[Preparation of a dual-specific antibody targeting human CD123 and exploration of its anti-acute myeloid leukemia effects].

Objective: To construct a novel dual-specific antibody targeting human CD123 (CD123 DuAb) and study its effects in acute myeloid leukemia (AML) . Methods: Based on the variable region of the CD123 monoclonal antibody independently developed at our institution, the CD123 DuAb expression plasmid was constructed by molecular cloning and transfected into ExpiCHO-S cells to prepare the antibody protein. Through a series of in vitro experiments, its activation and proliferation effect on T cells, as well as the effect of promoting T-cell killing of AML cells, were verified. Results: ① A novel CD123 DuAb plasmid targeting CD123 was successfully constructed and expressed in the Expi-CHO eukaryotic system. ②The CD123 DuAb could bind both CD3 on T cells and CD123 on CD123(+) tumor cells. ③When T cells were co-cultured with MV4-11 cells with addition of the CD123 DuAb at a concentration of 1 nmol/L, the positive expression rates of CD69 and CD25 on T cells were 68.0% and 44.3%, respectively, which were significantly higher than those of the control group (P<0.05). ④Co-culture with CD123 DuAb at 1 nmol/L promoted T-cell proliferation, and the absolute T-cell count increased from 5×10(5)/ml to 3.2×10(6)/ml on day 9, and CFSE fluorescence intensity decreased significantly. ⑤ With the increase in CD123 DuAb concentration in the culture system, T-cell exhaustion and apoptosis increased. When the CD123 DuAb was added at a concentration of 1 nmol/L to the culture system, the proportion of CD8(+) PD-1(+) LAG-3(+) T cells was 10.90%, and the proportion of propidium iodide (PI) (-) Annexin Ⅴ(+) T cells and PI(+) Annexin Ⅴ(+) T cells was 18.27% and 11.43%, respectively, which were significantly higher than those in the control group (P<0.05). ⑥ The CD123 DuAb significantly activated T cells, and the activation intensity was positively correlated with its concentration. The expression rate of CD107a on T cells reached 16.05% with 1 nmol/L CD123 DuAb, which was significantly higher than that of the control group (P<0.05). ⑦The CD123 DuAb promoted cytokine secretion by T cells at a concentration of 1 nmol/L, and the concentration of IFN-γ and TNF-α in the supernatant of the co-culture system reached 193.8 pg/ml and 169.8 pg/ml, respectively, which was significantly higher than that of the control group (P<0.05). ⑧When CD123 DuAb was added at a concentration of 1 nmol/L to the co-culture system of T cells and CD123(+) tumor cells, the killing intensity of T cells significantly increased, and the residual rates of CD123(+) MV4-11 cells, CD123(+) Molm13 cells, and CD123(+) THP-1 cells were 7.4%, 6.7%, and 14.6% on day 3, respectively, which were significantly lower than those in the control group (P<0.05) . Conclusion: In this study, a novel CD123 DuAb was constructed and expressed. In vitro experiments verified that the DuAb binds to CD123(+) tumor cells and T cells simultaneously, promotes T-cell activation and proliferation, and facilitates their anti-leukemia effect, which provides a basis for further clinical research.

Dual-targeting CD33/CD123 NANOBODY T-cell engager with potent anti-AML activity and good safety profile.

ABSTRACT: Novel therapies are needed for effective treatment of acute myeloid leukemia (AML). Relapse is common and salvage treatment with cytotoxic chemotherapy is rarely curative. CD123 and CD33, 2 clinically validated targets in AML, are jointly expressed on blasts and leukemic stem cells in >95% of patients with AML. However, their expression is heterogenous between subclones and between patients, which may affect the efficacy of single-targeting agents in certain patient populations. We present here a dual-targeting CD33/CD123 NANOBODY T-cell engager (CD33/CD123-TCE) that was designed to decrease the risk of relapse from possible single antigen-negative clones and to increase coverage within and across patients. CD33/CD123-TCE killed AML tumor cells expressing 1 or both antigens in vitro. Compared with single-targeting control compounds, CD33/CD123-TCE conferred equal or better ex vivo killing of AML blasts in most primary AML samples tested, suggesting a broader effectiveness across patients. In a disseminated cell-line-derived xenograft mouse model of AML, CD33/CD123-TCE cleared cancer cells in long bones and in soft tissues. As cytokine release syndrome is a well-documented adverse effect of TCE, the compound was tested in a cytokine release assay and shown to induce less cytokines compared to a CD123 single-targeting control. In an exploratory single-dose nonhuman primate study, CD33/CD123-TCE revealed a favorable PK profile. Depletion of CD123 and CD33 expressing cells was observed, but there were neither signs of cytokine release syndrome nor clinical signs of toxicity. Taken together, the CD33/CD123 dual-targeting NANOBODY TCE exhibits potent and safe anti-AML activity and promises a broad patient coverage.

