TCR Gene Transfer Research Articles

Abstract A047: Tumor specific γδ T cells expand and respond to PD-1 blockade

Abstract With the breakthrough of checkpoint blockade, immunotherapies targeting PD-1/PD-L1 have had remarkable success in the clinic and are now widely used to treat a variety of malignancies. While the majority of research on T cell exhaustion and PD-1 blockade has been focused on conventional αβ T cells, the contribution of innate-like T cells such as γδ T cells to PD-1 blockade is not clear. γδ T cells are generally not restricted to conventional MHC-peptide molecules and can recognize a diverse range of ligands including phosphoantigens presented on butyrophilins and MHC class I-like family members MR1, and CD1 isoforms. Using flow cytometry, we evaluated the γδ T cell responses in tumor biopsies and peripheral blood from six MCC patients treated with pembrolizumab. Furthermore, bulk and single cell γδ TCR sequencing was used to measure diversity and clonal expansion in the blood upon PD-1 blockade. TCRs were cloned and expressed in Jurkat cells to screen for reactivity against tumor cells lines. Finally, we used CRISPR-Cas9 to knockout known γδ T cell ligands to identify potential tumor antigen recognized by γδ T cells. We identified a MCC patient who experienced a complete response to pembrolizumab treatment and had a ten-fold expansion of γδ T cells in their tumor biopsies. Tumor-infiltrating γδ T cells expressed high levels of inhibitory receptors PD-1 and TIGIT. Furthermore, γδ T cells characterized in peripheral blood were predominantly Vδ1 cells and had increased Ki-67 expression upon pembrolizumab treatment. From TCR sequencing of peripheral blood, we observed the emergence of a dominant γδ T cell clonotype that was also found in the tumor. Upon TCR gene transfer into Jurkat 76 cells, we identified a γδ TCR that was able to recognize multiple Merkel cancer cell lines. CRISPR knockout of B2M in tumor cells still retained γδ TCR reactivity, suggesting that γδ TCR recognition is independent of B2M expression. Together, these results show innate-like T cells such as γδ T cells also have the capacity to respond to checkpoint blockade. As mutations in antigen presentation can be a resistance mechanism to PD-1 blockade, our study provides the rationale to utilize γδ T cells for treating cancer variants that have escaped the immune system and warrants further investigation of the interplay between γδ T cells and checkpoint blockade. Citation Format: Scott C. Lien, Dalam Ly, S.Y. Cindy Yang, Ben X. Wang, Michael St. Paul, Ramy Gadalla, Babak Noamani, Sarah Boross-Harmer, Trevor J. Pugh, Anna Spreafico, Naoto Hirano, Albiruni R.A. Razak, Pamela S. Ohashi. Tumor specific γδ T cells expand and respond to PD-1 blockade [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A047.

Therapeutic Advancements Across Clinical Stages in Melanoma, With a Focus on Targeted Immunotherapy.

Melanoma is the most fatal skin cancer. In the early stages, it can be safely treated with surgery alone. However, since 2011, there has been an important revolution in the treatment of melanoma with new effective treatments. Targeted therapy and immunotherapy with checkpoint inhibitors have changed the history of this disease. To date, more than half of advanced melanoma patients are alive at 5 years; despite this breakthrough, approximately half of the patients still do not respond to treatment. For these reasons, new therapeutic strategies are required to expand the number of patients who can benefit from immunotherapy or combination with targeted therapy. Current research aims at preventing primary and acquired resistance, which are both responsible for treatment failure in about 50% of patients. This could increase the effectiveness of available drugs and allow for the evaluation of new combinations and new targets. The main pathways and molecules under study are the IDO inhibitor, TLR9 agonist, STING, LAG-3, TIM-3, HDAC inhibitors, pegylated IL-2 (NKTR-214), GITR, and adenosine pathway inhibitors, among others (there are currently about 3000 trials that are evaluating immunotherapeutic combinations in different tumors). Other promising strategies are cancer vaccines and oncolytic viruses. Another approach is to isolate and remove immune cells (DCs, T cells, and NK cells) from the patient’s blood or tumor infiltrates, add specific gene fragments, expand them in culture with growth factors, and re-inoculate into the same patient. TILs, TCR gene transfer, and CAR-T therapy follow this approach. In this article, we give an overview over the current status of melanoma therapies, the clinical rationale for choosing treatments, and the new immunotherapy approaches.

A High-Avidity T-cell Receptor Redirects Natural Killer T-cell Specificity and Outcompetes the Endogenous Invariant T-cell Receptor.

