scholarly journals L4 Synthetic agonistic receptor-activating BiTEs – a modular platform for the efficient targeting of acute myeloid leukemia

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A2.2-A3
Author(s):  
M Benmebarek ◽  
BL Cadilha ◽  
M Hermann ◽  
S Lesch ◽  
C Augsburger ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
T Cell Activation ◽  
Tumour Cell ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Cell Lysis ◽  
Target Cells ◽  
Acute Myeloid

BackgroundTargeted immunotherapies have shown limited success in the context of acute myeloid leukemia (AML). Due to the mutational landscape and heterogeneity attributed to this malignancy and toxicities associated with the targeting of myeloid lineage antigens, it has become apparent that a modular and controllable cell therapy approach with the potential to target multiple antigens is required. We propose a controlled ACT approach, where T cells are armed with synthetic agonistic receptors (SARs) that are conditionally activated only in the presence of a target AML-associated antigen, and a cross-linking bispecific T cell engager (BiTE) specific for both (SAR) T cell and tumour cell.Materials and MethodsA SAR composed of an extracellular EGFRvIII, trans-membrane CD28, and intracellular CD28 and CD3z domains was fused via overlap-extension PCR cloning. T cells were retrovirally transduced to stably express our SAR construct. SAR-specific bispecific T cell engagers (BiTE) that target AML-associated antigens were designed and expressed in Expi293FTMcells and purified by nickel affinity and size exclusion chromatography (SEC). We validated our approach in three human cancer models and patient-derived AML blasts expressing our AML-associated target antigen CD33.ResultsCD33-EGFRvIII BiTE, monovalently selective for our SAR, induced conditional antigen-dependent activation, proliferation and differentiation of SAR-T cells. Further, SAR T cells bridged to their target cells by BiTE could form functional immunological synapses, resulting in efficient tumor cell lysis with specificity towards CD33-expressing AML cells. SAR.BiTE combination could also mediate specific cytotoxicity against patient-derived AML blasts whilst driving SAR T cell activation. In vivo, treatment with SAR.BiTE combination could efficiently eradicate leukemia and enhance survival in an AML xenograft model. Furthermore, we could show selective activation of SAR T cells, as well as a controllable reversibility of said activation upon depletion of the T cell engaging molecule.ConclusionsHere we apply the SAR x BiAb approach in efforts to deliver specific and conditional activation of agonistic receptor-transduced T cells, and targeted tumour cell lysis. The modularity of our platform will allow for a multi-targeting ACT approach with the potential to translate the ACT successes of B cell malignancies to AML. With a lack of truly specific AML antigens, it is invaluable that this approach possesses an intrinsic safety switch via its BiTE facet. Moreover, we are able to circumvent pan-T cell activation due to the specific targeting and activation of SAR T cells.Disclosure InformationM. Benmebarek: None. B.L. Cadilha: None. M. Hermann: None. S. Lesch: None. C. Augsburger: None. B. Brauchle: None. S. Stoiber: None. A. Darwich: None. F. Rataj: None. C. Klein: A. Employment (full or part-time); Significant; Roche. K. Hopfner: None. M. Subklewe: None. S. Endres: None. S. Kobold: None.

scholarly journals P07.01 A modular and controllable T cell therapy platform for AML

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A22.2-A23
Author(s):  
M Benmebarek ◽  
B Loureiro Cadilha ◽  
M Herrmann ◽  
S Schmitt ◽  
S Lesch ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Activation ◽  
Tumour Cell ◽  
Cell Activation ◽  
Cell Lysis ◽  
Target Cells ◽  
Single Chain

