Retroviral Gene Transfer of T Cell Receptors (TCR) Specific for Minor Histocompatibility Antigens to Virus-Specific T Cells as Cellular Immunotherapy of Patients with Relapsed Hematological Malignancies after Allogeneic Stem Cell Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5529-5529
Author(s):  
Marieke Griffioen ◽  
H.M. Esther van Egmond ◽  
Menno A.W.G. van der Hoorn ◽  
Renate S. Hagedoorn ◽  
Michel Kester ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematological Malignancies ◽  
Surface Expression ◽  
Retroviral Vector ◽  
Cellular Immunotherapy ◽  
Minor Histocompatibility Antigens ◽  
Vector Coding

Abstract Patients with hematological malignancies can be successfully treated by T cell-depleted allogeneic stem cell transplantation (alloSCT) followed by donor lymphocyte infusion (DLI). Failure of some relapsed hematological malignancies to respond to DLI is probably due to immune tolerance induced by regulatory and/or anergic T cells. We previously showed that functional T cells with redirected anti-leukemic reactivity can be generated by transfer of TCRs specific for minor histocompatibility antigens (mHags) to total peripheral blood mononuclear cells (PBMC) as well as CMV-specific T cells. By introducing TCRs into CMV or EBV specific T cells, T cells with proper memory/effector phenotypes are targeted, and due to virus persistence these T cells may show prolonged survival in vivo. The purpose of this study is to develop an efficient method for the isolation, retroviral transduction and expansion of TCR-transduced CMV- and EBV-specific T cells for cellular immunotherapy of patients with relapsed hematological malignancies after alloSCT. For clinical application, construction of single retroviral vectors coding for the α as well as β chains of mHag-specific TCRs is required. We used the MP71 retroviral vector for TCR gene transfer, since this vector contains Myeloproliferative Sarcoma Virus LTR sequences and a Mouse Embryonic Stem Cell Virus leader, which has been optimized for use in the clinic. The MP71 vector also contained a Woodchuck Hepatitis Response Element (WPRE). The WPRE, which is used as an element enhancing transgene expression at the post-transcriptional level, has recently been described to encode 60 amino acids of a protein with potential oncogenic activity. Therefore, we reconstructed the MP71 vector by introduction of a multiple cloning site (MCS) and, for safety issues, deleted the WPRE. The TCR α and β genes specific for the hematopoietic-restricted mHag HA-2 were linked by a 50-bp sequence encoding a “self-cleaving” 2A-like peptide and introduced into the MCS of the MP71 vector. Linkage of the TCR α and β genes by the 2A-like sequence allowed additional linkage of the low affinity nerve growth factor receptor (LNGFR) or human CD20 selection marker genes by an IRES sequence. The advantage of the human CD20 gene is that it can also function as suicide gene, allowing elimination of transduced cells in vivo if undesired side effects occur. Introduction of the single MP71 retroviral vector coding for the HA-2 TCR α and β chains as well as LNGFR into a TCR α- and β-deficient Jurkat T cell line led to high levels of TCR surface expression correlating with LNGFR marker gene expression. These data indicate proper cleavage and assembly of the transduced TCR α and β chains. Moreover, removal of the WPRE did not affect the surface expression level of the transduced TCR. Furthermore, CMV- and EBV-specific T cells were isolated from human individuals by the IFN-γ capture assay and subsequently transduced with a single retroviral vector coding for the HA-2-TCR α and β chains as well as LNGFR. CMV- and EBV-specific T cells from different human individuals could be successfully isolated to 60–90% purity and the TCR-transduced CMV- and EBV-specific T cells were shown to be fully functional, recognizing the viral peptides as well as the endogenously-processed HA-2 mHag.

scholarly journals Reprogramming of Virus-specific T Cells into Leukemia-reactive T Cells Using T Cell Receptor Gene Transfer

2004 ◽  
Vol 199 (7) ◽  
pp. 885-894 ◽  
Author(s):  
Mirjam H.M. Heemskerk ◽  
Manja Hoogeboom ◽  
Renate Hagedoorn ◽  
Michel G.D. Kester ◽  
Roel Willemze ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hematological Malignancies ◽  
Receptor Gene ◽  
Cell Receptor ◽  
Minor Histocompatibility Antigens ◽  
Leukocyte Antigen ◽  
Dual Specificity ◽  
T Cell Receptor Gene

