In vitro–expanded donor alloantigen–specific CD4+CD25+ regulatory T cells promote experimental transplantation tolerance

Blood ◽  
2006 ◽  
Vol 109 (2) ◽  
pp. 827-835 ◽  
Author(s):  
Dela Golshayan ◽  
Shuiping Jiang ◽  
Julia Tsang ◽  
Marina I. Garin ◽  
Christian Mottet ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Skin Graft ◽  
Graft Rejection ◽  
Treg Cells ◽  
Transplant Tolerance ◽  
Cba Mice

Abstract CD4+CD25+ regulatory T (Treg) cells play a critical role in the induction and maintenance of peripheral immune tolerance. In experimental transplantation models in which tolerance was induced, donor-specific Treg cells could be identified that were capable of transferring the tolerant state to naive animals. Furthermore, these cells appeared to have indirect allospecificity for donor antigens. Here we show that in vivo alloresponses can be regulated by donor alloantigen-specific Treg cells selected and expanded in vitro. Using autologous dendritic cells pulsed with an allopeptide from H2-Kb, we generated and expanded T-cell lines from purified Treg cells of CBA mice (H2k). Compared with fresh Treg cells, the cell lines maintained their characteristic phenotype, suppressive function, and homing capacities in vivo. When cotransferred with naive CD4+CD25− effector T cells after thymectomy and T-cell depletion in CBA mice that received CBK (H2k+Kb) skin grafts, the expanded Treg cells preferentially accumulated in the graft-draining lymph nodes and within the graft while preventing CBK but not third-party B10.A (H2k+Dd) skin graft rejection. In wild-type CBA, these donor-specific Treg cells significantly delayed CBK skin graft rejection without any other immunosuppression. Taken together, these data suggest that in vitro–generated tailored Treg cells could be considered a therapeutic tool to promote donor-specific transplant tolerance.

scholarly journals T-cell–intrinsic Tif1α/Trim24 regulates IL-1R expression on TH2 cells and TH2 cell-mediated airway allergy

2016 ◽  
Vol 113 (5) ◽  
pp. E568-E576 ◽  
Author(s):  
Jimena Perez-Lloret ◽  
Isobel S. Okoye ◽  
Riccardo Guidi ◽  
Yashaswini Kannan ◽  
Stephanie M. Coomes ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Allergic Diseases ◽  
Treg Cells ◽  
Th2 Cells ◽  
T Helper 2 ◽  
Cytokines And Chemokines ◽  
Th2 Cell

There is a paucity of new therapeutic targets to control allergic reactions and forestall the rising trend of allergic diseases. Although a variety of immune cells contribute to allergy, cytokine-secreting αβ+CD4+ T-helper 2 (TH2) cells orchestrate the type-2–driven immune response in a large proportion of atopic asthmatics. To identify previously unidentified putative targets in pathogenic TH2 cells, we performed in silico analyses of recently published transcriptional data from a wide variety of pathogenic TH cells [Okoye IS, et al. (2014) Proc Natl Acad Sci USA 111(30):E3081–E3090] and identified that transcription intermediary factor 1 regulator-alpha (Tif1α)/tripartite motif-containing 24 (Trim24) was predicted to be active in house dust mite (HDM)- and helminth-elicited Il4gfp+αβ+CD4+ TH2 cells but not in TH1, TH17, or Treg cells. Testing this prediction, we restricted Trim24 deficiency to T cells by using a mixed bone marrow chimera system and found that T-cell–intrinsic Trim24 is essential for HDM-mediated airway allergy and antihelminth immunity. Mechanistically, HDM-elicited Trim24−/− T cells have reduced expression of many TH2 cytokines and chemokines and were predicted to have compromised IL-1–regulated signaling. Following this prediction, we found that Trim24−/− T cells have reduced IL-1 receptor (IL-1R) expression, are refractory to IL-1β–mediated activation in vitro and in vivo, and fail to respond to IL-1β–exacerbated airway allergy. Collectively, these data identify a previously unappreciated Trim24-dependent requirement for IL-1R expression on TH2 cells and an important nonredundant role for T-cell–intrinsic Trim24 in TH2-mediated allergy and antihelminth immunity.

