Data from CD33–CD123 IF-THEN Gating Reduces Toxicity while Enhancing the Specificity and Memory Phenotype of AML-Targeting CAR-T Cells
Samy JambonJianping SunShawn BarmanSakunthala MuthugounderXue Rachel BitoArmita ShadfarAlexandra E. KovachBrent L. WoodVarsha Thoppey ManoharanA. Sorana MorrissyDeepa BhojwaniAlan S. WayneMichael A. PulsipherYong‐Mi KimShahab AsgharzadehChintan ParekhBabak Moghimi
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<div>Abstract<p>Chimeric antigen receptor (CAR) T-cell therapy has remarkably succeeded in treating lymphoblastic leukemia. However, its success in acute myeloid leukemia (AML) remains elusive because of the risk of on-target off-tumor toxicity to hematopoietic stem/progenitor cells (HSPC) and insufficient T-cell persistence and longevity. Using a SynNotch circuit, we generated a high-precision “IF-THEN” gated logical circuit against the combination of CD33 and CD123 AML antigens and demonstrated antitumor efficacy against AML cell lines and patient-derived xenografts. Unlike constitutively expressed CD123 CAR-T cells, those expressed through the CD33 SynNotch circuit could preserve HSPCs and lower the risk of on-target off-tumor hematopoietic toxicity. These gated CAR-T cells exhibited lower expression of exhaustion markers (PD-1, TIM-3, LAG-3, and CD39), higher frequency of memory T cells (CD62L<sup>+</sup>CD45RA<sup>+</sup>), and enhanced expansion. Although targeting AML, the moderated circuit CAR signal also helped mitigate cytokine release syndrome, potentially addressing one of the ongoing challenges in CAR-T immunotherapy.</p>Significance:<p>Our study demonstrates the use of “IF-THEN” SynNotch-gated CAR-T cells targeting CD33 and CD123 in AML reduces off-tumor toxicity. This strategy enhances T-cell phenotype, improves expansion, preserves HSPCs, and mitigates cytokine release syndrome—addressing critical limitations of existing AML CAR-T therapies.</p></div>Keywords:
Interleukin-3 receptor
Myeloid leukemia cutis (LC) and blastic plasmacytoid dendritic cell neoplasm (BPDCN) are morphologically indistinguishable malignancies that frequently manifest in the skin. Separating myeloperoxidase-negative LC from BPDCN may be particularly challenging. We identified a panel of immunohistochemical stains to distinguish myeloid LC (23 cases) from BPDCN (12 cases): myeloperoxidase, which stained 7 cases (30%) of LC and 0 cases (0%) of BPDCN; CD56, which stained 12 cases (52%) of LC and all 12 cases (100%) of BPDCN; CD4, which stained 2 cases (9%) of LC and all 12 cases (100%) of BPDCN; CD123, which stained 4 cases (17%) of LC and 10 cases (83%) of BPDCN; and Tcl-1, which stained 2 cases (9%) of LC and 9 (82%) of 11 cases of BPDCN. It is interesting that CD33 was not helpful; it stained 18 (78%) cases of LC and 11 cases (92%) of BPDCN. Our results indicate that a panel that includes CD4, CD56, CD123, and Tcl-1 can appropriately distinguish between these 2 entities.
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Owing to the more recent positive results with the anti-CD33 immunotoxin gemtuzumab ozogamicin, therapy against acute myeloid leukemias (AMLs) targeting CD33 holds many promises. Here, CD33 and CD123 expression on AML blasts was studied by flow cytometry in a cohort of 319 patients with detailed information on French-American-British/World Health Organization (FAB/WHO) classification, cytogenetics and molecular aberrations. AMLs of 87.8% express CD33 and would therefore be targetable with anti-CD33 therapies. Additionally, 9.4% of AMLs express CD123 without concomitant CD33 expression. Thus, nearly all AMLs could be either targeted via CD33 or CD123. Simultaneous presence of both antigens was observed in 69.5% of patients. Most importantly, even AMLs with adverse cytogenetics express CD33 and CD123 levels comparable to those with favorable and intermediate subtypes. Some patient groups with unfavorable alterations, such as FMS-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations, high FLT3-ITD mutant/wild-type ratios and monosomy 5 are even characterized by high expression of CD33 and CD123. In addition, blasts of patients with mutant nucleophosmin (NPM1) revealed significantly higher CD33 and CD123 expression pointing toward the possibility of minimal residual disease-guided interventions in mutated NPM1-positive AMLs. These results stimulate the development of novel concepts to redirect immune effector cells toward CD33- and CD123-expressing blasts using bi-specific antibodies or engineered T cells expressing chimeric antigen receptors.