T-cell receptor (TCR) gene transfer redirects T cells to target intracellular antigens. However, the potential autoreactivity generated by TCR mispairing and occurrence of graft-versus-host disease in the allogenic setting due to the retention of native TCRs remain major concerns. Natural killer T cells (NKT) have shown promise as a platform for adoptive T-cell therapy in cancer patients. Here, we showed their utility for TCR gene transfer. We successfully engineered and expanded NKTs expressing a functional TCR (TCR NKTs), showing HLA-restricted antitumor activity in xenogeneic mouse models in the absence of graft-versus-mouse reactions. We found that TCR NKTs downregulated the invariant TCR (iTCR), leading to iTCR+TCR+ and iTCR-TCR+ populations. In-depth analyses of these subsets revealed that in iTCR-TCR+ NKTs, the iTCR, although expressed at the mRNA and protein levels, was retained in the cytoplasm. This effect resulted from a competition for binding to CD3 molecules for cell-surface expression by the transgenic TCR. Overall, our results highlight the feasibility and advantages of using NKTs for TCR expression for adoptive cell immunotherapies. NKT-low intrinsic alloreactivity that associated with the observed iTCR displacement by the engineered TCR represents ideal characteristics for "off-the-shelf" products without further TCR gene editing.

Rapid Personalized Manufacture (RPM) of Sleeping Beauty System-Generated NY-ESO-1-Specific TCR-T Cells Co-Expressing Membrane-Bound IL-15 Yields Anti-Tumor Responses

CAR-redirected T cells have demonstrated clinical effectiveness in early phase clinical trials, with persistence of adoptively transferred CD19-specific T cells correlated with positive outcomes. Notwithstanding the successes for some hematological malignancies, CAR-T targets a limited number of cell-surface antigens that curtails their appeal for solid tumors. This can be overcome by TCR gene transfer with specificity for intracellular tumor-associated antigens such as NY-ESO-1 expressed by hematologic malignancies and solid tumors. The predominant technologies for both CAR- and TCR-redirection of T cells utilize viral genetic modification as well as require a lengthy period of in vitro propagation with resultant deleterious differentiation to achieve clinically relevant cell numbers. A major impediment with current TCR-T is the high-cost and lengthy time associated with a viral-based manufacture of a library of TCRs that can address a multitude of desired targets and match HLA restriction to meet the need to infuse personalized TCR-T products with multiple specificities in each recipient. The Sleeping Beauty (SB) platform is the most clinically advanced non-viral gene transfer technology and overcomes the issues of scalability with viral based manufacture of TCR-T. We initially showed in pre-clinical models that co-expression of membrane-bound interleukin-15 (mbIL15) enhanced in vivo persistence of CAR-T (PMID: 27849617). This technology has been recently advanced to produce CD19-specific T cells in ≤ 2 days after electro-transfer of DNA plasmids using so-called "rapid personalized manufacture" (RPM). This was based on the SB system to stably co-express CAR and mbIL15 with a kill switch (HER1t). We have now adapted these technologies to address current limitations for T-cell therapy by using the RPM process to very rapidly generate TCR-modified T cells. The rationale for RPM of TCR-T is based on: (i) SB to genetically modify resting T cells thus eliminating the need to propagate cells prior to, or after, genetic modification, (ii) introduction of TCR to redirect T-cell specificity to tumor-associated antigens, (iii) mbIL15-HER1t to support T-cell persistence and enable selective elimination to increase safety, and (iv) manufacture within two days of gene transfer which limits T-cell differentiation and decreases time to manufacture. Mononuclear cells were electroporated with SB-derived DNA plasmids expressing (a) HLA A2-restricted NY-ESO-1-specific TCR or (b) the TCR and mbIL15-HER1t in separate plasmids. Following electroporation, cells were directly (unpropagated) injected into NSG (immunocompromised) mice bearing established HLA A2+ NY-ESO-1+ tumor. Administration of RPM TCR-mbIL15 T cells exhibited superior anti-tumor activity compared with RPM TCR T cells (Figure). Though engraftment of TCR+ T cells was not significantly different between the two groups, the RPM TCR-mbIL15 T cell-treated mice exhibited increased frequency of CD27+TCR+ T cells (p = 0.035, n = 6-7, Mann Whitney test), a phenotype that is correlated with improved therapeutic responses in human subjects. The RPM technology can thus be adapted to co-express TCR with mbIL15 (and HER1t), which can now be scaled to provide a cost-effective approach to manufacturing a multitude of TCR-T products from a library of TCRs with the necessary complexity to manage the range of specificities and HLA restrictions to treat multiple patients. Disclosures Hurton: • M.D. Anderson Cancer Center: Patents & Royalties; Intrexon: Patents & Royalties: US 9,629,877 B2 ; Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties: US 9,629,877 B2 . Zhang:Intrexon: Patents & Royalties: US 9,629,877 B2; Ziopharm Oncology: Patents & Royalties: US 9,629,877 B2. Deniger:Ziopharm Oncology: Employment, Equity Ownership. Olivares:Ziopharm Oncology: Patents & Royalties: US9629877B2, US20160158285A1, WO2009091826A2, US20190085079A1, US20170158749A1, US20170333480A1, US20190055299A1; Intrexon: Patents & Royalties: US9629877B2, US20160158285A1, WO2009091826A2, US20190085079A1, US20170158749A1, US20170333480A1, US20190055299A1. Cooper:CytoSen: Equity Ownership; Targazyme: Equity Ownership; MD Anderson Cancer Center: Patents & Royalties; Sangamo BioSciences: Patents & Royalties; Immatics: Equity Ownership, Patents & Royalties; City of Hope: Patents & Royalties; Ziopharm Oncology: Employment, Equity Ownership, Other: Contracted research, Patents & Royalties; Secure Transfusion Services: Equity Ownership; CellChorus: Equity Ownership. Singh:Ziopharm Oncology: Patents & Royalties: US9629877B2, US20160096902A1, US20170333480A1, US10125193B2; Intrexon: Patents & Royalties: US9629877B2, US20160096902A1, US20170333480A1, US10125193B2.