Inhibition Of Cdk2 Promotes The Generation Of Inducible CD8+ T Regulatory Cells By Modulating The Epigenetic Regulator EZH2

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 138-138
Author(s):  
Lequn Li ◽  
Nikolaos Patsoukis ◽  
Anoma Nellore ◽  
Vassiliki A. Boussiotis
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fold Increase ◽  
Treg Cells ◽  
Foxp3 Mrna ◽  
Alloreactive T Cells ◽  
Epigenetic Regulator ◽  
Treg Differentiation

Abstract Graft versus host disease (GvHD) remains the main cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation. In spite of the intense research efforts, control of GvHD remains incomplete and novel therapeutic approaches are required. Cdk2 has a central role in cell cycle re-entry of mature T lymphocytes and inhibition of Cdk2 is mandatory for induction of T cell anergy in vitro and tolerance in vivo. While Cdk2 is essential for expansion of activated T cells, it is not critical for survival of resting lymphocytes, hematopoiesis or thymocyte development. These properties make Cdk2 an attractive target for control of GvHD. To determine the effects of Cdk2 inhibition on T cell alloresponses in vivo, we used the B6D2F1 mouse model of allogeneic BMT and two different Cdk2 inhibitors, CYC202 (IC50=0.1 uM) and CYC205 (IC50=1 nM). Lethally irradiated B6D2F1(Kd) recipients were infused with bone marrow from C57BL/6(Kb) donors with (BMT) or without splenocytes (BM) and were subsequently treated with each Cdk2 inhibitor for three weeks. Treatment was administered daily during week 1, every other day on week 2, and twice a week on week 3. Effects of treatment on GvHD were assessed by body weight and survival during a 70-day period. Although BMT recipients treated with Cdk2 inhibitor displayed a transient initial weight loss, subsequently regained weight to levels comparable to control BM recipients. Furthermore, treated BMT recipient groups displayed significantly delayed GvHD mortality (p=0.0054). Recently, it was determined that inducible CD8+ Treg cells, have a central role in mediating protection from GvHD. Some immunosuppressive drugs have detrimental effects on Treg whereas others spare these cells or may even be beneficial to their proportional increase. To examine whether Cdk2 inhibitors induced Treg cells, we used GFP- T cells from Foxp3.GFP-KI mice (C57BL/6 background) as a source of T cells during BMT. Assessment of peripheral blood lymphocytes, splenocytes, peripheral lymph nodes and intestinal lymphoid cells (ILC) in BMT recipients revealed no differences in CD4+GFP+ Treg between treated and control groups. In contrast, the treated group displayed an increase of CD8+GFP+ Treg cells in these cell populations, predominantly ILC, which displayed a 5-fold increase of CD8+ Treg (p=0.05). To further investigate whether Cdk2 inhibitors had a selective effect on CD8+ Treg differentiation, we isolated CD4+GFP- and CD8+GFP- T cells from Foxp3.GFP-KI mice and subjected them to in vitro Treg polarizing with or without Cdk2 inhibitors. Inhibition of Cdk2 had almost no effect on CD4+GFP+ cells but induced a 2-4 fold increase of CD8+GFP+ cells. To determine whether Cdk2 inhibition induced its effect on CD8+ Treg differentiation by reducing the threshold of TGF-β-mediated signaling, we cultured CD8+GFP- cells with stable concentrations of Cdk2 inhibitors and decreasing concentrations of TGF-β. Cdk2 inhibition induced CD8+ Treg differentiation in the presence of TGF-β concentrations that failed to induce any significant numbers of CD8+ Treg cells when used alone. Expression of FOX family genes is regulated by transcriptional and epigenetic mechanisms. A critical epigenetic regulator of FOX transcription factors in cancer cells is the Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), which promotes histone H3 lysine 27 trimethylation (H3K27me3) and induces epigenetic gene silencing. Cdk1 and Cdk2 phosphorylate EZH2 at Thr350 in an evolutionarily conserved motif. Phosphorylation of Thr350 is important for EZH2 recruitment and maintenance of H3K27me3 levels at EZH2-target loci. We examined whether EZH2 becomes phosphorylated in CD8+ T cells and whether Cdk2 inhibition might affect this event. Upon polarizing CD8+ T cell culture, EZH2 displayed robust phosphorylation on Thr350, which was blocked by Cdk2 inhibition. This event temporally coincided with a 44-fold increase in Foxp3 mRNA expression compared to base line levels in control T cells. These results reveal an unexpected mechanism via which Cdk2 inhibitors mediate suppression of alloreactive T cells and protection from GvHD by inducing CD8+ Treg. Because Cdk-mediated EZH2 phosphorylation is a key mechanism governing EZH2 function to regulate epigenetic silencing, Cdk2 inhibition might have additional, yet unidentified implications on gene expression programs of alloreactive T cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Clinical Development of a Non-Gene-Edited Allogeneic Bcma-Targeting CAR T-Cell Product in Relapsed or Refractory Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 27-28