BackgroundTargeted immunotherapies have shown limited success in the context of acute myeloid leukemia (AML). The mutational landscape, heterogeneity attributed to this malignancy and toxicities associated with the targeting of myeloid lineage antigens, it has become apparent that a modular and controllable cell therapy approach with the potential to target multiple antigens is required. We propose a controlled ACT approach, where T cells are equipped with synthetic agonistic receptors (SARs) that are selectively activated only in the presence of a target AML-associated antigen, and a cross-linking tandem single chain variable fragment (taFv) specific for both (SAR) T cell and tumour cell.Materials and MethodsA SAR composed of an extracellular EGFRvIII, trans- membrane CD28, and intracellular CD28 and CD3z domains was fused via overlap- extension PCR cloning. T cells were retrovirally transduced to stably express our SAR construct. SAR-specific taFvs that target AML-associated antigens were designed and expressed in Expi293FTM cells and purified by nickel affinity and size exclusion chromatography (SEC). We validated our approach in three human cancer models and patient-derived AML blasts expressing our AML-associated target antigens CD33 and CD123.ResultsAnti-CD33-EGFRvIII and anti-CD123 EGFRvIII taFv, monovalently selective for our SAR, induced conditional antigen-dependent activation, proliferation and differentiation of SAR-T cells. Further, SAR T cells bridged to their target cells by taFv could form functional immunological synapses, resulting in efficient tumor cell lysis with specificity towards CD33-expressing AML cells. SAR-taFv combination could also mediate specific cytotoxicity against patient-derived AML blasts and leukemic stem cells whilst driving SAR T cell activation. In vivo, treatment with SAR-taFv combination could efficiently eradicate leukemia and enhance survival in an AML xenograft models. Furthermore, we could show selective activation of SAR T cells, as well as a controllable reversibility and modularity of said activation upon depletion of the T cell engaging molecule, both in vitro and in vivo.ConclusionsHere we apply the SAR-taFv platform in efforts to deliver specific and conditional activation of SAR-transduced T cells, and targeted tumour cell lysis. The modularity of our platform will allow for a multi-targeting ACT approach with the potential to translate the ACT successes of B cell malignancies to AML. With a lack of truly specific AML antigens, it is invaluable that this approach possesses an intrinsic safety switch via its taFv facet. Moreover, we are able to circumvent pan-T cell activation due to the specific targeting and activation of SAR T cells.Disclosure InformationM. Benmebarek: None. B. Loureiro Cadilha: None. M. Herrmann: None. S. Schmitt: None. S. Lesch: None. S. Stoiber: None. A. Darwich: None. C. Augsberger: None. B. Brauchle: None. M. Schwerdtfeger: None. A. Gottschlich: None. A. Gottschlich Rataj: None. N.C. Fenn: None. C. Klein: None. M. Subklewe: None. S. Endres: None. K. Hopfner: None. S. Kobold: None.

scholarly journals Characterization of the DNAM-1, TIGIT and TACTILE Axis on Circulating NK, NKT-Like and T Cell Subsets in Patients with Acute Myeloid Leukemia

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2171
Author(s):  
Isabel Valhondo ◽  
Fakhri Hassouneh ◽  
Nelson Lopez-Sejas ◽  
Alejandra Pera ◽  
Beatriz Sanchez-Correa ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Healthy Volunteers ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Nk Cell ◽  
T Cell Subsets ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) remains a major clinical challenge due to poor overall survival, which is even more dramatic in elderly patients. TIGIT, an inhibitory receptor that interacts with CD155 and CD112 molecules, is considered as a checkpoint in T and NK cell activation. This receptor shares ligands with the co-stimulatory receptor DNAM-1 and with TACTILE. The aim of this work was to analyze the expression of DNAM-1, TIGIT and TACTILE in NK cells and T cell subsets in AML patients. Methods: We have studied 36 patients at the time of diagnosis of AML and 20 healthy volunteers. The expression of DNAM-1, TIGIT and TACTILE in NK cells and T cells, according to the expression of CD3 and CD56, was performed by flow cytometry. Results: NK cells, CD56− T cells and CD56+ T (NKT-like) cells from AML patients presented a reduced expression of DNAM-1 compared with healthy volunteers. An increased expression of TIGIT was observed in mainstream CD56− T cells. No differences were observed in the expression of TACTILE. Simplified presentation of incredibly complex evaluations (SPICE) analysis of the co-expression of DNAM-1, TIGIT and TACTILE showed an increase in NK and T cells lacking DNAM-1 and co-expressing TIGIT and TACTILE. Low percentages of DNAM-1−TIGIT+TACTILE+ NK cells and DNAM-1− TIGIT+TACTILE+ CD56− T cells were associated with a better survival of AML patients. Conclusions: The expression of DNAM-1 is reduced in NK cells and in CD4+ and CD8+ T cells from AML patients compared with those from healthy volunteers. An increased percentage of NK and T cells lacking DNAM-1 and co-expressing TIGIT and TACTILE is associated with patient survival, supporting the role of TIGIT as a novel candidate for checkpoint blockade.