Simultaneous Deletion of Endogenous TCRαβ for TCR Gene Therapy Creates an Improved and Safe Cellular Therapeutic.

Generation of an optimal Tcell therapeutic expressing high frequencies of transgenic Tcell receptor (tgTCR) is essential for improving TCR gene therapy. Upon TCR gene transfer, presence of endogenous TCRαβ reduces expression of tgTCR due to TCR mixed-dimer formation and competition for binding CD3. Knockout (KO) of endogenous TCRαβ was recently achieved using CRISPR/Cas9 editing of the TRAC or TRBC loci, resulting in increased expression and function of tgTCR. Here, we adopt this approach into current protocols for generating Tcell populations expressing tgTCR to validate this strategy in the context of four clinically relevant TCRs. First, simultaneous editing of TRAC and TRBC loci was reproducible and resulted in high double KO efficiencies in bulk CD8 Tcells. Next, tgTCR expression was significantly higher in double TRAC/BC KO conditions for all TCRs tested, including those that contained structural modifications to encourage preferential pairing. Finally, increased expression of tgTCR in edited Tcell populations allowed for increased recognition of antigen expressing tumor targets and prolonged control of tumor outgrowth in a preclinical model of multiple myeloma. In conclusion, CRISPR/Cas9-mediated KO of both endogenous TCRαβ chains can be incorporated in current Tcell production protocols and is preferential to ensure an improved and safe clinical therapeutic.

Abstract A038: Effective rerouting of NK cell cytotoxicity against B-cell malignancies upon TCR gene transfer

Abstract T-cell receptor (TCR) gene transfer involves ex-vivo introduction of a tumour-reactive TCR into patient-derived CD8 T-cells enabling specific-targeting of tumour cells. Competition for expression with CD3 from the endogenous TCR and the potential for TCR mixed- dimer formation necessitate optimisation of cellular therapeutics with sustained potency and increased safety. NK-cells (CD3-CD56+) are potent short-lived effector cells that lyse abnormal or stressed cells independent of antigen. Efficacy and safety of adoptive NK therapy has been demonstrated in the treatment of hematologic malignancies in both the autologous and allogeneic setting. Here,we aimed to exploit NK-cell cytotoxicity and redirect it toward antigen-specific recognition of tumors without the limitation of TCR mixed- dimer formation and competition for CD3.Firstly, peripheral blood derived NK-cells were expanded and retrovirally transduced to express BOB1-specific TCR, restricted to HLA- B*07:02, in combination with CD3 alongside CD8 co-receptor.BOB1 is a B-cell restricted transcription factor, important for B-cell survival. Purified BOB1-TCR expressing NK-cells demonstrated antigen-specific binding of BOB1-specific pMHC-tetramer and proliferated upon co-culture with HLA-B*07:02 positive B-lymphoblastic cell lines (B-LCL) but not with HLA-B*07:02 negative B-LCL. Furthermore, BOB1-TCR expressing NK-cells demonstrated in vitro cytotoxicity against HLA-B*07:02 positive B-LCL, multiple myeloma and B-ALL cell lines. Conversely, these tumor cell lines remained resistant to NK-cell mediated lysis when co-cultured with mock transduced NK-cells. Finally, NK sensitive cell line K562 was comparably lysed by both BOB1-TCR or mock transduced NK-cells demonstrating retained NK-cell mediated activity.These data demonstrated that NK-cell cytotoxicity can be redirected toward antigen-specific recognition of tumors and is TCR-dependent. Retention of NK-cell function in genetically modified cells allows for a double-hit therapeutic approach that can offer advantages over current cellular approaches. Citation Format: Laura T. Morton, Anne K. Wouters, Dennis F. Remst, Renate S. Hagedoorn, Marleen M. Van Loenen, Renate de Boer, J. H.F. Falkenberg, Mirjam H.M. Heemskerk. Effective rerouting of NK cell cytotoxicity against B-cell malignancies upon TCR gene transfer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A038.