Author(s):  
A. Samer Al-Homsi ◽  
Sebastien Anguille ◽  
Jason Brayer ◽  
Dries Deeren ◽  
Nathalie Meuleman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Gene Editing ◽  
Cell Product ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Background Autologous CAR T-cell therapy targeting the B-cell maturation antigen (BCMA) has shown impressive objective response rates in patients with advanced multiple myeloma (MM). Clinical grade manufacturing of autologous CAR T-cells has limitations including vein-to-vein delivery time delay and potentially sub-optimal immunological capability of T-cells isolated from patients with advanced disease. Allogeneic CAR T-cell products, whereby cells from healthy third-party donors are used to generate an "off-the-shelf" CAR T-cell product, have the potential to overcome some of these issues. To circumvent the primary potential risk of graft-versus-host disease (GvHD) associated with the use of allogeneic T-cells, abrogation of the T-cell receptor (TCR) expression in the CAR T-cells, via gene editing, is being actively pursued. To avoid the potential safety risks and manufacturing challenges associated with gene editing, the allogeneic CYAD-211 CAR T-cell product exploits short hairpin RNA (shRNA) interference technology to down-regulate TCR expression thus avoiding the risk of life-threatening GvHD. Aim The aim is to generate a BCMA-specific allogeneic CAR T-cell product using a non-gene editing approach and study its activity both in vitro and in vivo. CYAD-211 combines a BCMA-specific CAR with a single optimized shRNA targeting the TCR CD3ζ subunit. Downregulation of CD3ζ impairs the TCR expression on the surface of the donor T-cells, preventing their reactivity with the normal host tissue cells and potential GvHD induction. Maintaining all the elements required for the therapy within a single vector (all-in-one vector) provides some significant manufacturing advantages, as a solitary selection step will isolate cells expressing all the desired traits. Results CYAD-211 cells produce high amounts of interferon-gamma (IFN-γ) during in vitro co-cultures with various BCMA-expressing MM cell lines (i.e., RPMI-8226, OPM-2, U266, and KMS-11). Cytotoxicity experiments confirmed that CYAD-211 efficiently kills MM cell lines in a BCMA-specific manner. The anti-tumor efficacy of CYAD-211 was further confirmed in vivo, in xenograft MM models using the RPMI-8226 and KMS-11 cell lines. Preclinical data also showed no demonstrable evidence of GvHD when CYAD-211 was infused in NSG mice confirming efficient inhibition of TCR-induced activation. Following FDA acceptance of the IND application, IMMUNICY-1, a first-in-human, open-label dose-escalation phase I clinical study evaluating the safety and clinical activity of CYAD-211 for the treatment of relapsed or refractory MM patients to at least two prior MM treatment regimens, is scheduled to begin recruitment. IMMUNICY-1 will evaluate three dose-levels of CYAD-211 (3x107, 1x108 and 3x108 cells/infusion) administered as a single infusion after a non-myeloablative conditioning (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, daily for 3 days) according to a classical Fibonacci 3+3 design. Description of the study design and preliminary safety and clinical data from the first cohort will be presented at ASH 2020. Conclusion CYAD-211 is the first generation of non-gene edited allogeneic CAR T-cell product based on shRNA technology. The IMMUNICY-1 clinical study seeks to provide proof of principle that single shRNA-mediated knockdown can generate fully functional allogeneic CAR T-cells in humans without GvHD-inducing potential. We anticipate that subsequent generations of this technology will incorporate multiple shRNA hairpins within a single vector system. This will enable the production of allogeneic CAR T-cells in which multiple genes of interest are modulated simultaneously thereby providing a platform approach that can underpin the future of this therapeutic modality. Figure 1 Disclosures Al-Homsi: Celyad: Membership on an entity's Board of Directors or advisory committees. Brayer:Janssen: Consultancy; Bristol-Myers Squibb, WindMIL Therapeutics: Research Funding; Bristol-Myers Squibb, Janssen, Amgen: Speakers Bureau. Nishihori:Novartis: Other: Research support to institution; Karyopharm: Other: Research support to institution. Sotiropoulou:Celyad Oncology: Current Employment. Twyffels:Celyad Oncology: Current Employment. Bolsee:Celyad Oncology: Current Employment. Braun:Celyad Oncology: Current Employment. Lonez:Celyad Oncology: Current Employment. Gilham:Celyad Oncology: Current Employment. Flament:Celyad Oncology: Current Employment. Lehmann:Celyad Oncology: Current Employment.