T cells directed against minor histocompatibility antigens (mHags) might be responsible for eradication of hematological malignancies after allogeneic stem cell transplantation. We investigated whether transfer of T cell receptors (TCRs) directed against mHags, exclusively expressed on hematopoietic cells, could redirect virus-specific T cells toward antileukemic reactivity, without the loss of their original specificity. Generation of T cells with dual specificity may lead to survival of these TCR-transferred T cells for prolonged periods of time in vivo due to transactivation of the endogenous TCR of the tumor-reactive T cells by the latent presence of viral antigens. Furthermore, TCR transfer into restricted T cell populations, which are nonself reactive, will minimize the risk of autoimmunity. We demonstrate that cytomegalovirus (CMV)-specific T cells can be efficiently reprogrammed into leukemia-reactive T cells by transfer of TCRs directed against the mHag HA-2. HA-2-TCR–transferred CMV-specific T cells derived from human histocompatibility leukocyte antigen (HLA)-A2+ or HLA-A2− individuals exerted potent antileukemic as well as CMV reactivity, without signs of anti–HLA-A2 alloreactivity. The dual specificity of these mHag-specific, TCR-redirected virus-specific T cells opens new possibilities for the treatment of hematological malignancies of HLA-A2+ HA-2–expressing patients transplanted with HLA-A2–matched or –mismatched donors.

Improved Vaccine Design For Adoptive Immunotherapy In Hematological Malignancies Through Chemically Modified Minor Histocompatibility Antigen Epitopes

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5435-5435
Author(s):  
Rimke Oostvogels ◽  
Rieuwert Hoppes ◽  
Henk Lokhorst ◽  
Robbert M Spaapen ◽  
Huib Ovaa ◽  
...  
Keyword(s):  
Amino Acids ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Hematological Malignancies ◽  
Ex Vivo

Abstract Allogeneic stem cell transplantation (allo-SCT), alone or followed by donor lymphocyte infusion (DLI), is a potentially curative treatment for various hematological malignancies. In an HLA-matched transplantation setting, the therapeutic graft-versus-tumor (GvT) effect is mediated by donor T-cells directed at minor histocompatibility antigens (mHags), which are HLA-bound polymorphic peptides. Unfortunately, most patients don’t achieve complete response or relapse after allogeneic stem cell transplantation and thus still require additional therapies. Immunotherapy aimed at hematopoietically restricted mHags could theoretically provide an ideal method to augment the GvT effect, without causing GvHD. The most relevant mHags for immunotherapy are those antigens that are only expressed on hematopoietic tissue, are presented by frequent HLA molecules and display an equally balanced population frequency. UTA2-1 and HA-1 are two of these most broadly applicable mHags identified up until now and are therefore included in on-going clinical trials of mHag-peptide loaded dendritic cell vaccination in patients with various hematological malignancies. Another method for mHag-based immunotherapy could be adoptive transfer of ex vivo cultured mHag-specific cytotoxic T lymphocytes (CTL). However, initial results of both methods, also from preclinical models and trials in patients with solid tumors, postulate the necessity for improved strategies for efficient ex vivo and in vivo induction of tumour specific CTLs. We here show for the HLA-A*02 restricted epitopes UTA2-1 and HA-1 that their MHC binding and consequent T cell reactivity can be improved through the incorporation of certain newly designed non-proteogenic amino acids at crucial MHC anchoring positions. With this novel approach we designed superior altered peptide ligands (APLs) for both epitopes, of which the best modifications not only increased MHC binding and stability, but also improved recognition by antigen specific T cells. Most importantly, these optimised peptides gave rise to superior antitumor T cell responses in vitro and in vivo in comparison to the native epitope, as they induced significantly enhanced proliferation of peptide-specific T cells with retained cytotoxic potential against malignant targets expressing the natural UTA2-1 antigen. Hence, these APLs designed with non-proteogenic amino acids with enhanced MHC-affinity and immunogenicity may improve the therapeutic outcome of mHag-based vaccination strategies, or can be utilized for ex vivo antigen-specific T cell enrichment and expansion for transfer into patients with haematological malignancies. Disclosures: Lokhorst: Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria.