T-cells "à la CAR-T(e)" - Genetically engineering T-cell response against cancer.

The last decade will be remembered as the dawn of the immunotherapy era during which we have witnessed the approval by regulatory agencies of genetically engineered CAR T-cells and of checkpoint inhibitors for cancer treatment. Understandably, T-lymphocytes represent the essential player in these approaches. These cells can mediate impressive tumor regression in terminally-ill cancer patients. Moreover, they are amenable to genetic engineering to improve their function and specificity. In the present review, we will give an overview of the most recent developments in the field of T-cell genetic engineering including TCR-gene transfer and CAR T-cells strategies. We will also elaborate on the development of other types of genetic modifications to enhance their anti-tumor immune response such as the use of co-stimulatory chimeric receptors (CCRs) and unconventional CARs built on non-antibody molecules. Finally, we will discuss recent advances in genome editing and synthetic biology applied to T-cell engineering and comment on the next challenges ahead.

Generation of Memory Stem T Cells Specific for Tumor Antigens and Resistant to Inhibitory Signals By Genome Editing

Tumor-infiltrating lymphocytes (TILs) may express different inhibitory receptors (IRs) on the cell surface that are exploited by cancer cells to evade the immune attack. Thus, exhausted T cells display an unresponsive functional profile and fail to kill tumor cells. TIM-3 and LAG-3 represent highly expressed inhibitory receptors in exhausted T cells infiltrating solid tumors. We recently showed that a large fraction of bone marrow infiltrating CD8+ cells co-express multiple inhibitory receptors, including TIM-3, in patients with acute myeloid leukemia relapsing after allogeneic hematopoietic stem cell transplantation. These observations advocate for strategies to generate innovative tumor-specific T cell products resistant to inhibitory signals and able to persist long-term in treated patients. To reach these objectives we developed a strategy to simultaneously redirect T cell specificity by TCR gene transfer and permanently disrupt IRs by CRISPR/Cas9 system in long-living memory stem T cells (TSCM) for adoptive cell therapy.Primary T cells were activated with CD3/CD28-conjugated beads and cultured with low doses of IL7 and IL15, to preserve their TSCM phenotype, and electroporated with Cas9/gRNA ribonucleoproteins (RNPs) targeting a coding sequence of TIM-3,LAG3, 2B4 and of the TCR α and β chain constant region (TRAC and TRBC1/2) genes. Each gene was edited alone or in combination in a multiplex approach. The efficiencies of NHEJ-mediated inactivation of each gene were assessed by cytofluorimetric analysis and confirmed at molecular level by surveyor assay or ddPCR.In order to avoid mispairing between the endogenous TCR and the tumor-specific TCR, we simultaneously inactivated the constant regions of α and β chain of the endogenous TCR genes. Efficiency of TRAC and/or TRBC1/2 gene disruption was 98%, when measured as % of CD3neg cells. TCR knocked-out cells could be efficiently (70-85%) transduced with a lentiviral vector encoding for TCR specific for an HLA-A2 restricted peptide from NY-ESO1.We then focused on IR gene inactivation. Single gene editing produced up to 98%, 93% and 63% genetic knock-out of the TIM-3 , the LAG-3 and the 2B4 genes, respectively. We finally combined one IR inactivation with TCR gene editing in a single protocol. We obtained an average of 68% and 69% of TIM3neg and LAG3neg NY-ESO1-TCR redirected T cells, respectively, and more than 90% of edited cells showed a TSCM phenotype.When tested in functional assays, TCR-IR-edited TSCM cells proved highly effective and specific in killing HLA-A2+ NY-ESO1+ multiple myeloma cells. Of notice, TIM-3neg and LAG-3neg cells proved more effective in producing IFNg, TNFa and CD107a, once challenged with tumor cells.In conclusion, by combining the versatility of multiplex gene editing by CRISPR/Cas9 with culture conditions designed to engineer TSCM cells, we can generate innovative tumor-specific cellular products redirected against tumor antigens and resistant to inhibitory signals. Ongoing in vivo evaluation of tumor-specific IR disrupted memory stem T cells will be reported and discussed. DisclosuresBonini:Intellia Therapeutics: Research Funding.