scholarly journals Prostaglandin E2 directly inhibits the conversion of inducible regulatory T cells through EP2 and EP4 receptors via antagonizing TGF-β signalling

2021 ◽  
Author(s):  
Marie Goepp ◽  
Siobhan Crittenden ◽  
You Zhou ◽  
Adriano G Rossi ◽  
Shuh Narumiya ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Prostaglandin E2 ◽  
Th17 Cells ◽  
Foxp3 Expression ◽  
Treg Cells ◽  
Itreg Cell

Background and Purpose: Regulatory T (Treg) cells are essential for control of inflammatory processes by suppressing Th1 and Th17 cells. The bioactive lipid mediator prostaglandin E2 (PGE2) promotes inflammatory Th1 and Th17 cells and exacerbates T cell-mediated autoimmune diseases. However, the actions of PGE2 on the development and function of Treg cells, particularly under inflammatory conditions, are debated. In this study, we examined whether PGE2 had a direct action on T cells to modulate de novo differentiation of Treg cells. Experimental Approach: We employed an in vitro T cell culture system of TGF-β-dependent Treg induction from naive T cells. PGE2 and selective agonists for its receptors, and other small molecular inhibitors were used. Mice with specific lack of EP4 receptors in T cells were used to assess Treg cell differentiation in vivo. Human peripheral blood T cells from healthy individuals were used to induce differentiation of inducible Treg cells. Key Results: TGF-β-induced Foxp3 expression and Treg cell differentiation in vitro was markedly inhibited by PGE2, which was due to interrupting TGF-β signalling. EP2 or EP4 agonism mimicked suppression of Foxp3 expression in WT T cells, but not in T cells deficient in EP2 or EP4, respectively. Moreover, deficiency of EP4 in T cells impaired iTreg cell differentiation in vivo. PGE2 also appeared to inhibit the conversion of human iTreg cells. Conclusion and Implications: Our results show a direct, negative regulation of iTreg cell differentiation by PGE2, highlighting the potential for selectively targeting the PGE2-EP2/EP4 pathway to control T cell-mediated inflammation.

Expansion and Transformation of Primary Acute Lymphoblastic Leukemia Cells into Antigen-Presenting Cells using a Novel Culturing System Enables the Generation of Leukemia-Reactive T Cell Responses that Are Effective in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 303-303
Author(s):  
Bart A. Nijmeijer ◽  
Marianke L.J. Van Schie ◽  
Roel Willemze ◽  
J.H. Frederik Falkenburg
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Primary Cells ◽  
T Cell Anergy ◽  
Cell Anergy ◽  
Antigen Presenting