scholarly journals A novel C2 domain binding CD33xCD3 bispecific antibody with potent T-cell redirection activity against acute myeloid leukemia

2020 ◽  
Vol 4 (5) ◽  
pp. 906-919 ◽  
Author(s):  
Priyanka Nair-Gupta ◽  
Michael Diem ◽  
Dara Reeves ◽  
Weirong Wang ◽  
Robert Schulingkamp ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
T Cell ◽  
T Cell Activation ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Bispecific Antibody ◽  
C2 Domain ◽  
Cytokine Release ◽  
Cynomolgus Monkeys ◽  
Acute Myeloid

Abstract CD33 is expressed in 90% of patients with acute myeloid leukemia (AML), and its extracellular portion consists of a V domain and a C2 domain. A recent study showed that a single nucleotide polymorphism (SNP), rs12459419 (C > T), results in the reduced expression of V domain–containing CD33 and limited efficacy of V domain–binding anti-CD33 antibodies. We developed JNJ-67571244, a novel human bispecific antibody capable of binding to the C2 domain of CD33 and to CD3, to induce T-cell recruitment and CD33+ tumor cell cytotoxicity independently of their SNP genotype status. JNJ-67571244 specifically binds to CD33-expressing target cells and induces cytotoxicity of CD33+ AML cell lines in vitro along with T-cell activation and cytokine release. JNJ-67571244 also exhibited statistically significant antitumor activity in vivo in established disseminated and subcutaneous mouse models of human AML. Furthermore, this antibody depletes CD33+ blasts in AML patient blood samples with concurrent T-cell activation. JNJ-67571244 also cross-reacts with cynomolgus monkey CD33 and CD3, and dosing of JNJ-67571244 in cynomolgus monkeys resulted in T-cell activation, transient cytokine release, and sustained reduction in CD33+ leukocyte populations. JNJ-67571244 was well tolerated in cynomolgus monkeys up to 30 mg/kg. Lastly, JNJ-67571244 mediated efficient cytotoxicity of cell lines and primary samples regardless of their SNP genotype status, suggesting a potential therapeutic benefit over other V-binding antibodies. JNJ-67571244 is currently in phase 1 clinical trials in patients with relapsed/refractory AML and high-risk myelodysplastic syndrome.

Activation of T Cells by Bispecific Single-Chain Construct MT103 (MEDI-538) Is Strictly Target Cell-Dependent.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3889-3889
Author(s):  
Klaus Brischwein ◽  
Scott A. Hammond ◽  
Larissa Parr ◽  
Schlereth Bernd ◽  
Mathias Locher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Target Cell ◽  
Cell Activation ◽  
Cell Lysis ◽  
Target Cells ◽  
Single Chain ◽  
Activation Markers ◽  
Human T Cells

Abstract Background: Bispecific antibodies have been extensively studied in vitro and in vivo for their use in redirected tumor cell lysis. A particular challenge of bispecific antibody constructs recognizing the CD3 signaling complex is to achieve a controlled polyclonal activation of T-cells that, ideally, is entirely dependent on the presence of target cells. If this is not the case, systemic production of inflammatory cytokines and secondary endothelial reactions may occur as side effects, as are observed with the murine anti-human CD3e antibody OKT-3 (muromab, Orthoclone®). Here we present evidence that MT103 (or MEDI-538), a bispecific single chain antibody of the BiTE class that targets CD19 and CD3, induces T-cell activation exclusively in the presence of target cells. Material and methods: Peripheral blood mononuclear cells from healthy donors were prepared by Ficoll density centrifugation. PBMC were incubated for 24 hours with MT103 in presence or absence of specific target cells. Target cell lysis was determined by measurement of adenylate kinase activity released from lysed cells. De novo expression of activation markers CD69 and CD25 on T-cells was assessed by flow cytometry using directly conjugated monoclonal antibodies, and the concentration of cytokines in the supernatant was determined by a commercial FACS-based bead array. Results: MT103 was analyzed for conditional T-cell activation. In the presence of target-expressing cell lines, low picomolar concentrations of MT103 were sufficient to stimulate a high percentage of peripheral human T-cells to express cytokines and surface activation markers, to enter into the cell cycle and to induce redirected lysis of target cells. However, in the absence of target cells, the BiTE molecules no longer detectably activated human T-cells even at concentrations exceeding the ED50 for redirected lysis and conditional T-cell activation by more than five orders of magnitude. Conclusion: Our data show that T-cell activation by MT103 is highly conditional in that it is strictly dependent on the presence.

