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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Abstract CAR T-cell therapy has remarkably succeeded in treating lymphoblastic leukemia. However, its success in AML remains elusive due to the risk of on-target off-tumor toxicity to hematopoietic stem and 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 anti-tumor 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 (PD1, Tim3, LAG3, and CD39), higher frequency of memory T cells (CD62L+CD45RA+), and enhanced expansion. While targeting AML, the moderated circuit CAR signal also helped to mitigate cytokine release syndrome, potentially addressing one of the ongoing challenges in CAR-T immunotherapy.Keywords:
Interleukin-3 receptor
Cytokine Release Syndrome
CD15
Precursor cell
Lineage (genetic)
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Objectives In myelodysplastic syndromes (MDS), neoplastic myeloblast (CD34+CD13+CD33+ cells) numbers often increase over time, leading to secondary acute myeloid leukemia (AML). In recent studies, blasts in some MDS patients have been found to express a megakaryocyte-lineage molecule, CD41, and such patients show extremely poor prognosis. This is the first study to evaluate whether myeloblasts transition to CD41+ blasts over time and to investigate the detailed immunophenotypic features of CD41+ blasts in MDS. Methods We performed a retrospective cohort study, in which time-dependent changes in blast immunophenotypes were analyzed using multidimensional flow cytometry (MDF) in 74 patients with MDS and AML (which progressed from MDS). Results CD41+ blasts (at least 20% of CD34+ blasts expressing CD41) were detected in 12 patients. In five of these 12 patients, blasts were CD41+ from the first MDF analysis. In the other seven patients, myeloblasts (CD34+CD33+CD41- cells) transitioned to megakaryoblasts (CD34+CD41+ cells) over time, which was often accompanied by disease progression (including leukemic transformation). These CD41+ patients were more frequently observed among patients with monosomal and complex karyotypes. CD41+ blasts were negative for the erythroid antigen, CD235a, and positive for CD33 in all cases, but CD33 expression levels were lower in three cases when compared with CD34+CD41- blasts. Among the five CD41+ patients who underwent extensive immunophenotyping, CD41+ blasts all expressed CD61, but two cases had reduced CD42b expression, three had reduced/absent CD13 expression, and three also expressed CD7. Conclusions Myeloblasts become megakaryoblastic over time in some MDS patients, and examining the megakaryocyte lineage (not only as a diagnostic work-up but also as follow-up) is needed to detect CD41+ MDS. The immunophenotypic features revealed in this study may have diagnostic relevance for CD41+ MDS patients.
Immunophenotyping
Acute megakaryoblastic leukemia
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CD117
Immunophenotyping
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We examined the multidrug resistant P‐glycoprotein (P‐gp) on normal bone marrow (BM) cells and acute myeloid leukaemia (AML) cells, using newly devised flow cytometric multi‐parameter analysis with CD33, CD34 and MRK16 monoclonal antibodies. In both normal BM cells and AML cells, CD34 + CD33 − cells expressed P‐gp strongly, CD34 + CD33 + cells moderately, and CD34 − CD33 + cells weakly. Acute promyelocytic leukaemia, mainly expressing CD34 − CD33 + but not CD34 + CD33 − at diagnosis, expressed less P‐gp. P‐gp expression of AML cells at diagnosis was increased as compared with normal cells of the same phenotype. P‐gp expression was more increased in relapsed cases, especially in immature subpopulations.
P-glycoprotein
Immunophenotyping
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Summary We have analysed the immunological characteristics of blasts from 89 acute lymphoblastic leukaemia (ALL) cases (62 adults and 27 children), by using a panel of antilymphoid and myeloid associated monoclonal antibodies (McAb) and the APAAP method, which detects membrane and cytoplasmic expression of antigens. The McAb CD19 was the marker most consistently expressed in B lineage ALL, being positive in 100% of cases, compared to CD24 and CD22 expressed in 82% and 79%, respectively. Similarly, for the T lymphoid lineage, the McAb CD3 was the most reliable and specific marker, being expressed in all T‐ALL cases including those with an early thymic phenotype (CD7 +, TdT+). Lymphoblasts from eight adults (12.9%) and three children (11.1%) expressed one to four myeloid associated antigens recognized by CD13, CD14, CD33 and anti‐myeloperoxidase. There were no substantial clinical and morphological differences between the two ALL groups with or without myeloid associated markers. However, the presence of myeloid associated markers in adult ALL was associated with a significantly lower complete remission (CR) rate ( P =0.05) and with a shorter survival ( P = 0.001): this variable was independent of advanced age and high WBC. It is concluded that immunophenotypic analysis in ALL should include myeloid markers for its probable, prognostic implications.