Abstract Allogeneic cellular immunotherapy is generally ineffective in acute lymphoblastic leukemia (ALL). In vitro studies have suggested that this inefficacy may be the result of a lack of costimulatory molecule expression by ALL cells, resulting in the induction of T cell anergy. Activation of T cells by ALL cells that are transformed into adequate antigen-presenting cells (ALL-APC) may prevent the induction of T cell anergy and result in the generation of competent leukemia-reactive T cell responses for adoptive immunotherapy. However, in vitro modification of ALL cells was hampered by the fact that ALL cells from adult patients could not be cultured in vitro for prolonged periods of time. We have developed a novel serum-free culturing system for B-lineage ALL in which proliferation is initiated and sustained by ALL-cell derived growth factors. Long-term (>2 yrs) proliferation was induced in 12 out of 26 randomly selected primary samples from patients with ALL. The cell cultures ( Leiden cell lines) proliferated with a mean doubling time of 3.0 days (range 2.7–3.6 days). All Leiden cell lines presented the chromosomal abberations observed in the primary cells. The Leiden cell lines displayed an immune phenotype similar to the primary cells, exept for loss of CD34 expression. In vivo characteristics of Leiden cells were evaluated in NOD/scid mice. After intravenous inoculation, Leiden cell lines and primary cells showed identical homing patterns initially involving spleen and bone marrow, followed by the development of overt and progressive leukemia. A comparison of in vivo progression kinetics was performed for one of the Leiden cell lines and the corresponding primary cells. Weekly determination of leukemic cell counts in the blood of engrafted animals revealed that the cell line and the primary cells displayed similar doubling times in vivo of 6.3 and 7.7 days, respectively. To generate cells with improved antigen presentation function, Leiden cell lines were exposed to various activating agents. Stimulation with CpG containing oligonucleotides resulted in induction of CD40 in 9 out of 10 lines. Subsequent ligation of CD40 by culturing CpG-activated Leiden cells on fibroblasts expressing human CD40 ligand resulted in the induction of CD80 or CD86 in 7 of these 10 cell lines. To study the immune stimulatory properties of these Leiden ALL-APC, allogeneic HLA-identical T cells were first activated in vitro by coculturing these cells with either unmodified Leiden cells or with the corresponding Leiden ALL-APC for 3 days, and subsequently infused into groups of 6 leukemic NOD/scid mice. While T cells cocultured with unmodified Leiden cells did not expand in vivo, T cells cocultured with Leiden ALL-APC expanded after infusion in 5 out of 6 animals. This expansion coincided with a 20–75% decrease in leukemic cell numbers in the blood. In conclusion, the novel serum-free culturing system enables long-term culture and manipulation of a significant fraction of primary human ALL. These Leiden cell lines can be modified into ALL-APC that display adequate antigen presenting function, preventing the induction of T cell anergy as demonstrated in vivo in the NOD/scid mouse model.

Overcoming Rejection of Bone Marrow Allografts by Treg Cells in a Stringent Mouse Model for T Cell Mediated Rejection: Tolerance Induction by Third Party “Off-the-Shelf” Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 576-576
Author(s):  
David Steiner ◽  
Noga Brunicki ◽  
Esther Bachar-Lustig ◽  
Yair Reisner
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Graft Rejection ◽  
Ex Vivo ◽  
Tolerance Induction ◽  
Treg Cells ◽  
Third Party ◽  
Donor Type

Abstract Recent reports have shown that donor or host CD4+CD25+ Treg cells can be used to control GVHD or graft rejection following allogeneic BMT in mice. More recent data suggests that in the context of T cell depleted BM allografting, engraftment was only mildly improved by Treg cells alone, or by Rapamycin (RAPA) alone, but it was markedly enhanced by using Treg cells in conjunction with RAPA. These studies were carried out in a mouse model specifically designed to measure T cell mediated graft rejection. In this model, lethally irradiated (11Gy) C3H mice were infused with 1x104 purified host type T cells (HTC) and were transplanted one day later with 2x106 BM cells from Balb-Nude donors, which are markedly depleted of T cells and do not induce GVHD. Rejection mediated by the HTC is manifested by severe aplasia and lethality within 21 days posttransplant. In 10 independent experiments none of the mice in the irradiation control survived (0/62), the majority of the mice receiving BM survived (58/63) while marked rejection, associated with poor survival (2/62) was found in the group receiving purified HTC prior to the BM transplant. In the present study we further tested in this model whether third party Treg cells could be used instead of donor or host Treg cells to overcome rejection of BM allografts. We initially tested freshly isolated lymph node CD4+CD25+ cells. C3H (H2k) recipients received BM from Balb- Nude (H2d) donors and the Treg cells were obtained from Balb/c or FVB (H2q) donors. As in our previous study, while none of the recipients survived upon treatment with RAPA alone, using third party or donor type Treg cells in conjunction with RAPA led to survival of 9 of 13 and 7 of 10 mice respectively. Thus, the third party fresh Treg cells were as effective as the donor type cells in preventing graft rejection (P>0.05). Considering the low levels of CD4+CD25+ cells in peripheral blood or spleen, new strategies for growing these cells ex-vivo have been developed. Although, Treg cells exhibit low proliferative potential in-vitro upon TCR stimulation, the feasibility of growing mouse or human regulatory cells has been demonstrated mainly using the combination of TCR stimulation (either with an anti-TCR antibody or with allogeneic stimulator cells), costimulatory signals and high doses of IL-2. When tested in the same model, Treg cells ex-vivo expanded by stimulation against 4th party allogeneic cells, exhibited effective enhancement of engraftment of Balb-Nude BM. Thus, in four independent experiments, when assessing treatment with expanded Treg cells, of third party or donor type origin, the survival rate was 19 of 35 (54%) and 25 of 40 (62%) mice, respectively. Again, in both instances the marked potential of Treg cells to overcome T cell mediated rejection was exhibited only when co-administered with RAPA. In conclusion, our data strongly indicate that, at least in the bone marrow transplantation setting, third party Treg cells could afford a new viable ‘off-the-shelf’ source for tolerance induction. The use of third party Treg cells in contrast to donor type cells could allow advanced preparation of a large bank of Treg cells, with all the appropriate quality controls required for cell therapy. Further studies with human Treg cells in-vitro are required to ascertain the potential of third party cells as a valuable source for clinical transplantation.