scholarly journals Multi-Antigen Primed T Cells Promote Apoptosis of Acute Myeloid Leukemia (AML)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4794-4794
Author(s):  
Ebtesam Nafie ◽  
Mathias Oelke ◽  
Melissa Valerio ◽  
Sojung Kim ◽  
Ivan Rodriguez ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Time Dependent ◽  
Target Cells ◽  
Ifn Γ ◽  
Acute Myeloid

Abstract Introduction Acute myeloid leukemia (AML), the most common acute leukemia in adults, is characterized by uncontrolled proliferation of immuature myeloid cells. Despite newly approved drugs, AML remains largely incurable due to the persistence of the leukemia stem cell (LSC) population which lie quiescent in the bone marrow niche. Immunotherapy has potential to eradicate LSCs, however, no unique LSC immunophenotype has been identified. Moreover, it is necessary to simultaneously target multiple antigens (Ags) to prevent immune escape and to overcome refractory disease. We present in vitro studies in support of a therapeutic platform capable of targeting multiple intracellular Ags which could meet this challenge. The adoptive transfer of activated T cells primed to engage diverse AML associated epitopes by ex vivo exposure to artificial Ag presenting cells (aAPC) has the potential to eliminate both primary leukemia blasts and LSCs. Hypothesis Ex vivo enrichment and expansion (E+E) of antigen-educated CD8+ T cells recognizing 5 peptides derived from 3 proteins, Cyclin A1, PRAME and WT1, can selectively identify, engage, and kill AML cell lines or patient-derived (PD) AML blasts in a HLA A*02:01 restricted manner in vitro. Materials and Methods T cells from the peripheral blood mononuclear cell fraction of a healthy HLA A*02:01 donor were enriched for antigen-educated CD8+/CD4 -T cells. These cells were cultured with nanoparticles decorated with the 5 peptides and a costimulatory protein, resulting in the activation and expansion of those T cells expressing the cognate T cell receptors. These cells are composed of ~97% abT cells, 3% gdT cells and ~13% T scm, 41.5% T cm, 39.5%T em, 6%T emra and 1% T n. Results Ex vivo expanded educated T cells exhibit target-specific anti-AML activity. T cell mediated cell apoptosis of HLA-matched THP1 cells is dose and time-dependent. At 10:1 effector to target (E:T) ratio, ~28% apoptosis occurred at 24 hrs, while apoptosis was at basal levels when antigen non-educated T cells were used (data not shown). Studies were extended to PD AML cells (Fig. 1A: 012; Fig. 1B: 415) where antigen educated T cells elicited rapid (<16 hrs) and extensive (~90%) apoptosis of target PD AML cells at all E:T ratios examined. Time lapse photography of T cell/PD AML incubations revealed antigen-educated T cells clustered around AML cells (Fig. 2A), a fraction of the latter disappearing over the course of 12 hrs while PD AML cells incubated with non-educated T cells (Fig. 2B) remained viable over 12 hrs. Furthermore, there is little or no T cell movement or clustering in the wells with unprimed, non-active T cells. Release of IFN-γ by educated T cells. T cells (Fig.3A: antigen-educated through E+E) were incubated at E:T::5:1 for 24 to 48 hrs and IFN-γ in supernatants measured. The fold difference over non-educated T cells incubated with AML cells for the same time is shown and can reach over 5-fold. IFN-γ accumulation was time-dependent where antigen-educated T cells were combined with HLA-A2 matched THP1 or PD AML cells (012, 415, 470). Educated T cells were not active against target cells lacking HLA-A2 (K562) demonstrating HLA restricted killing (Fig. 3B). Additionally, antigen-educated T cells incubated without any target released slightly more IFN-γ than non-educated T cells under similar conditions but AML cells fail to stimulate IFN-γ release from non-educated T cells (data not shown). Conclusions We demonstrate HLA restricted cytotoxic activity of antigen-educated T cells against THP1 AML cells and PD AML blasts as shown by flow cytometry and microscopy. Consistent with target cell death, the supernatants from assays with antigen-educated T cells and HLA A*02:01 AML target cells exhibited over 5-fold more IFN-γ than media from assays of non-educated cells under identical conditions. Under these in vitro conditions, PD AML blasts were more readily killed than THP1 cells perhaps due to higher target antigen density (data not shown). These results support the use of multi-antigen-educated T cells for adoptive transfer to treat AML. To investigate the safety and establish the recommended phase II dose, a multi-center Phase I clinical study is underway in relapsed AML post-allo-HCT (NCT 04284228). Future studies will incorporate new antigens to enable broader targeting of a heterogeneous population of AML within and across patients Figure 1 Figure 1. Disclosures Oelke: Neximmune, Inc: Current Employment. Kim: Neximmune, Inc: Current Employment. Marcucci: Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings. Al Malki: Jazz Pharmaceuticals, Inc.: Consultancy; Hansa Biopharma: Consultancy; Neximmune: Consultancy; CareDx: Consultancy; Rigel Pharma: Consultancy.