scholarly journals Early Reconstitution of Antibody Secreting Cells after Allogeneic Stem Cell Transplantation

2022 ◽  
Vol 11 (1) ◽  
pp. 270
Author(s):  
Martina Hinterleitner ◽  
Clemens Hinterleitner ◽  
Elke Malenke ◽  
Birgit Federmann ◽  
Ursula Holzer ◽  
...  
Keyword(s):  
Innate Immunity ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
B Cells ◽  
Stem Cell Transplantation ◽  
B Cell ◽  
Cell Transplantation ◽  
Antibody Secreting Cells

Immune cell reconstitution after stem cell transplantation is allocated over several stages. Whereas cells mediating innate immunity recover rapidly, adaptive immune cells, including T and B cells, recover slowly over several months. In this study we investigated kinetics and reconstitution of de novo B cell formation in patients receiving CD3 and CD19 depleted haploidentical stem cell transplantation with additional in vivo T cell depletion with monoclonal anti-CD3 antibody. This model enables a detailed in vivo evaluation of hierarchy and attribution of defined lymphocyte populations without skewing by mTOR- or NFAT-inhibitors. As expected CD3+ T cells and their subsets had delayed reconstitution (<100 cells/μL at day +90). Well defined CD19+ B lymphocytes of naïve and memory phenotype were detected at day +60. Remarkably, we observed a very early reconstitution of antibody-secreting cells (ASC) at day +14. These ASC carried the HLA-haplotype of the donor and secreted the isotypes IgM and IgA more prevalent than IgG. They correlated with a population of CD19− CD27− CD38low/+ CD138− cells. Of note, reconstitution of this ASC occurred without detectable circulating T cells and before increase of BAFF or other B cell stimulating factors. In summary, we describe a rapid reconstitution of peripheral blood ASC after CD3 and CD19 depleted haploidentical stem cell transplantation, far preceding detection of naïve and memory type B cells. Incidence before T cell reconstitution and spontaneous secretion of immunoglobulins allocate these early ASC to innate immunity, eventually maintaining natural antibody levels.

Adoptive Transfer of Adenovirus Specific T-Cells in Children with Systemic Adenovirus Infection after Allogeneic Stem Cell Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 80-80
Author(s):  
Tobias F. Feuchtinger ◽  
Susanne Matthes-Martin ◽  
Celine Richard ◽  
Thomas Lion ◽  
Klaus Hamprecht ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Adoptive Transfer ◽  
Cell Transfer ◽  
New Treatment ◽  
T Cell Transfer

Abstract Allogeneic stem cell transplantation (SCT) has become an increasing treatment option for a variety of malignant and non-malignant disease. During immune reconstitution the host is at significant risk for viral infections. Human adenovirus (HAdV) infection is especially in children an important and serious complication. Virus-specific T-cells are essential for the clearance of HAdV, since antiviral chemotherapy has been insufficient to date. We present a new treatment option using virus-specific donor T-cells for adoptive transfer of immunity to patients with systemic HAdV-infection. We isolated in 6 patients with systemic HAdV-infection after SCT virus-specific T-cells of the donor, according to INF-γ secretion after short in vitro stimulation with viral antigen, resulting in a combination of CD4+ and CD8+ T-cells. Between 5-50x103/kg T-cells were infused for adoptive transfer. For follow-up, the infection and the in-vivo expansion of infused T-cells were evaluated. Isolated cells showed high specificity and markedly reduced but residual alloreactivity in-vitro. In three of four evaluable patients the infused T-cells underwent an in-vivo expansion and in these three patients the viral load decreased in peripheral blood after adoptive T-cell transfer. In-vivo expansion of specific T-cells was dose-independent. T-cell infusion was well tolerated. One patient experienced GvHD°II of the skin after T-cell transfer. In conclusion specific T-cell immunotherapy as a new treatment approach for children was performed in 6 cases of systemic HAdV-infection after allogeneic SCT. Induction of a specific T-cell response through adoptive transfer has been shown feasible and effective to protect from HAdV-related complications.