Phase 1 Clinical Trial of Adoptive Immunotherapy for Acute Myelogenous Leukemia and Myelodysplastic Syndrome, Using Gene-Modified Autologous Lymphocytes Expressing WT1-Specific T-Cell Receptor

Elderly patients (pts) with acute myelogenous leukemia (AML) and high-risk myelodysplastic syndrome (MDS) particularly who are ineligible for allogeneic hematopoietic stem cell transplantation (allo-HSCT) and have disease relapse after allo-HSCT still have a poor prognosis. Thus, the development of novel treatment option providing higher response rate and prolonged survival time for those patients still remains an unmet need. Because not a few clinical and preclinical studies have described a promising value of Wilms Tumor 1 (WT1), a leukemia-associated antigen as a therapeutic target of antileukemia immunotherapy, we conducted a clinical study of novel adoptive immunotherapy using gene-modified autologous lymphocytes expressing WT1-specific T-cell receptor (TCR) for the treatment of refractory AML and high-risk MDS.A multicenter phase 1 study was conducted to assess feasibility, safety and preliminary antileukemia reactivity of patient-derived gene-modified lymphocytes expressing WT1-specific TCR. Antigen-specific TCR-gene transfer may cause a serious autoimmune disease mediated by mispaired TCR between introduced and endogenous TCR α/β chains. To avoid that, we established a retroviral vector system encoding siRNAs for endogenous TCR genes (siTCR vector). We conducted a first-in-human clinical trial employing this siTCR vector. After given written informed consents, mononuclear cells were collected from at most 200ml of peripheral blood (PB) from each patient. Then, proliferating lymphocytes pre-cultured with IL-2, anti-CD3 antibody and RetroNectinTM were infected with a retroviral vector, MS3-WT1-siTCR composed of DNAs encoding WT1235-243/HLA-A*24:02 complex specific TCR-α/β chains and siRNAs against endogenous TCR genes. Expanded gene-modified lymphocytes (WT1-siTCR/T cells) in additional culture for 13-14 more days were harvested and frozen until use. Eligibility included HLA-A*24:02 positive pts with refractory AML or high-risk MDS, > 20 y.o, ineligible for allo-HSCT and performance status of 0 to 2. WT1-siTCR/T cells were intravenously infused twice on days 0 and 28. Heteroclitic WT1235-243 ninemer peptide (300mg) emulsified with MontanideTM was given subcutaneously on day 2 and 16 after the second infusion. Besides safety assays, kinetics of WT1-siTCR/T cells in PB, immunological responses and residual leukemia burden determined by qRT-PCR for WT1 mRNA were serially measured until day 58 since the first infusion.Among 12 pts enrolled, 8 pts (5 AML, 3 MDS) with a median age of 68.5 y. received study treatment. Three pts received 2x108 cells/ infusion (cohort 1), 3 received 1x109 cells/ infusion (cohort 2), and 2 received extra-cohort doses. Median follow-up time after the first infusion was 257 days (as June 13, 2016). At the first infusion, all pts contracted progressive disease. Circulatory WT1-siTCR/T cells retaining the target reactivity appeared immediately after each infusion, peaked between 1 to 3 days, and declined thereafter. WT1 peptide vaccination did not seem to affect the transition of infused cells. Values of WT1 mRNA in PB were transiently suppressed in all pts and declined in 4 pts thereafter. Clinical outcomes included one with stable disease and 2 pts with partial remission (PR). In one with PR, the epitope-spreading phenomenon was suggested. In all pts, no serious adverse events associated with infused WT1-siTCR/T cells were observed.Adoptive transfer of autologous WT1-siTCR/T cells was feasible and safe. Although the persistence of infused WT1-siTCR/T cells was limited, infused WT1-siTCR/T cells at least seemed to be involved in the antileukemia reactivity. DisclosuresTawara:Astellas: Honoraria. Akatsuka:Takara Bio Inc.: Consultancy. Nukaya:Takara Bio Inc.: Employment. Takesako:Takara Bio Inc.: Employment.

Novel immunotherapy for adult T-cell leukemia/lymphoma: Targeting aurora kinase A

ABSTRACTAdult T-cell leukemia/lymphoma is caused by infection with HTLV-1, following a long latent period. Immunotherapy targeting Aurora kinase A, a tumor-associated antigen over-expressed in adult T-cell leukemia/lymphoma, holds great therapeutic potential. We review the evidence in favor of a therapeutic strategy combining vaccination and TCR-gene transfer against this target.

A CD22-reactive TCR from the T-cell allorepertoire for the treatment of acute lymphoblastic leukemia by TCR gene transfer.