scholarly journals Identification and Isolation of Tumor-Specific Cytotoxic T Lymphocytes in Acute Myeloid Leukemia Patients through the Identification of T-Cells Capable to Establish Stable Interactions with Leukemic Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3336-3336
Author(s):  
Estefania Garcia-Guerrero ◽  
Luis I. Sanchez-Abarca ◽  
Esther Domingo ◽  
Teresa Ramos ◽  
Jose Antonio Bejarano-García ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
T Lymphocytes ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Myeloid Leukemia ◽  
Target Cells ◽  
Acute Myeloid

Abstract Introduction Autologous adoptive T cell therapies, based on the use of tumor infiltrating lymphocytes (TILs), have made great progress in recent years for the treatment of solid tumors, especially melanoma. However, further work is needed to isolate tumor-reactive T cells among patients diagnosed with hematologic malignancies. The dynamics of the interaction between T cells and antigen presenting cells (APC) dictate the quality of the immune responses. While stable joints between target cells and T lymphocytes lead to the induction of T cell activation and immune response, brief contacts contribute to the induction of immune-tolerance. Taking advantage of the strong interaction between target cell and activated T-cells, we show the feasibility to identify and isolate tumor-specific cytotoxic T lymphocytes (CTLs) from acute myeloid leukemia (AML) patients. Using this approach, CTLs stably bound through T cell receptor to tumor cells (doublet forming T-cells) can be identified in peripheral blood and bone marrow and subsequently selected and isolated by FACS-based cell sorting. Methods Co-cultures between PBMC from AML patients in complete remission and AML tumor cells (PKH-stained) from the same patient were performed to study the percentage of doublet-forming T cells (CD3+PKH+) (T cell bound to a tumor cell). After 15 hours of co-culture, cells were stained and sorted. Secondary co-cultures with autologous tumor cells (used in primary co-culture) were performed to study the cytotoxic activity and cytokine production of T-cells capable or not to form stable joints with the leukemic cells (doublet population vs non-doublet population). Results Doublet-forming T cells from AML patients were identified in a range of 2% to 6% (mean=3.83%, n=5). Immunophenotyping analysis showed differences between doublet-forming T cells (CD3+PKH+) and those T cells which did not form stable and strong interactions with target cells (CD3+PKH-). Doublet T cells displayed a higher percentage of CD8+ T cells and higher percentage of effector CD4+ and CD8+ T cells compared to non-doublet T cells. Next, we explored, among effector CD4+ and CD8+ cells, those with cytotoxic phenotype. As expected, a high percentage of effector CD8+ doublet T cells showed Granzyme B and perforin expression, thus corresponding with a cytotoxic immune-phenotype (n=3, mean 65.51%). Within effector CD4+ doublet T cells, a mean of 9.053 % showed expression of both Granzyme B and perforin corresponding with CD4+ CTL (n=3). Regarding CD57 and CD16 markers, a mean of 18.62% of effector CD4+ doublet T cells were positive for both markers, compared to 65.84% of effector CD8+ doublet T cells (n=3). Further, we performed secondary co-cultures to analyze the CD69 activation marker after 24h of co-culture. A high percentage of CD69+ cells was observed in co-cultures with doublet-forming T cells against target cells as compared to non-doublet T cells (n=3, p=0.0053). Finally, analysis of supernatants of co-culture of doublet T cells and non-doublet T cells with target cells revealed specific secretion of IFNγ and IL-2 (n=3, p=0.0001; p=0.0005, respectively). The cytolytic activity was evaluated comparing the viability of tumor cells cultured alone or with doublet-forming T cells or non-doublet T cells from the same patient. A significant increase of the specific lysis of AML cells was observed when doublet T cells were co-cultured as compared to non-doublet T cells (p=0.0424, n=5). This encouraged us to examine whether we were able to identify doublet-forming T cells from bone marrow of AML patients at diagnosis. Analyses of bone marrow by flow cytometry reveled a small percentage of CD3+CD34+ population corresponding with bone marrow-doublet-forming T cells (n=3, mean=2.9%). Interestingly, bone marrow-doublet-forming T cells show a higher percentage of CD4+ T cells, whereas bone marrow-non-doublet T cells show a higher percentage of CD8+ T cells. Conclusions Our data demonstrate that when T cells from AML patients are co-cultured with tumor cells, a "doublet T cell" population appears. This population consists of T cells capable to bind tumor cells. These CTLs display higher percentage of effector cells and a marked cytotoxic activity against AML blasts. In conclusion, we have developed a new procedure to identify and select specific cytotoxic T cells in both bone marrow and peripheral blood from patients diagnosed with acute myeloid leukemia. Figure. Figure. Disclosures Sanchez-Abarca: Virgen del Rocio University Hospital: Patents & Royalties. Ramos:Takeda Oncology: Research Funding.

Chimeric Antigen Receptor for Specific Targeting of Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4225-4225
Author(s):  
Irene Pizzitola ◽  
Fernando Anjos-Afonso ◽  
Kevin Rouault-Pierre ◽  
Francois Lassailly ◽  
Sarah Tettamanti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Cord Blood ◽  
Myeloid Leukemia ◽  
Genetically Modified ◽  
Target Cells ◽  
Cik Cells ◽  
Acute Myeloid

