HLA-A*01:01-transduced TAP2-deficient T2 cells, loaded with EBV-LMP2-ESE peptide. This specific LMP2-derived peptide showed to be functionally processed, presented and recognized by EBV-LMP2-ESE-specific T cells when using HLA-A*01:01/LMP2-transduced K562 cells. To assess the suitability for TCR gene-therapy, the TCRs from the 4 functional T-cell populations were sequenced and cloned into a retroviral vector. Surprisingly, all 4 EBV-LMP2-ESE-specific T-cell populations used the TRBV6-2 gene for TCR beta-chain expression. Additionally, for TCRalpha-chain expression these populations used either TRAV12 or TRAV30. These TCRs contained small differences in the CDR3 region. Despite differences in tetramer binding, all TCRs were functional when transduced into primary CD8 T cells, CD4 T cells, and CD8 negative TCR knock-out Jurkat cells, implying CD8 independent recognition. Finally, recognition of HLA-A*01:01+ EBV-LCLs demonstrated the potential of these EBV-LMP2-ESE-specific TCRs to recognize naturally occurring endogenous LMP2. In conclusion, we isolated and validated the first functional HLA-A*01:01-restricted EBV-LMP2-specific T-cell populations and TCRs, which can be used for adoptive transfer or retro/lentiviral TCR gene therapy to treat EBV-associated type II/III lymphomas, EBV+ malignancies of epithelial origin and PTLDs. Disclosures No relevant conflicts of interest to declare.

112 Rational design of chimeric antigen receptor T cells against glypican 3 decouples toxicity from therapeutic efficacy

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A124-A124
Author(s):  
Letizia Giardino ◽  
Ryan Gilbreth ◽  
Cui Chen ◽  
Erin Sult ◽  
Noel Monks ◽  
...  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Single Chain ◽  
High Affinity ◽  
Car T Cells ◽  
Car T ◽  
Animal Care ◽  
Cytotoxic Function

BackgroundChimeric antigen receptor (CAR)-T therapy has yielded impressive clinical results in hematological malignancies and it is a promising approach for solid tumor treatment. However, toxicity, including on-target off-tumor antigen binding, is a concern hampering its broader use.MethodsIn selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CAR bearing a low and high affinity single-chain variable fragment (scFv,) binding to the same epitope and cross-reactive with murine GPC3. We characterized low and high affinity CAR-T cells immunophenotype and effector function in vitro, followed by in vivo efficacy and safety studies in hepatocellular carcinoma (HCC) xenograft models.ResultsCompared to the high-affinity construct, the low-affinity CAR maintained cytotoxic function but did not show in vivo toxicity. High-affinity CAR-induced toxicity was caused by on-target off-tumor binding, based on the evidence that high-affinity but not low-affinity CAR, were toxic in non-tumor bearing mice and accumulated in organs with low expression of GPC3. To add another layer of safety, we developed a mean to target and eliminate CAR-T cells using anti-TNFα antibody therapy post-CAR-T infusion. This antibody functioned by eliminating early antigen-activated CAR-T cells, but not all CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from anti-tumor efficacy.ConclusionsSelecting a domain with higher off-rate improved the quality of the CAR-T cells by maintaining cytotoxic function while reducing cytokine production and activation upon antigen engagement. By exploring additional traits of the CAR-T cells post-activation, we further identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that would eliminate early activated CAR-T following antigen engagement in vivo. By combining the reduced affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.Ethics ApprovalAll animal experiments were conducted in a facility accredited by the Association for Assessment of Laboratory Animal Care (AALAC) under Institutional Animal Care and Use Committee (IACUC) guidelines and appropriate animal research approval.

scholarly journals Alpha-Fetoprotein- and CD40Ligand-Expressing Dendritic Cells for Immunotherapy of Hepatocellular Carcinoma

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3375
Author(s):  
Annabelle Vogt ◽  
Farsaneh Sadeghlar ◽  
Tiyasha H. Ayub ◽  
Carlo Schneider ◽  
Christian Möhring ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
Dendritic Cells ◽  
Tumor Regression ◽  
Combined Therapy ◽  
Term Survival ◽  
Effector Cells ◽  
Tumor Environment ◽  
The Impact