CD22 is currently evaluated as a target-antigen for the treatment of B-cell malignancies using chimeric antigen receptor (CAR)-engineered T-cells or monoclonal antibodies (mAbs). CAR- and mAbs-based immunotherapies have been successfully applied targeting other antigens, however, occurrence of refractory disease to these interventions urges the identification of additional strategies. Here, we identified a TCR recognizing the CD22-derived peptide RPFPPHIQL (CD22RPF) presented in human leukocyte antigen (HLA)-B*07:02. To overcome tolerance to self-antigens such as CD22, we exploited the immunogenicity of allogeneic HLA. CD22RPF-specific T-cell clone 9D4 was isolated from a healthy HLA-B*07:02neg individual, efficiently produced cytokines upon stimulation with primary acute lymphoblastic leukemia and healthy B-cells, but did not react towards healthy hematopoietic and nonhematopoietic cell subsets, including dendritic cells (DCs) and macrophages expressing low levels of CD22. Gene transfer of TCR-9D4 installed potent CD22-specificity onto recipient CD8+ T-cells that recognized and lysed primary B-cell leukemia. TCR-transduced T-cells spared healthy CD22neg hematopoietic cell subsets but weakly lysed CD22low-expressing DCs and macrophages. CD22-specific TCR-engineered T-cells could form an additional immunotherapeutic strategy with a complementary role to CAR- and antibody-based interventions in the treatment of B-cell malignancies. However, CD22 expression on non-B-cells may limit the attractiveness of CD22 as target-antigen in cellular immunotherapy.

A novel SIV gag-specific CD4+T-cell clone suppresses SIVmac239 replication in CD4+T cells revealing the interplay between antiviral effector cells and their infected targets

To study CD4+T-cell suppression of AIDS virus replication, we isolated nine rhesus macaque SIVGag-specific CD4+T-cell clones. One responding clone, Gag68, produced a typical cytotoxic CD8+T-cell response: induction of intracellular IFN-γ, MIP-1α, MIP-1β, and CD107a degranulation. Gag68 effectively suppressed the spread of SIVmac239 in CD4+T cells with a corresponding reduction of infected Gag68 effector cells, suggesting that CD4+effectors need to suppress their own infection in addition to their targets to be effective. Gag68 TCR cloning and gene transfer into CD4+T cells enabled additional experiments with this unique specificity after the original clone senesced. Our data supports the idea that CD4+T cells can directly limit AIDS virus spread in T cells. Furthermore, Gag68 TCR transfer into CD4+T-cell clones with differing properties holds promise to better understand the suppressive effector mechanisms used by this important component of the antiviral response using the rhesus macaque model.

Adoptive Transfer of WT1-Specific TCR Gene-Transduced Lymphocytes in Patients with Myelodysplastic Syndrome and Acute Myeloid Leukemia

Wilms' Tumor 1 (WT1) is expressed in a majority of MDS and AML cells and mRNA of WT1 in peripheral blood and bone marrow is monitored as a marker of minimal residual disease of AML and MDS. Several WT1 protein-derived epitopes that are recognized by cytotoxic T lymphocytes (CTLs) along with HLA molecules are determined. In vitro study and WT1 peptide vaccine trials have demonstrated that WT1-specfic CD8+ T cells with cytotoxic activity can be induced. Adoptive T cell therapy using ex vivo expanded WT1-specific CTLs or WT1-specific T-cell receptor (TCR)-gene transduced cells are potentially effective to refractory MDS and AML.Antigen-specific TCR-gene transfer may cause serious autoimmune disease by mispairing of introduced and endogenous TCR chains that recognize auto-antigens. We established a retroviral vector system encoding siRNAs for endogenous TCR genes to eliminate TCR-mispairing. Using the siRNA-encoding viral vector, we have conducted a first-in-man trial of WT1-specfic TCR-gene transduced T cell transfer. In the trial, we evaluate the safety of the TCR T cell transfer in patients with MDS and AML, and assess in vivo kinetics of the transferred cells. The study was designed as cell-dose escalation with three cohorts of 2x108, 1x109, and 5x109cells per infusion.Peripheral blood mononuclear cells were collected from each patient. Then, the cells were cultured with IL-2, anti-CD3 antibody, and RetroNectin®. Proliferating lymphocytes were infected with a retroviral vector, MS3-WT1-siTCR, which was constructed from DNA encoding WT1235-243/HLA*A24:02 specific TCR-α and -β chains and siRNAs for endogenous TCR genes. After 13-14 days in culture, the lymphocytes were harvested and frozen until infusion.Patients were enrolled to the clinical trial if they were refractory AML or MDS ineligible for allogeneic stem cell transplant, positive for HLA-A*24:02, had performance status of 0 to 2, and had normal organ function. WT1-TCR T cells were infused intravenously twice on days 0 and 28. Modified WT1235-243peptide (300μg) emulsified with Montanide, was given subcutaneously on day 2 and 16 after the second infusion.To date, 5 patients (4 MDS and 1 AML cases) with a median age of 69 years, received WT1-TCR T cells. Three received 2x108 cells (cohort 1) and 2 received 1x109 cells (cohort 2) per infusion, respectively. We did not see any severe adverse events related to the cell infusion or peptide vaccination. No renal or mesothelial damages were observed. We then assessed transduced TCR-gene copy numbers in peripheral blood samples collected at multiple pre-determined time points until day 58.TCR-gene marked cells were detected in all patients after the cell infusion. They appeared immediately after the infusion, reaching peak levels between 1 and 3 days. Then, the levels gradually declined. After the second infusion, which was followed by peptide vaccination, the cells appeared in the similar way to the first cycle. The peptide vaccine did not seem to affect the peripheral cell kinetics. Dose-dependent kinetics were shown between the cohort 1(2 x108 cells) and the cohort 2 (1x109cells). In two patients, transient decline of peripheral abnormal cells that were MDS-related erythroblasts, and decrease of bone marrow blasts were observed, respectively.Although the clinical trial is still ongoing, transfer of WT1-TCR-gene transduced lymphocyte to MDS and AML patients is safe and tolerable. TCR- T cells appeared in peripheral blood with cell-dose dependent manner. DisclosuresFujiwara:Celgene: Honoraria, Other: Travel, Acomodations, Expenses. Akatsuka:Takara Bio. Inc.: Other: Advisor to the CAR project. Tomura:TAKARO BIO INC.: Employment. Nukaya:TAKARA BIO INC.: Employment. Takesako:TAKARA BIO INC.: Employment.