Abstract Abstract 4225 Despite the progress in the treatment of acute myeloid leukemia (AML) achieved in the last decades, a significant number of patients are still refractory to or relapse after conventional chemotherapy regimens. Therefore it is necessary to develop novel alternative approaches. Immunotherapy with T cells genetically modified to express chimeric antigen receptors (CARs) represent a valid option in this sense. CARs are artificial T-cell receptors constituted by a specific antigen-binding domain, and a signaling region, that, upon antigen recognition, leads to T-cell activation, and lysis of the target cells. AML is a potential optimal target for CAR strategy because of the over-expression of a number of surface antigens like CD33, CD123. Since CD33 is also expressed on normal hematopoietic stem/progenitors cells (HSPCs) resulting in a potential severe impairment of normal myelopoiesis, CD123 has recently emerged as new potential attractive molecules based on its differential expression pattern, being still wildly overexpressed by AML population, and at the same time less expressed on HSPCs. Here we describe the in vivo efficacy and the safety of this approach based on Cytokine-Induced-Killers (CIK) cells genetically modified to express CAR molecules specific for the CD33 or CD123 antigen. Once injected into low-level AML engrafted NSG mice (median of hCD45+CD33+ 0.6% before treatment), genetically modify T cells had a potent antitumor effect. Indeed, the bone marrow of control untreated animals or mice treated with un-manipulated CIK cells, was infiltrated by leukemic cells (86% and 81% leukemic engraftment), while in 7/8 anti-CD33-CD28-OX40-ζ and 8/10 anti-CD123-CD28-OX40-ζ treated mice we couldn't detect any AML cells. Similar results have been obtained when the treatment via T cell injection start when high AML burden has been obtained (median of hCD45+CD33+ 70% before treatment). One week after the last CIK's injection the level of AML engraftment was 96%, 87%, 0.35% and 0.34% for untreated mice, mice treated with un-manipulated CIK cells and with anti-CD33-CD28-OX40-ζ and anti-CD123-CD28-OX40-ζ transduced CIK-cells respectively. We performed secondary transplantation on the residual AML cells present in these animals and mice were treated again with transduced CIK cells. Residual AML cells were still sensitive to CARs approach, leading once again to an almost complete eradication of the disease (median level of hCD45+CD33+ engraftment was 98%, 0.02% and 0.04% respectively for untreated mice, anti-CD33-CD28-OX40-ζ and anti-CD123-CD28-OX40-ζ transduced CIK-cells). Furthermore, a fundamental issue was to determine the safety profile of such approach against normal hematopoietic precursors. In untreated mice injected with primary cord blood derived CD34+ cells the level of engraftment of hCD45 compartment was 42% whilst in mice treated with un-manipulated, anti-CD33-CD28-OX40-ζ or anti-CD123-CD28-OX40-ζ transduced CIK-cells the levels of human compartment was 40%, 11.7% and 26.3% respectively. Moreover when we consider specifically the CD34+CD38- compartment, enriched