MARCH3 negatively regulates IL-3-triggered inflammatory response by mediating K48-linked polyubiquitination and degradation of IL-3Rα.

Interleukin-3 (IL-3) is a hematopoietic growth factor and critical regulator of inflammatory response such as sepsis. IL-3 binds to IL-3 receptor α (IL-3Rα), which is then associated with IL-3Rß to initiate signaling. How IL-3-triggered physiological and pathological effects are regulated at the receptor level is unclear. Here, we show that the plasma membrane-associated E3 ubiquitin ligase MARCH3 negatively regulates IL-3-triggered signaling. MARCH3 is associated with IL-3Rα, mediates its K48-linked polyubiquitination at K377 and promotes its proteasomal degradation. MARCH3-deficiency promotes IL-3-triggered transcription of downstream effector genes and IL-3-induced expansion of myeloid cells. In the cecal ligation and puncture (CLP) model of sepsis, MARCH3-deficiency aggravates IL-3-ampified expression of inflammatory cytokines, organ damage and inflammatory death. Our findings suggest that regulation of IL-3Rα by MARCH3 plays an important role in IL-3-triggered physiological functions and inflammatory diseases.

Engineering CAR-NK cells to secrete IL-15 sustains their anti-AML functionality but is associated with systemic toxicities.

BACKGROUND: The prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. Natural Killer (NK) cells can elicit an antileukemic response via a repertoire of activating receptors that bind AML surface ligands. NK-cell adoptive transfer is safe but thus far has shown limited anti-AML efficacy. Here, we aimed to overcome this limitation by engineering NK cells to express chimeric antigen receptors (CARs) to boost their anti-AML activity and interleukin (IL)-15 to enhance their persistence. METHODS: We characterized in detail NK-cell populations expressing a panel of AML (CD123)-specific CARs and/or IL-15 in vitro and in AML xenograft models. RESULTS: CARs with 2B4.ζ or 4-1BB.ζ signaling domains demonstrated greater cell surface expression and endowed NK cells with improved anti-AML activity in vitro. Initial in vivo testing revealed that only 2B4.ζ Chimeric Antigen Receptor (CAR)-NK cells had improved anti-AML activity in comparison to untransduced (UTD) and 4-1BB.ζ CAR-NK cells. However, the benefit was transient due to limited CAR-NK-cell persistence. Transgenic expression of secretory interleukin (sIL)-15 in 2B4.ζ CAR and UTD NK cells improved their effector function in the setting of chronic antigen simulation in vitro. Multiparameter flow analysis after chronic antigen exposure identified the expansion of unique NK-cell subsets. 2B4.ζ/sIL-15 CAR and sIL-15 NK cells maintained an overall activated NK-cell phenotype. This was confirmed by transcriptomic analysis, which revealed a highly proliferative and activated signature in these NK-cell groups. In vivo, 2B4.ζ/sIL-15 CAR-NK cells had potent anti-AML activity in one model, while 2B4.ζ/sIL-15 CAR and sIL-15 NK cells induced lethal toxicity in a second model. CONCLUSION: Transgenic expression of CD123-CARs and sIL-15 enabled NK cells to function in the setting of chronic antigen exposure but was associated with systemic toxicities. Thus, our study provides the impetus to explore inducible and controllable expression systems to provide cytokine signals to AML-specific CAR-NK cells before embarking on early-phase clinical testing.

Demethylating therapy increases anti-CD123 CAR T cell cytotoxicity against acute myeloid leukemia.

Successful treatment of acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) T cells is hampered by toxicity on normal hematopoietic progenitor cells and low CAR T cell persistence. Here, we develop third-generation anti-CD123 CAR T cells with a humanized CSL362-based ScFv and a CD28-OX40-CD3ζ intracellular signaling domain. This CAR demonstrates anti-AML activity without affecting the healthy hematopoietic system, or causing epithelial tissue damage in a xenograft model. CD123 expression on leukemia cells increases upon 5'-Azacitidine (AZA) treatment. AZA treatment of leukemia-bearing mice causes an increase in CTLA-4negative anti-CD123 CAR T cell numbers following infusion. Functionally, the CTLA-4negative anti-CD123 CAR T cells exhibit superior cytotoxicity against AML cells, accompanied by higher TNFα production and enhanced downstream phosphorylation of key T cell activation molecules. Our findings indicate that AZA increases the immunogenicity of AML cells, enhancing recognition and elimination of malignant cells by highly efficient CTLA-4negative anti-CD123 CAR T cells.

LOX-1-Expressing Immature Neutrophils Identify Critically-Ill COVID-19 Patients at Risk of Thrombotic Complications.