Immunotherapy with TCR-Redirected T Cells: Comparison of TCR-Transduced and TCR-Engineered Hematopoietic Stem Cell–Derived T Cells

Redirecting Ag specificity by transfer of TCR genes into PBLs is an attractive method to generate large numbers of cytotoxic T cells for immunotherapy of cancer and viral diseases. However, transferred TCR chains can pair with endogenous TCR chains, resulting in the formation of mispaired TCR dimers and decreased or unspecific reactivity. TCR gene transfer into hematopoietic stem cells (HSCs) is an alternative to create T cells with desired Ag specificity, because in this case expression of endogenous TCR chains is then less likely owing to allelic exclusion. We generated TCR-transduced T cells from peripheral T cells using the lymphocytic choriomeningitis virus-specific P14 TCR. After transfer of the P14 TCR genes into HSCs and subsequent reconstitution of irradiated mice, TCR-engineered HSC-derived T cells were produced. We then compared the Ag-specific T cell populations with P14 TCR-transgenic T cells for their therapeutic efficiency in three in vivo models. In this study, we demonstrate that TCR-transduced T cells and TCR-engineered HSC-derived T cells are comparable in controlling lymphocytic choriomeningitis virus infection in mice and suppress growth of B16 tumor cells expressing the cognate Ag in a comparable manner.

Redirected T cell-based antileukemia adoptive immunotherapy using tumor antigen-specific TCR gene transfer

Redirected T cell-based antileukemia adoptive immunotherapy using tumor antigen-specific TCR gene transfer

Influenza virus-specific TCR-transduced T cells as a model for adoptive immunotherapy

Adoptive transfer of T lymphocytes equipped with tumor-antigen specific T-cell receptors (TCRs) represents a promising strategy in cancer immunotherapy, but the approach remains technically demanding. Using influenza virus (Flu)-specific T-cell responses as a model system we compared different methods for the generation of T-cell clones and isolation of antigen-specific TCRs. Altogether, we generated 12 CD8+ T-cell clones reacting to the Flu matrix protein (Flu-M) and 6 CD4+ T-cell clones reacting to the Flu nucleoprotein (Flu-NP) from 4 healthy donors. IFN-γ-secretion-based enrichment of antigen-specific cells, optionally combined with tetramer staining, was the most efficient way for generating T-cell clones. In contrast, the commonly used limiting dilution approach was least efficient. TCR genes were isolated from T-cell clones and cloned into both a previously used gammaretroviral LTR-vector, MP91 and the novel lentiviral self-inactivating vector LeGO-MP that contains MP91-derived promotor and regulatory elements. To directly compare their functional efficiencies, we in parallel transduced T-cell lines and primary T cells with the two vectors encoding identical TCRs. Transduction efficiencies were approximately twice higher with the gammaretroviral vector. Secretion of high amounts of IFN-γ, IL-2 and TNF-α by transduced cells after exposure to the respective influenza target epitope proved efficient specificity transfer of the isolated TCRs to primary T-cells for both vectors, at the same time indicating superior functionality of MP91-transduced cells. In conclusion, we have developed optimized strategies to obtain and transfer antigen-specific TCRs as well as designed a novel lentiviral vector for TCR-gene transfer. Our data may help to improve adoptive T-cell therapies.