Dendritic cells (DC) as professional antigen presenting cells are able to prime T-cells against the tumor-associated antigen α-fetoprotein (AFP) for immunotherapy of hepatocellular carcinoma (HCC). However, a strong immunosuppressive tumor environment limits their efficacy in patients. The co-stimulation with CD40Ligand (CD40L) is critical in the maturation of DC and T-cell priming. In this study, the impact of intratumoral (i.t.) CD40L-expressing DC to improve vaccination with murine (m)AFP-transduced DC (Ad-mAFP-DC) was analyzed in subcutaneous (s.c.) and orthotopic murine HCC. Murine DC were adenovirally transduced with Ad-mAFP or Ad-CD40L. Hepa129-mAFP-cells were injected into the right flank or the liver of C3H-mice to induce subcutaneous (s.c.) and orthotopic HCC. For treatments, 106 Ad-mAFP-transduced DC were inoculated s.c. followed by 106 CD40L-expressing DC injected intratumorally (i.t.). S.c. inoculation with Ad-mAFP-transduced DC, as vaccine, induced a delay of tumor-growth of AFP-positive HCC compared to controls. When s.c.-inoculation of Ad-mAFP-DC was combined with i.t.-application of Ad-CD40L-DC synergistic antitumoral effects were observed and complete remissions and long-term survival in 62% of tumor-bearing animals were achieved. Analysis of the tumor environment at different time points revealed that s.c.-vaccination with Ad-mAFP-DC seems to stimulate tumor-specific effector cells, allowing an earlier recruitment of effector T-cells and a Th1 shift within the tumors. After i.t. co-stimulation with Ad-CD40L-DC, production of Th1-cytokines was strongly increased and accompanied by a robust tumor infiltration of mature DC, activated CD4+-, CD8+-T-cells as well as reduction of regulatory T-cells. Moreover, Ad-CD40L-DC induced tumor cell apoptosis. Intratumoral co-stimulation with CD40L-expressing DC significantly improves vaccination with Ad-mAFP-DC in pre-established HCC in vivo. Combined therapy caused an early and strong Th1-shift in the tumor environment as well as higher tumor apoptosis, leading to synergistic tumor regression of HCC. Thus, CD40L co-stimulation represents a promising tool for improving DC-based immunotherapy of HCC.

CMVpp65-Specific T cells generated from naïve T cell populations recognize atypical but not canonical epitopes and may be protective in Vivo

Cytotherapy ◽  
2015 ◽  
Vol 17 (6) ◽  
pp. S9-S10
Author(s):  
Patrick Hanley ◽  
Joseph Melenhorst ◽  
Russell Cruz ◽  
Caridad Martinez ◽  
Helen Heslop ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Populations ◽  
Naïve T Cell ◽  
Naive T Cell

scholarly journals Induction of idiotype-bearing, nuclease-specific helper T cells by in vivo treatment with anti-idiotype.

1981 ◽  
Vol 154 (1) ◽  
pp. 24-34 ◽  
Author(s):  
G G Miller ◽  
P I Nadler ◽  
Y Asano ◽  
R J Hodes ◽  
D H Sachs
Keyword(s):  
T Cells ◽  
Network Theory ◽  
Individual Cell ◽  
Binding Activity ◽  
Helper T Cells ◽  
Antigen Binding ◽  
Receptor Interactions ◽  
Detectable Antigen

Treatment of BALB/c mice with purified pig anti-(BALB/c anti-nuclease) anti-idiotypic antibodies has been found to induce the appearance of idiotype-bearing immunoglobulins (Id') in the serum of these mice in the absence of detectable antigen binding activity. This phenomenon appeared to require T cells in the hosts because no Id' was detected in the serum of nude mice similarly treated. Furthermore, the spleens of BALB/c mice treated with anti-idiotype were found to contain helper T cells capable of providing help in an in vitro plaque-forming cell response to trinitrophenyl-nuclease equivalent to that provided by helper T cells from the spleens of nuclease-primed animals. Helper T cells from both anti-idiotype-treated and nuclease-treated animals were found to be antigen-specific and to be similarly susceptible to elimination by treatment with anti-idiotype plus complement. Therefore, treatment with both antigen and anti-idiotype appeared to prime similar populations of antigen-specific helper T cells, while having different effects on the induction of antibody. These findings are consistent with the network theory of receptor interactions in the immune response, and may provide a means for studying individual cell populations involved in such interactions.

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