in HSC, the level of engraftment was 1.92%, 1.02%, 0.55% and 0.83%. Secondary transplantations are now ongoing to give a more complete profile about the remaining HSC repopulating capability after treatment. To more closely mimic a physiological context, similar experiments are ongoing using mice engrafted with normal adult bone marrow instead of umbilical cord blood. These experiments should offer relevant information concerning the efficacy and safety of the proposed strategy particularly in the context of minimal residual disease in high-risk transplanted AML patients. Moreover CAR approach could be potentially used to treat patients resistant to conventional chemotherapeutic approaches or for whom high dose chemotherapy treatment could not be proposed. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Bifunctional PD-1 × αCD3 × αCD33 fusion protein reverses adaptive immune escape in acute myeloid leukemia

Blood ◽  
2018 ◽  
Vol 132 (23) ◽  
pp. 2484-2494 ◽  
Author(s):  
Monika Herrmann ◽  
Christina Krupka ◽  
Katrin Deiser ◽  
Bettina Brauchle ◽  
Anetta Marcinek ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
T Cell ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Immune Checkpoint Blockade ◽  
Checkpoint Blockade ◽  
Adaptive Immune ◽  
Acute Myeloid

Abstract The CD33-targeting bispecific T-cell engager (BiTE) AMG 330 proved to be highly efficient in mediating cytolysis of acute myeloid leukemia (AML) cells in vitro and in mouse models. Yet, T-cell activation is correlated with upregulation of programmed cell death-ligand 1 (PD-L1) and other inhibitory checkpoints on AML cells that confer adaptive immune resistance. PD-1 and PD-L1 blocking agents may counteract T-cell dysfunction, however, at the expense of broadly distributed immune-related adverse events (irAEs). We developed a bifunctional checkpoint inhibitory T cell–engaging (CiTE) antibody that combines T-cell redirection to CD33 on AML cells with locally restricted immune checkpoint blockade. This is accomplished by fusing the extracellular domain of PD-1 (PD-1ex), which naturally holds a low affinity to PD-L1, to an αCD3.αCD33 BiTE-like scaffold. By a synergistic effect of checkpoint blockade and avidity-dependent binding, the PD-1ex attachment increases T-cell activation (3.3-fold elevation of interferon-γ) and leads to efficient and highly selective cytotoxicity against CD33+PD-L1+ cell lines (50% effective concentration = 2.3-26.9 pM) as well as patient-derived AML cells (n = 8). In a murine xenograft model, the CiTE induces complete AML eradication without initial signs of irAEs as measured by body weight loss. We conclude that our molecule preferentially targets AML cells, whereas high-affinity blockers, such as clinically approved anticancer agents, also address PD-L1+ non-AML cells. By combining the high efficacy of T-cell engagers with immune checkpoint blockade in a single molecule, we expect to minimize irAEs associated with the systemic application of immune checkpoint inhibitors and suggest high therapeutic potential, particularly for patients with relapsed/ refractory AML.