RhIL-15 and rhIL-2 together enhances growth and function of TCR transduced T cells for adoptive immunotherapy (P4358)

Abstract The use of TCR transduced T cells for the treatment of melanoma has shown promise in recent years. However, there is a need to improve the function and growth of transduced T cells to allow for better clinical outcomes. We wanted to evaluate the impact of rhIL-15 on TCR transduced T cells. T cells from multiple donors were transduced with a retrovirus encoding the TIL 1383I T cell receptor. The transduced T cells were cultured and expanded for approximately 21 days under several different cytokine conditions including 300IU/mL of rhIL-2, 100ng/mL of rhIL-15 or both rhIL-2 and rhIL-15. For each culture condition, data was collected throughout the process for growth, function, phenotype and TCR expression. We found that the T cells cultured in rhIL-15 had a greater TCR expression and produced more IFN-γ in response to tumor than T cells cultured only in rhIL-2. rhIL-15 alone did not increase the growth of TCR transduced T cells when compared to rhIL-2 whereas the use of both rhIL-2 and rhIL-15 not only enhanced the TCR expression and anti-tumor activity but also improved the growth of the T cells. We also noticed that T cells cultured with rhIL-15 increased resistance to apoptosis during oxidative stress. Based on these results, we have initiated our TCR gene transfer clinical trial using rhIL-15 and rhIL-2 to transduce and expand our T cells.

Development of genetically engineered TCR-transduced T cells for immunotherapy of chronic hepatitis B and C virus infections (P4222)

Abstract More than 350 million people worldwide are chronically infected with hepatitis B (HBV) or hepatitis C virus (HCV). While vigorous CD8 T cell responses are required to spontaneously clear acute HBV and HCV infections, these virus-specific T cells are typically diminished in number and functionally impaired in chronic HBV and HCV infections. Here, we set out to use adoptive therapy with TCR-engineered T cells to restore HBV and HCV-specific immune responses. Three HBV-specific and four HCV-specific TCRs with high avidity were cloned from patients and chimpanzees that cleared the respective infection, expressed in retroviral vectors and used to transduce lymphocytes of chimpanzees with chronic HBV and HCV infection. The transduced T cells gained a broad spectrum of antigen-specific effector functions (IFN-γ secretion, cytotoxicity and proliferation). They were expanded to high numbers via short-term anti-CD3 and IL-15/IL-21 stimulation, which resulted in a preservation of their differentiation status and a lower frequency of regulatory T cells than the conventional expansion method with anti-CD3 and IL-2. Collectively, we show that retroviral TCR gene transfer followed by short-term expansion in the presence of IL-15/IL-21 efficiently redirects the antigen specificity of resting primary T cells and generates a large number of functional effector T cells. These will be used in adoptive T cell therapy protocols to restore T cell function in chronic viral hepatitis.

Cord Blood T Cells Retain Early Differentiation Phenotype Suitable for Immunotherapy After TCR Gene Transfer to Confer EBV Specificity

Adoptive T cell therapy can be effective for Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease and melanoma. Transducing high-affinity TCR genes into T lymphocytes is an emerging method to improve potency and specificity of tumor-specific T cells. However, both methods necessitate in vitro lymphocyte proliferation, generating highly differentiated effector cells that display reduced survival and antitumor efficacy postinfusion. TCR-transduction of naive lymphocytes isolated from peripheral blood is reported to provide superior in vivo survival and function. We utilized cord blood (CB) lymphocytes, which comprise mainly naive cells, for transducing EBV-specific TCR. Comparable TCR expression was achieved in adult and CB cells, but the latter expressed an earlier differentiation profile. Further antigen-driven stimulation skewed adult lymphocytes to a late differentiation phenotype associated with immune exhaustion. In contrast, CB T cells retained a less differentiated phenotype after antigen stimulation, remaining CD57-negative but were still capable of antigen-specific polyfunctional cytokine expression and cytotoxicity in response to EBV antigen. CB T cells also retained longer telomeres and in general possessed higher telomerase activity indicative of greater proliferative potential. CB lymphocytes therefore have qualities indicating prolonged survival and effector function favorable to immunotherapy, especially in settings where donor lymphocytes are unavailable such as in solid organ and CB transplantation.

Improving TCR Gene Therapy for Treatment of Haematological Malignancies

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.