Attenuated Acute Graft-Versus-Host Disease Following Allogeneic Stem Cell Transplantation In the Absence of RORγt.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3742-3742
Author(s):  
LeShara M Fulton ◽  
Michael J Carlson ◽  
James Coghill ◽  
Michelle L. West ◽  
Angela Panoskaltisis-Mortari ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Graft Versus Host Disease ◽  
Allogeneic Stem Cell Transplantation ◽  
Graft Versus Host ◽  
Donor T Cells

Abstract Abstract 3742 CD4+ T helper (Th) cells play a critical role in the development of Graft-versus-Host Disease (GvHD). The relative contributions of particular Th subsets to GVHD pathogenesis, however, are incompletely understood. In order to clarify the contribution of the Th17 subset to GVHD induction, we made use of mice knocked out at the RORgt locus (RORgt−/−), a transcription factor crucial for Th17 polarization. Methods: Haplotype matched and complete MHC mismatched murine HSCT models were used. For the haploidentical model C57BL/6 (H-2b, B6) mice served as donors while C57BL/6 × DBA2 F1 (H-2bxd, B6D2) mice functioned as recipients. Effector T cells (Teffs) were isolated from the spleens of wild type (WT) B6 and RORgt knockout mice backcrossed 7–8 generations onto a B6 background. B6D2 mice were lethally irradiated with 900 rads on day -1 and injected intravenously with 4 × 106 Teffs from WT or RORgt−/− mice supplemented with 3 × 106 WT T cell depleted bone marrow cells (TCD BM) on day 0. For the completely MHC mismatched model, BALB/c mice (H-2d) were lethally irradiated with 800 rads on day -1 and administered 5 × 105 WT or RORgt−/− Teffs supplemented with 5 × 106 B6 TCD BM on day 0. Results: B6D2 mice that received RORgt−/− Teffs displayed significantly attenuated GvHD, recovering from weight loss by day +31 and demonstrating 100% survival on day +60. Conversely, mice that received WT Teffs showed intense disease progression with 100% mortality by day +31 (Figure A, p<0.0001 for survival comparison between WT and RORgt−/− recipients using Fisher's exact test). Similar results were seen using the completely MHC mismatched model, with superior overall survival noted in those animals receiving RORgt −/− Teffs (put in p value here). Recipients of RORgt −/− T cells demonstrated statistically significant decreased TNF in serum compared to WT recipients (Figure B, p=0.001 comparing WT and RORgt−/− recipients using student's t test). Interestingly, despite the decreased severity of GvHD, serum concentrations of IFN-g were increased in recipients transplanted with RORgt −/− T cells. Chimerism studies post-transplant revealed complete donor reconstitution in recipients of both RORgt−/− and WT Teffs. Donor Teffs isolated from recipient livers post-transplant consistently demonstrated an activated phenotype, with low L selectin and high CD25 expression. Conclusions: T cell expression of the Th17 transcription factor, RORgt, is critical for the development of lethal GvHD following allogeneic stem cell transplantation in both the haploidentical and MHC complete mismatch models. GvHD attenuation in the absence of RORgt is not the result of an inability for donor T cells to undergo activation or to engraft in vivo. Interestingly, the absence of RORgt from donor T cells led to enhanced IFN-g in serum. Thus, in vivo, the Th17 pathway is critical for the induction of GvHD. Disclosures: No relevant conflicts of interest to declare.

Effect of a Novel Nucleoside Analogue, Triciribine Phosphate (TCN-P) on Murine Acute Graft-Vrs-Host Disease (aGvHD)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2977-2977
Author(s):  
Edward Dela Ziga ◽  
Jaebok Choi ◽  
Mark Needles ◽  
Julie Ritchey ◽  
John F. DiPersio
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nucleoside Analogue ◽  
Foxp3 Expression ◽  
Negative Control ◽  
Jurkat Cells ◽  
Acute Graft