scholarly journals Host CD4+CD25+ T cells can expand and comprise a major component of the Treg compartment after experimental HCT

Blood ◽  
2009 ◽  
Vol 113 (3) ◽  
pp. 733-743 ◽  
Author(s):  
Allison L. Bayer ◽  
Monica Jones ◽  
Jackeline Chirinos ◽  
Lesley de Armas ◽  
Taylor H. Schreiber ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hematopoietic Cell Transplantation ◽  
Extended Period ◽  
Treg Cells ◽  
Allogeneic Bone ◽  
Vaccine Responses ◽  
Antitumor Vaccine

Abstract Reconstitution of the recipient lymphoid compartment following hematopoietic cell transplantation (HCT) is typically delayed. The present studies investigated the residual host CD4+CD25+Foxp3+ (Treg) compartment after several conditioning regimens, including T cell–depleted and T cell–replete HCT and observed (1) a small number of recipient Treg cells survived aggressive conditioning; (2) the surviving, that is, residual Tregs underwent marked expansion; and (3) recipient CD4+FoxP3+ cells composed the majority of the Treg compartment for several months post-syngeneic HCT. Notably, residual Tregs also dominated the compartment post-HCT with T cell–depleted (TCD) major histocompatibility complex–matched allogeneic bone marrow but not following T cell–replete transplantations. The residual Treg cell compartment was functionally competent as assessed by in vitro lymphoid suppression and in vivo autoimmune disease transfer assay. These observations support the notion that functional host Tregs initially occupy a niche in lymphopenic transplantation recipients, undergo significant expansion, and contribute to the compartment for an extended period before donor-derived CD4+FoxP3+ T cells eventually compose the majority of the compartment. In total, the findings suggest that the presence of host Tregs may be important to consider regarding elicitation of immune (eg, antitumor, vaccine) responses in recipients during the early post-transplant period involving autologous and certain allogeneic HCT regimens.

858 A bispecific antibody targeting CD40 and EpCAM induces superior anti-tumor effects compared to the combination of the monospecific antibodies

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A911-A911
Author(s):  
Peter Ellmark ◽  
Karin Hägerbrand ◽  
Mattias Levin ◽  
Laura Von Schantz ◽  
Adnan Deronic ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Cell Lines ◽  
Tumor Target ◽  
T Cell Infiltration ◽  
Antigen Presenting ◽  
Monospecific Antibodies