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NPM1
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The aim of the study was to investigate the clinical characteristics and diagnosis of acute myeloid leukemia with CD56- blastic plasmacytoid dendritic cell neoplasm.The clinical characteristics and diagnosis of CD56-blastic plasmacytoid dendritic cell neoplasm and review related literature of 3 patients with acute myeloid leukemia were retrospectively analyzed.This paper reports 3 cases and all were elderly men. The bone marrow features of three patients suggested the diagnosis of acute myeloid leukemia with blastic plasmacytoid dendritic cell neoplasm. Case 1: Flow cytometry showed that visible abnormalities in myeloid cells, accounting for 19.25% of nucleated cells, the phenotypes were CD117+, CD38+, CD33+, CD13+, CD123+, HLA-DR+, CD34 partial+, CD64 partial+ and TDT partial+, CD7-, CD11b-, CD22-, CD15-, CD5-, CD2-, CD20-, CD19-, CD10-, CD4-, CD14-, CD36, MPO-, CD9-, cCD79a-, cCD3-, mCD3-, CD5-. In addition, a group of abnormal plasmacytoid dendritic cells was observed, accounting for 13.83% of nuclear cells (CD2-, TDT partial+, CD303+, CD304+, CD123+, CD34-, HLA-DR+, and CD56-). Second generation sequencing: RUNX1 mutation 41.7%, DNMT3A mutation 41.3%. Case 2: Flow cytometry showed that visible abnormalities in myeloid cells, accounting for 33.66% of nucleated cells, express the strong expression of CD34 expression of CD117, HLA - DR, CD38, CD13, CD33, CD123, the TDT, no expression of MPO, cCD3, and cCD79a, conform to the AML phenotype. In addition, a group of abnormal plasmacytoid dendritic cells was observed, accounting for 26.87% of nucleated cells (CD303+, CD304+, CD123++, HLA-DR+, CD33+, CD36+, CD7 dim, CD4+, CD56-, TDT-). Second generation sequencing: The mutations of FLT3, CBL, RUNX1, and SRSF2 were 7.4%, 7.5%, 53.3%, and 29.9%. Case 3: Flow cytometry showed that visible abnormalities in myeloid cells, accounting for 23.76% of nucleated cells, the phenotypes were CD117++, HLA-DR++, CD34++, CD38+, CD13+, CD123+, CD7 partial+, and CD33 partial+, MPO-, TDT-, cCD3-, cCD79a-. In addition, a group of abnormal plasmacytoid dendritic cells was observed, accounting for 16.66% of nuclear cells (TDT+, CD303+, CD304+, CD 123++, HLA-DR+, CD38+, CD7+, CD56-, CD34-).Acute myeloid leukemia with CD56-blastic plasmacytoid dendritic cell neoplasm is extremely rare and no special clinical manifestations are found, and the diagnosis is based on bone marrow cytology and immunophenotyping. There is no standard regimen for treatment of acute myeloid leukemia with mature blastic plasmacytoid dendritic cell neoplasm, and the prognosis depends on the progression of acute myeloid leukemia.
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CD117
Myeloproliferative neoplasm
CD5
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Immunotherapy, such as chimeric antigen receptor T (CAR-T) cells targeting CD33 or CD123, has been well developed over the past decade for the treatment of acute myeloid leukemia (AML). However, the inability to sustain tumor-free survival and the possibility of relapse due to antigen loss have raised concerns. A dual targeting of CD33 and CD123 is needed for better outcomes. Based on our previously constructed CD33 and CD123 monovalent CAR-T, Loop33 × 123 and Loop123 × 33 CAR-T were constructed with molecular cloning techniques. All CAR-T cells were generated by lentivirus transduction of T cells from healthy donors. Phenotype detection was evaluated on day 7 concerning activation, exhaustion, and subtype proportions. Coculture killing assays were conducted using various AML cell lines and primary AML cells. Degranulation and cytokine secretion levels were detected by flow cytometry. Cell-derived xenograft models were established using wild-type Molm 13 cell lines, or a mixture of Molm 13-KO33 and Molm 13-KO123 cells as an ideal model of immune escape. By monitoring body weight and survival of tumor-bearing mice, Loop33 × 123 and Loop123 × 33 CAR-T cells were further assessed for their efficacy in vivo. In vitro study, our results demonstrated that Loop33 × 123 CAR-T cells could efficiently eliminate AML cell lines and primary AML cells with elevated degranulation and cytokine secretion levels. Compared with our previously constructed monovalent CD33 or CD123 CAR-T cells, Loop33 × 123 CAR-T cells showed superior advantages in an immune escape model. In vivo studies further confirmed that Loop33 × 123 CAR-T cells could effectively prolong the survival of mice without significant toxicity. However, Loop123 × 33 CAR-T cells failed to show the same effects. Furthermore, Loop33 × 123 CAR-T cells efficiently circumvented potential immune escape, a challenge where monovalent CAR-T cells failed. Loop33 × 123 CAR-T targeting CD33 and CD123 could efficiently eliminate AML cells and prolong survival of tumor-bearing mice, while addressing the issue of immune escape.