Abstract Abstract 2977 BACKGROUND: The successful establishment of donor registries and development of improved conditioning regimens among others, has led to the increased use of hematopoietic stem cell transplant (HSCT) as a key component in the treatment of some malignant and benign hematopoietic/lymphoid disorders as well as some metabolic disorders. Although a potential curative therapy for many hematologic diseases, allogeneic stem cell transplantation is associated with considerable morbidity and mortality primarily from acute graft-versus-host disease (aGvHD). Furthermore, graft-versus-leukemia (GVL) mediated by donor T cells can be abrogated with T cell depletion or suppression in vivo resulting in disease relapse with treatment of aGvHD. Moreso, modern therapies for aGvHD are limited and often toxic, thus there is a need for novel treatments and approaches that control aGvHD without compromising GVL. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor has been shown to decrease the severity of aGvHD (Reddy et al, PNAS 2004) through its effect on pro-inflammatory cytokines while maintaining GVL in a murine GvHD model. Also, previous work from our lab demonstrated that treatment of mice with the hypomethylating agent azacitidine (AzaC) after allogeneic HSCT mitigates aGvHD while preserving GVL by inducing FOXP3 expression in activated non-T regulatory cells (Choi et al, Blood 2010). However, the myelosuppression mediated by AzaC is a potential limitation that results in delayed donor engraftment. This led us to explore alternate options for single or combination drug therapy in the treatment of aGvHD. We screened a library of 2000 chemical agents obtained from the National Institutes of Health. Screening resulted in a single hit identified as Triciribine phosphate (TCN-P), an Akt inhibitor with structural similarity to the nucleoside analogue AzaC. In this experiment, a Foxp3 promoter-luciferase construct was designed and transfected into Jurkat cells. Cells were incubated for 2 days and then treated with three concentrations (0.1uM, 1umM and 10uM) of each chemical agent in the library. Bioluminescence imaging (BLI) was done on day 4 with AzaC as positive control (Choi et al, Blood 2010) and PBS as negative control. Only wells treated with TCN-P 10uM showed a signal, suggesting luciferase activity secondary to the Foxp3-promoter activation. We therefore hypothesized that TCN-P as a single agent or in combination with SAHA and or AzaC would mitigate GvHD by inducing FOXP3 without interfering with engraftment or immune reconstitution. METHODS: Using a C57BL/6(H2b) into Balb/c (H2d) murine MHC mismatch bone marrow transplant (BMT) model, we transplanted 5 × 106 T cell-depleted (TCD) bone marrow cells obtained from C57BL/6 (H2b, CD45.1+) mice into Balb/c (H2d, CD45.2+) mice after 900cGy of TBI. Delayed donor infusions of 2 × 106 pan-T cells/mouse obtained from FOXP3/GFP KI: B6 CD45.2+ H2b mice were infused on Day +11 in order to induce GvHD. Azacitidine 2mg/kg, SAHA 35mg/kg and TCN-P 10mg/kg were injected intraperitoneally every other day from Day +15 to Day +21(total of 4 doses). Acute GvHD was assessed by a standardized scoring developed by Cooke and Ferrara. (Blood, 1996) RESULTS : 1. Using our Foxp3-reporter system, both AzaC and TCN-P induced significant luciferase expression in Jurkat cells. SAHA had no effect. 2. Only AzaC but neither SAHA nor TCN-P induced significant Foxp3 expression in WT bead activated T cells. 3. In vivo, both AzaC 2mg/kg and TCN-P 10mg/kg but not SAHA 35mg/kg significantly improved survival of mice with less weight loss and clinical signs of aGvHD in a MHC mismatched aGvHD model. CONCLUSION: A novel nucleoside analogue TCN-P that was previously FDA approved for treatment of multiple myeloma and structurally related to AzaC, induces Foxp3 using a luciferase reporter construct in Jurkat cells and improves survival in mice after MHC mismatched allogeneic transplant. Though the 100 day survival between TCN-P and PBS (as negative control) in our murine aGvHD model was not quite statistically significant, the findings suggest a therapeutic potential for TCN-P and possibly other Akt inhibitors in the mitigation of aGvHD. Disclosures: No relevant conflicts of interest to declare.