BackgroundAlligator has developed a new concept, Neo-X’, to enable antigen presenting cells to efficiently enhance priming of neoantigen-specific T cells, which may be the missing aspect in tumors that lack T cell infiltration. We hypothesize that binding of the CD40 x EpCAM bsAb (4224) to CD40 on DCs and EpCAM on tumor exosomes or tumor debris leads to i) activation of the DC, ii) uptake of the tumor material, iii) cross-presentation of tumor-derived neoantigen (present in exosomes or debris) and iiii) priming of tumor neoantigen-specific T cells, resulting in an increased quantity and/or quality of the tumor-targeting T cell pool. CD40 crosslinking by engagement with a tumor antigen on a tumor cell is required to achieve a functional agonistic effect, and subsequent DC activation will therefore only be achieved in the presence of tumor antigens.Methods4224 evaluated in vitro using human monocyte-derived DC, co-cultured with cells expressing EpCAM. In addition the functional effects were evaluated using tumor cell lines and B-cell lines expressing CD40. In vivo, the anti-tumor efficacy of the CD40 x EpCAM bsAb was determined in human CD40 transgenic mice bearing MB49 bladder carcinoma tumors transfected with human EpCAM or controls.ResultsIn vitro, we have demonstrated that the CD40 x EpCAM bsAb induces tumor target dependent activation of dendritic cells, as analyzed by flow cytometry measuring HLA-DR and CD86 expression on the DC and by measuring IL-12p40 levels in the supernatant. Further, the ability of bsAbs within the Neo-X’ concept to mediate co-localization of tumor debris and CD40 expressing antigen presenting cells depends on the receptor density of the tumor target. In vivo, 4224 displayed a potent, EpCAM-dependent anti-tumor effect with significantly reduced tumor growth and improved survival compared to an equivalent dose of the combination of the monospecific CD40 Ab and EpCAM targeting antibody. The tumor-localizing property of 4224 also shows potential for improved safety compared to CD40 monospecific antibodies. A biodistribution analysis demonstrated that the bispecific 4224 in the RUBY-format displayed similar half-life as the monospecific CD40 mAb in mice.ConclusionsIn conclusion, the Neo-X’ concept, by targeting CD40 and a tumor specific antigen, has the potential to mediate an expansion of the tumor-specific T cell repertoire, resulting in increased T cell infiltration and potent anti-tumor effects.

scholarly journals GD2 or HER2 targeting T cell engaging bispecific antibodies to treat osteosarcoma

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Jeong A. Park ◽  
Nai-Kong V. Cheung
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Immune Checkpoint ◽  
Immune Checkpoint Inhibitors ◽  
Checkpoint Inhibitors ◽  
Tumor Control ◽  
Osteosarcoma Cell

Abstract Background The cure rate for metastatic osteosarcoma has not substantially improved over the past decades. Clinical trials of anti-HER2 trastuzumab or anti-GD2 dinutuximab for metastatic or refractory osteosarcoma were not successful, and neither was immune checkpoint inhibitors (ICIs). Methods We tested various target antigen expressions on osteosarcoma cell lines using flow cytometry and analyzed in vitro T cell engaging BsAb (T-BsAb)-dependent T cell-mediated cytotoxicity using 4-h 51Cr release assay. We tested in vivo anti-tumor activities of T-BsAb targeting GD2 or HER2 in established osteosarcoma cell line or patient-derived xenograft (PDX) mouse models carried out in BALB-Rag2−/−IL-2R-γc-KO (BRG) mice. We also generated ex vivo BsAb-armed T cells (EATs) and studied their tumor-suppressive effect against osteosarcoma xenografts. In order to improve the anti-tumor response, ICIs, anti-human PD-1 (pembrolizumab) or anti-human PD-L1 (atezolizumab) antibodies were tested their synergy with GD2- or HER2-BsAb against osteosarcoma. Results GD2 and HER2 were chosen from a panel of surface markers on osteosarcoma cell lines and PDXs. Anti-GD2 BsAb or anti-HER2 BsAb exerted potent anti-tumor effect against osteosarcoma tumors in vitro and in vivo. T cells armed with anti-GD2-BsAb (GD2-EATs) or anti-HER2-BsAb (HER2-EATs) showed significant anti-tumor activities as well. Anti-PD-L1 combination treatment enhanced BsAb-armed T cell function in vivo and improved tumor control and survival of the mice, when given sequentially and continuously. Conclusion Anti-GD2 and anti-HER2 BsAbs were effective in controlling osteosarcoma. These data support the clinical investigation of GD2 and HER2 targeted T-BsAb treatment in combination with immune checkpoint inhibitors, particularly anti-PD-L1, in patients with osteosarcoma to improve their treatment outcome.