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FLT3 internal tandem duplication (ITD) mutations are found in around 25% of all acute myeloid leukaemia (AML) cases and is associated with shorter disease-free and overall survival. Previous reports have shown that FLT3-ITD induces a specific phenotype in leukemic blasts, which is characterized by high levels of CD33 and CD123, and that expression of CD33 and CD123 is directly influenced by the DNA FLT3-ITD/wild-type FLT3 allelic ratio (AR).A total of 42 FLT3-ITD and 104 FLT3-ITD-negative AML patients were analysed. Immunophenotyping data were used to calculate antigen expression levels as the ratio between the geometric mean fluorescence intensities (MFIs) of leukemic blasts and MFIs of negative lymphocyte populations. FLT3-ITD-DNA and RNA analysis was performed, under the same conditions, by capillary electrophoresis.Compared with the control group, the FLT3-ITD cohort presented significantly higher CD7, CD33 and CD123 levels. In order to assess the impact of FLT3-ITD abundance on antigen expression, the patients were grouped for each parameter into two cohorts using the following threshold values: (a) 0.5 for the AR, according to current AML guidelines; (b) 0.7 for the FLT3-ITD/FLT3-WT mRNA ratio (RR); and (c) 1.3 for the FLT3-ITD RR/AR ratio. We found higher values of CD33 for RR/AR ≥1.3, and no other statistical differences between CD7, CD33 and CD123 levels of the other FLT3-ITD groups. In terms of correlations between MFI values and FLT3-ITD parameters, we only observed a moderate interdependence between CD33 MFI and the RR/AR ratio, and a weak negative correlation between CD123 MFI and AR.FLT3-ITD mutations induce a specific antigen profile in AML blasts, and our data do not onfirm previous reports of FLT3-ITD AR influencing both CD33 and CD123 expression.
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In the January/February 2013 edition, Maio et al describe a patient with CD4/CD56/CD123 ascribed as blastic plasmacytoid dendritic cell neoplasia (BDCN). However, since CD4/CD56/CD123 neoplasms are highly heterogeneous, the precise diagnosis requires an extensive immunophenotypic panel. 3 Although highly suggestive, the cytochemical positivity for CD4, CD56 together with CD123 in the absence of myeloperoxidase, CD3, CD2, CD5, and CD7, is not sufficient to determine the BDCN malignant nature. Despite the expression of CD123, the aforementioned phenotype could also correspond to acute myeloid dendritic cell leukemia or acute myeloid leukemia (myeloid leukemia cutis), especially with monocytic differentiation. The diagnostic work-up of these entities relies on a comprehensive antibody panel that should also include CD13, CD33, CD15, CD14, CD64, CD16, CD34, CD117, BDCA-2 (CD303), BDCA-4 (CD304), BDCA-3 (CD141) and TCL1. BDCN are phenotypically recognized by expression of specific plasmacytoid dendritic cell proteins (CD303 and CD304) in the absence or dim expression of myeloid markers. Conversely, acute myeloid dendritic cell leukemias specifically express CD141 along with some myeloid markers. By exclusion, the absence of CD303, CD304 and CD141, along with the presence of myeloid and monocytic markers, supports the diagnosis of myelomonocytic leukemia. Additional molecular tests targeting well-characterized abnormalities could serve as ancillary diagnostic tools. Although the panel herein proposed could not be entirely performed on skin biopsies, it could be easily applied by flow cytometry on circulating cells during the disseminated phase. It is clear that strong collaborative efforts are required to improve diagnosis and management of these rare diseases.
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Plasmacytoid dendritic cell
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CD117
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Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare subtype of acute leukemia that typically follows a highly aggressive clinical course in adults, whereas experience in children with this disease is very limited. We report cases of two children in whom bone marrow showed infiltration by large atypical monocytoid 'blast-like' cells which on immunophenotyping expressed CD4, CD56, HLA-DR and CD33 while were negative for CD34 other T-cell, B-cell and myeloid markers. The differential diagnoses considered were AML, T/NK-cell leukemia and acute undifferentiated leukemia. Additional markers CD303/BDCA-2 and CD123 which are recently validated plasmacytoid dendritic cell markers were done which helped us clinch the diagnosis of this rare neoplasm. An accurate diagnosis of BPDCN is essential in order to provide prompt treatment. Due to its rarity and only recent recognition as a distinct clinicopathological entity, no standardized therapeutic approach has been established for BPDCN.
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