Interruption of IFNγR Signaling Results in Altered T Cell Trafficking in Vivo and Abrogation of GvHD While Maintaining a Robust Gvl Response

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 455-455
Author(s):  
Jaebok Choi ◽  
Edward Dela Ziga ◽  
Julie Ritchey ◽  
Lynne Collins ◽  
Julie Prior ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell ◽  
Cell Trafficking ◽  
Hematopoietic Stem ◽  
T Cell Trafficking ◽  
Target Organs ◽  
Donor T Cells

Abstract Abstract 455 Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for patients with relapsed/refractory leukemia, and marrow failure states such as myelodysplasia and aplastic anemia. However, allo-HSCT is complicated by allogeneic donor T cell-mediated graft-versus-host disease (GvHD) which can be life-threatening especially in recipients of unrelated or HLA-mismatched hematopoietic stem cell products. These same alloreactive donor T cells also mediate a beneficial graft-versus-leukemia (GvL) effect. Thus, the clinical goal in allo-HSCT is to minimize GvHD while maintaining GvL. Recent studies have suggested that this might be achieved by infusing regulatory T cells (Tregs) which in some preclinical models suppress GvHD-causing alloreactive donor T cells but have only limited effects on GvL-promoting alloreactive donor T cells. Unfortunately, Tregs exist in low frequency in the peripheral blood, are costly to purify and expand, and after expansion are difficult to isolate due to the lack of cell surface markers, all of which prevent their routine use in the clinic. Thus, alternative therapeutic approaches that do not require Tregs are needed. We have found that interferon gamma receptor deficient (IFNγR−/−) allogeneic donor T cells induce significantly less GvHD in both a MHC fully-mismatched (B6 (H-2b) → Balb/c (H-2d)) and a minor-mismatched (B6 (H-2b) → B6×129(H-2b)) allo-HSCT models compared to WT T cells. In addition, IFNγR−/− donor T cells maintain a beneficial GvL effect, which has been examined in both systemic leukemia and solid tumor models using luciferase-expressing A20 cells derived from Balb/c. We find that IFNγR−/− T cells migrate primarily to the spleen while WT T cells to GI tract and peripheral lymph nodes (LNs) using bioluminescence imaging (BLI), suggesting that altered T cell trafficking of IFNγR−/− T cells to GvHD target organs might be the major reason for the reduced GvHD. We further demonstrate that the IFNγR-mediated signaling in alloreactive donor T cells is required for expression of CXCR3 which has been implicated in trafficking of T cells to areas of inflammation and target organs, commonly known to be the sites of GvHD. Indeed, CXCR3−/− T cells recapitulate the reduced GvHD potential of IFNγR−/− T cells. In addition, forced overexpression of CXCR3 in IFNγR−/− T cells via retroviral transduction partially rescues the GvHD defect observed in IFNγR−/− T cells. We next examine if inhibition of IFNγR signaling using a small molecule inhibitor can recapitulate the anti-GVHD effects seen in IFNγR−/− T cells. We find that INCB018424, an inhibitor of JAK1/JAK2 which are the mediators of IFNγR signaling, blocks CXCR3 expression in vitro. Most importantly, in vivo administration of INCB018424 after allo-HSCT alters T cell trafficking and significantly reduces GvHD. Thus, the IFNγR signaling pathway represents a promising therapeutic target for future efforts to mitigate GvHD while maintaining GvL after allo-HSCT. Moreover, this pathway can be exploited in other diseases besides GvHD such as those from organ transplantation, chronic inflammatory diseases and autoimmune diseases. Disclosures: DiPersio: genzyme: Honoraria.

Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1999-1999
Author(s):  
Annie L. Oh ◽  
Dolores Mahmud ◽  
Benedetta Nicolini ◽  
Nadim Mahmud ◽  
Elisa Bonetti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

CD3 limits the efficacy of TCR gene therapy in vivo

Blood ◽  
2011 ◽  
Vol 118 (13) ◽  
pp. 3528-3537 ◽  
Author(s):  
Maryam Ahmadi ◽  
Judith W. King ◽  
Shao-An Xue ◽  
Cécile Voisine ◽  
Angelika Holler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Cell Receptor ◽  
Surface Expression ◽  
T Cell Function ◽  
Engineered T Cells ◽  
Rate Limiting