100 Drug-regulatable engineered T cells eliminate CD33+ and CD33ΔE2+ AML

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A111-A111
Author(s):  
Jacob Appelbaum ◽  
Wai-Hang Leung ◽  
Unja Martin ◽  
Kaori Oda ◽  
Giacomo Tampella ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Splice Variants ◽  
Antigen Binding ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Car T

BackgroundBioengineered T cell treatments for acute myeloid leukemia (AML) are challenged by near universal expression of leukemia antigens on normal hematopoietic stem/progenitor cells:1 2 ‘on target/off tumor‘ activity may cause myelosuppression while sustained antigen exposure can lead to T cell exhaustion.3 In addition, splicing variants may allow antigen escape. We hypothesize that by using a novel CD33-C2-specific single domain VHH antibody as the antigen targeting domain in dimerizing agent-regulated immunoreceptor complex T cells (DARIC T cells), we will enable pharmacologically-controllable targeting of CD33, allowing eradication of leukemia expressing either of the major splice variants of CD33: i.e., full-length CD33 or CD33ΔE2.MethodsWe engineered DARIC-expressing lentiviral vectors containing encoding separated CD33-C2-specific antigen binding and 41BB-CD3zeta signaling chains that heterodimerize following addition of rapamycin via embedded FKBP12 and FRB* domains.4 Peripheral blood mononuclear cells were stimulated with IL-2, anti-CD3, and anti-CD28 antibodies 24h prior to transduction with DARIC33 lentiviral vector. Surface expression of antigen binding or signaling chains was assessed using biotinylated CD33, or antibodies to VHH-domains or FRB* respectively. Rapamycin-dependent in vitro activity was measured by IFNg release. To evaluate in vivo activity, NSG mice injected with 1 × 105 MOLM-14/luc cells were treated 5-7 days later with 1 × 107 DARIC33 T cells in the presence or absence of rapamycin and tumor progression followed by luciferase activity.ResultsDARIC33+ T cells bound biotinylated-CD33, anti-VHH and anti-FRB* antibodies. Rapamycin addition increased expression of both signaling and antigen-recognition chains, suggesting augmented receptor stability in the presence of dimerizing drug. In the presence of rapamycin, coculture of DARIC33 T cells with cell lines expressing either full length or CD33ΔE25 showed equivalent rapamycin-dependent activation, demonstrating DARIC33 responds to both splice variants. Titration experiments showed rapamycin-dependent activation with EC50 = 25pM. Negligible IFNg release was observed in the absence of drug. DARIC33 T cells significantly extended survival of AML-bearing mice, but only when treated with rapamycin. The DARIC33 T cells were activated in vivo by sub-immunosuppressive rapamycin dosing, as weekly or 0.1 mg/kg QOD dosing led to similar levels of tumor suppression.ConclusionsDARIC33 T cells appear to be potent antileukemic agents: they are activated by AML cell lines in vitro as demonstrated by cytokine release and cytotoxicity, and significantly extend survival in an aggressive xenograft model. Temporal control provided by the DARIC architecture promises to enhance safety and potentially efficacy of CAR T therapy for AML, for example by enabling hematopoietic recovery or providing T cell rest.ReferencesPerna F, Berman SH, Soni RK, Mansilla-Soto J, Eyquem J, Hamieh M, et al. Integrating proteomics and transcriptomics for systematic combinatorial chimeric antigen receptor therapy of AML. Cancer Cell 2017 Oct 9;32(4):506–519.e5.Haubner S, Perna F, Köhnke T, Schmidt C, Berman S, Augsberger C, et al. Coexpression profile of leukemic stem cell markers for combinatorial targeted therapy in AML. Leukemia. 2019 Jan;33(1):64.Lamarche C, Novakovsky GE, Qi CN, Weber EW, Mackall CL, Levings MK. Repeated stimulation or tonic-signaling chimeric antigen receptors drive regulatory T cell exhaustion. bioRxiv. 2020 Jun 28;2020.06.27.175158.Leung W-H, Gay J, Martin U, Garrett TE, Horton HM, Certo MT, et al. Sensitive and adaptable pharmacological control of CAR T cells through extracellular receptor dimerization. JCI Insight [Internet]. 2019 Jun 6 [cited 2019 Jun 11];4(11). Available from: https://insight.jci.org/articles/view/124430Pérez-Oliva AB, Martínez-Esparza M, Vicente-Fernández JJ, Corral-San Miguel R, García-Peñarrubia P, Hernández-Caselles T. Epitope mapping, expression and post-translational modifications of two isoforms of CD33 (CD33M and CD33m) on lymphoid and myeloid human cells. Glycobiology 2011;21(6):757–770.

Sign in / Sign up

Export Citation Format

Share Document