Lymphoblast
Immunophenotyping
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Precursor cell
Lymphoblast
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The function of human myeloid derived suppressor cells (MDSCs) cannot be precisely measured without the proper identification of homogeneous populations. Indeed, many published accounts of human MDSCs are really describing multiple distinct populations confused either by sample processing, poor gating strategies, and/or lack of appropriate cell surface markers. To improve the standardization of the identification of human myeloid derived suppressor cells and other myeloid phenotypes, we developed a method for the identification and analysis of human myeloid populations by the use of several 10-color flow cytometric protocols in combination with novel software analyses. This method utilizes the direct staining of peripheral blood that allows for the quantitation of myeloid cells and the delineation of non-overlapping immunophenotypes. The 10-color protocols, tested in both healthy volunteer controls and cancer patients, allow us to define diverse phenotypes that include mature monocytes, granulocytes, circulating dendritic cells, immature myeloid cells, in addition to MDSCs. We demonstrate that human MDSC subsets fall into three distinct populations: CD14+HLA-DRlo/neg monocytes, LIN-CD33+HLA-DR- cells, and CD15+ granulocytes. We additionally identify CD123 as a marker uniquely expressed on LIN-CD33+HLA-DR- MDSCs. Our method permits us to measure myeloid and MDSC phenotypes in relation to total mononuclear cells, total myeloid cells, total leukocytes and as cell counts. The data generated from our method will allow for more uniform reporting of myeloid and MDSC phenotypes for biomarker development.
Myeloid-derived Suppressor Cell
Interleukin-3 receptor
Myelopoiesis
CD15
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Objective To study the expression of CD19, CD56 and CD2 in acute myeloid leukemia and their significance in detecting minimal residual disease(MRD). Methods One hundred and thirty bone marrow specimens of new AML patients were selected,and immunophenotypes were assayed using three sets of 4-color antibodies combination(CD34/CD13/CD45/CD19, CD34/CD56/CD33/CD45 and CD34/CD2/CD33/CD45)by multiparameter flow cytometry(MFC). Results Expression frenquence of CD19 was 2.31%, CD56 was 3.75%, and CD2 was 1.25% in AML.None of them were detected in M3. Conclusion Expression frenquence of CD19, CD56, CD2 in AML is low. CD56, CD2 may be markers in detecting MRD in AML, but CD19 could be not.
Minimal Residual Disease
Immunophenotyping
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Objective To analyze expression of differentiation antigen of acute myeloid leukemia(AML) and its relationship with clinical characteristics and prognosis.MethodsThe expression of differentiation antigen of 111 cases of AML was investigated by flow cytometry assay.Results(1)The frequency of antigen expression on blast cells in AML were 89.2%(CD13),79.3%(CD33),50.5%(CD34),37.8%(CD15),36.9%(HLA-DR) and 21.6%(CD14) respectively.HLA-DR was negative in M3 patients.CD7,one of the lymphoid antigens,was found to be expressed frequently in the AML blast cells.(2)The expression of CD15+ of AML was related with the blasts' count and white blood cell count.Those patients with CD7+ usually had spleno-hepatomegaly.(3)The CR rate in the cases with CD34+ was significantly lower than those with CD34-,but the time to CR was longer.CR rate in CD7+ cases was significantly lower than those with CD7-.However,patients with CD19-had lower CR rate than those with CD19+.But interestingly,CR rate in cases with CD7+CD34+ was significantly lower than those with CD7-CD34-.ConclusionCD7,CD19 and CD34 are relating with the clinical characteristics and prognosis of AML,and could be the one of important factors in predicting prognosis.
CD15
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The World Health Organization (WHO) characterization of the immunophenotype of precursor B-cell acute lymphoblastic leukemia (BALL)/lymphoblastic lymphoma (LBL) includes the possible expression of myeloid cluster of differentiation (CD) markers CD13 and CD33. In precursor T-cell ALL/LBL, myeloid markers CD13 and CD33 are frequent while CD117 is rare. In the present investigation, 58 cases of confirmed precursor B-cell ALL/LBL were evaluated for the presence of CD13, CD33 and CD117. Of the 13 (22.4%) cases that positively expressed myeloid markers, 8 (62%) expressed CD13; 10 (77%) expressed CD33; and 1 (8%) expressed CD117. Four (31%) expressed both CD13 and CD33, and 1 (8%) expressed CD13, CD33, and CD117. 18 cases of confirmed precursor T-cell ALL/LBL were analyzed for myeloid markers CD13, CD33, CD117 and MPO. Of the 5 (28%) expressing myeloid markers, 3 (60%) were positive for CD13; 3 (60%) for CD33; 3 (60%) for CD117; and 1 (20%) for MPO. One (20%) was positive for both CD13 and CD117; 1 (20%) for CD13, CD33 and CD117; and 1 (20%) for CD13, CD33 and MPO. These markers portend a poor prognosis compared to ALL/LBL cases without myeloid antigens, and a poor response to drug therapies targeting conventional ALL/LBL. Future studies will be directed to correlation of these markers with prognosis and therapeutic response, as well as whether drug therapies targeting myeloid antigens could be of use in treatment.
CD117
Immunophenotyping
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