Abstract The function of T-cell receptor (TCR) gene modified T cells is dependent on efficient surface expression of the introduced TCR α/β heterodimer. We tested whether endogenous CD3 chains are rate-limiting for TCR expression and antigen-specific T-cell function. We show that co-transfer of CD3 and TCR genes into primary murine T cells enhanced TCR expression and antigen-specific T-cell function in vitro. Peptide titration experiments showed that T cells expressing introduced CD3 and TCR genes recognized lower concentration of antigen than T cells expressing TCR only. In vivo imaging revealed that TCR+CD3 gene modified T cells infiltrated tumors faster and in larger numbers, which resulted in more rapid tumor elimination compared with T cells modified by TCR only. After tumor clearance, TCR+CD3 engineered T cells persisted in larger numbers than TCR-only T cells and mounted a more effective memory response when rechallenged with antigen. The data demonstrate that provision of additional CD3 molecules is an effective strategy to enhance the avidity, anti-tumor activity and functional memory formation of TCR gene modified T cells in vivo.

scholarly journals Discovery of TSC-101: A First-in-Class Natural HA-2-Specific TCR to Treat Leukemia Following Hematopoietic Stem Cell Transplant Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1704-1704
Author(s):  
Ribhu Nayar ◽  
Mollie M Jurewicz ◽  
Sonal Jangalwe ◽  
Hannah Bader ◽  
Kimberly M Cirelli ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplant ◽  
Stem Cell Transplant ◽  
Surface Expression ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Genome Wide ◽  
Clinical Candidate

Abstract Background Approximately 50% of AML patients relapse following allogeneic hematopoietic stem cell transplant therapy, leaving them with very few treatment options (Rautenberg et al. (2019) Int. J. Mol. Sci. 20:228). Rare patients who naturally develop a minor antigen-specific graft-versus-leukemia T cell response show substantially lower relapse rates (Marijt et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100:2742-2747; Spierings et al. (2013) Biol. Blood Marrow Transplant. 19:1244-1253). HA-2 (YIGEVLVSV, genotype RS_61739531 C/C or T/C) is an HLA-A*02:01- and haematopoietically-restricted minor histocompatibility antigen derived from the class I myosin protein, MYO1G (Pierce et al. (2001) J. Immunol. 167:3223-3230). Patients receiving donor lymphocyte infusion from HA-2-mismatched donors who develop HA-2-specific T cells show a graft vs leukemia response and often experience long-term remission (Marijt et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100:2742-2747), making HA-2 an ideal candidate for TCR-engineered T cell immunotherapy of liquid tumors. Methods Using TScan's proprietary ReceptorScan platform, we discovered 1,302 HA-2-specific TCRs by screening 237 million naïve CD8 + T cells from 5 healthy HA-2-negative donors. We evaluated these TCRs using our proprietary DexScan platform to select the 15 TCRs with the highest surface expression and greatest affinity for the HA-2 peptide when transferred into primary human T cells. We further tested each TCR individually in our clinical vector backbone for surface expression, selective cytotoxicity, cytokine production, and proliferation using a panel of cell lines that express varying levels of HLA-A*02:01 and MYO1G. Finally, the top 5 TCRs were evaluated for alloreactivity using an array-based screen assessing 108 MHC-I molecules individually, and for off-target cross-reactivity using our proprietary genome-wide TargetScan platform. A lead TCR with limited alloreactivity and a narrow off-target profile was selected as our lead TSC-101 TCR. The avidity of TSC-101 for its putative off-targets was further measured in peptide-pulsed experiments to better appreciate the toxicity risks associated with our lead clinical candidate. Results and Conclusion Of the 1,302 HA-2-specific TCRs identified by our ReceptorScan platform, we identified TSC-101 as the most active TCR. TSC-101 displayed no alloreactivity to 107/108 HLAs tested and limited off-target risks in a genome-wide screens. Potential off-target peptides identified for TSC-101 displayed extremely weak avidities, predicting an absence of toxicity risks for our clinical candidate. Based on these results, TSC-101 has been advanced to IND-enabling activities to prepare for first-in-human testing in 2022. To our knowledge, this is the first clinical grade HA-2-specifc TCR being developed for immunotherapy for liquid tumors. Disclosures Macbeath: TScan Therapeutics: Current Employment, Current equity holder in publicly-traded company.

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