scholarly journals Cyclin-A1 represents a new immunogenic targetable antigen expressed in acute myeloid leukemia stem cells with characteristics of a cancer-testis antigen

Blood ◽  
2012 ◽  
Vol 119 (23) ◽  
pp. 5492-5501 ◽  
Author(s):  
Sebastian Ochsenreither ◽  
Ravindra Majeti ◽  
Thomas Schmitt ◽  
Derek Stirewalt ◽  
Ulrich Keilholz ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Myeloid Leukemia ◽  
High Expression ◽  
Target Cells ◽  
Cyclin A1 ◽  
Peripheral Tissues ◽  
Acute Myeloid

Abstract Targeted T-cell therapy is a potentially less toxic strategy than allogeneic stem cell transplantation for providing a cytotoxic antileukemic response to eliminate leukemic stem cells (LSCs) in acute myeloid leukemia (AML). However, this strategy requires identification of leukemia-associated antigens that are immunogenic and exhibit selective high expression in AML LSCs. Using microarray expression analysis of LSCs, hematopoietic cell subpopulations, and peripheral tissues to screen for candidate antigens, cyclin-A1 was identified as a candidate gene. Cyclin-A1 promotes cell proliferation and survival, has been shown to be leukemogenic in mice, is detected in LSCs of more than 50% of AML patients, and is minimally expressed in normal tissues with exception of testis. Using dendritic cells pulsed with a cyclin-A1 peptide library, we generated T cells against several cyclin-A1 oligopeptides. Two HLA A*0201-restricted epitopes were further characterized, and specific CD8 T-cell clones recognized both peptide-pulsed target cells and the HLA A*0201-positive AML line THP-1, which expresses cyclin-A1. Furthermore, cyclin-A1–specific CD8 T cells lysed primary AML cells. Thus, cyclin-A1 is the first prototypic leukemia-testis-antigen to be expressed in AML LSCs. The pro-oncogenic activity, high expression levels, and multitude of immunogenic epitopes make it a viable target for pursuing T cell–based therapy approaches.

scholarly journals Immunosuppression and Outcomes in Acute Myeloid Leukemia

2021 ◽  
Author(s):  
Francesca Ferraro ◽  
Christopher Miller ◽  
Keegan Christensen ◽  
Nichole M. Helton ◽  
Margareth O'Laughlin ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Rna Sequencing ◽  
Mhc Class Ii ◽  
T Cell Activation ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Independent Set ◽  
Normal Karyotype ◽  
Acute Myeloid

Acute myeloid leukemia (AML) patients rarely have long first remissions (> 5 years) after standard-of-care chemotherapy, unless classified as favorable risk at presentation. Identification of the mechanisms responsible for long vs. more typical, short remissions may help to define prognostic determinants for chemotherapy responses. Using exome sequencing, RNA sequencing and functional immunologic studies, we characterized 28 Normal Karyotype (NK)-AML patients with >5 year first remissions after chemotherapy (Long First Remissions, LFR) and compared them to a well-matched group of 31 NK-AML patients who relapsed within 2 years (Standard First Remissions, SFR). Our combined analyses indicated that genetic risk profiling at presentation (as defined by ELN 2017 Criteria) was not sufficient to explain the outcomes of many SFR cases. Single cell RNA-sequencing studies of 15 AML samples showed that SFR AML cells differentially expressed many genes associated with immune suppression. The bone marrow of SFR cases had significantly fewer CD4+ Th1 cells; these T cells expressed an exhaustion signature and were resistant to activation by T-cell receptor stimulation in the presence of autologous AML cells. T cell activation could be restored by removing the AML cells, or blocking the inhibitory MHC Class II receptor, LAG3. Most LFR cases did not display these features, suggesting that their AML cells were not as immunosuppressive. These findings were confirmed and extended in an independent set of 50 AML cases representing all ELN 2017 risk groups. AML cell-mediated suppression of CD4+ Tcell activation at presentation is strongly associated with unfavorable outcomes in AML patients treated with standard chemotherapy.

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