Background: Lymphopenia and the neutrophil/lymphocyte ratio may have prognostic value in COVID-19 severity. Objective: We investigated neutrophil subsets and functions in blood and bronchoalveolar lavage (BAL) of COVID-19 patients on the basis of patients' clinical characteristics. Methods: We used a multiparametric cytometry profiling based to mature and immature neutrophil markers in 146 critical or severe COVID-19 patients. Results: The Discovery study (38 patients, first pandemic wave) showed that 80% of Intensive Care Unit (ICU) patients develop strong myelemia with CD10-CD64+ immature neutrophils (ImNs). Cellular profiling revealed three distinct neutrophil subsets expressing either the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the interleukin-3 receptor alpha (CD123), or programmed death-ligand 1 (PD-L1) overrepresented in ICU patients compared to non-ICU patients. The proportion of LOX-1- or CD123-expressing ImNs is positively correlated with clinical severity, cytokine storm (IL-1ß, IL-6, IL-8, TNFα), acute respiratory distress syndrome (ARDS), and thrombosis. BALs of patients with ARDS were highly enriched in LOX-1-expressing ImN subsets and in antimicrobial neutrophil factors. A validation study (118 patients, second pandemic wave) confirmed and strengthened the association of the proportion of ImN subsets with disease severity, invasive ventilation, and death. Only high proportions of LOX-1-expressing ImNs remained strongly associated with a high risk of severe thrombosis independently of the plasma antimicrobial neutrophil factors, suggesting an independent association of ImN markers with their functions. Conclusion: LOX-1-expressing ImNs may help identifying COVID-19 patients at high risk of severity and thrombosis complications.

SIRPα-αCD123 fusion antibodies targeting CD123 in conjunction with CD47 blockade enhance the clearance of AML-initiating cells.

BACKGROUND: Acute myeloid leukaemia (AML) stem cells (LSCs) cause disease relapse. The CD47 "don't eat me signal" is upregulated on LSCs and contributes to immune evasion by inhibiting phagocytosis through interacting with myeloid-specific signal regulatory protein alpha (SIRPα). Activation of macrophages by blocking CD47 has been successful, but the ubiquitous expression of CD47 on healthy cells poses potential limitations for such therapies. In contrast, CD123 is a well-known LSC-specific surface marker utilized as a therapeutic target. Here, we report the development of SIRPα-αCD123 fusion antibodies that localize the disruption of CD47/SIRPα signalling to AML while specifically enhancing LSC clearance. METHODS: SIRPα-αCD123 antibodies were generated by fusing the extracellular domain of SIRPα to an αCD123 antibody. The binding properties of the antibodies were analysed by flow cytometry and surface plasmon resonance. The functional characteristics of the fusion antibodies were determined by antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity assays using primary AML patient cells. Finally, an in vivo engraftment assay was utilized to assess LSC targeting. RESULTS: SIRPα-αCD123 fusion antibodies exhibited increased binding and preferential targeting of CD123+ CD47+ AML cells even in the presence of CD47+ healthy cells. Furthermore, SIRPα-αCD123 fusion antibodies confined disruption of the CD47-SIRPα axis locally to AML cells. In vitro experiments demonstrated that SIRPα-αCD123 antibodies greatly enhanced AML cell phagocytosis mediated by allogeneic and autologous macrophages. Moreover, SIRPα-αCD123 fusion antibodies efficiently targeted LSCs with in vivo engraftment potential. CONCLUSIONS: SIRPα-αCD123 antibodies combine local CD47 blockade with specific LSC targeting in a single molecule, minimize the risk of targeting healthy cells and efficiently eliminate AML LSCs. These results validate SIRPα-αCD123 antibodies as promising therapeutic interventions for AML.

Improved Activity against Acute Myeloid Leukemia with Chimeric Antigen Receptor (CAR)-NK-92 Cells Designed to Target CD123.

Anti-cancer activity can be improved by engineering immune cells to express chimeric antigen receptors (CARs) that recognize tumor-associated antigens. Retroviral vector gene transfer strategies allow stable and durable transgene expression. Here, we used alpharetroviral vectors to modify NK-92 cells, a natural killer cell line, with a third-generation CAR designed to target the IL-3 receptor subunit alpha (CD123), which is strongly expressed on the surface of acute myeloid leukemia (AML) cells. Alpharetroviral vectors also contained a transgene cassette to allow constitutive expression of human IL-15 for increased NK cell persistence in vivo. The anti-AML activity of CAR-NK-92 cells was tested via in vitro cytotoxicity assays with the CD123+ AML cell line KG-1a and in vivo in a patient-derived xenotransplantation CD123+ AML model. Unmodified NK-92 cells or NK-92 cells modified with a truncated version of the CAR that lacked the signaling domain served as controls. Alpharetroviral vector-modified NK-92 cells stably expressed the transgenes and secreted IL-15. Anti-CD123-CAR-NK-92 cells exhibited enhanced anti-AML activity in vitro and in vivo as compared to control NK-92 cells. Our data (1) shows the importance of IL-15 expression for in vivo persistence of NK-92 cells, (2) supports continued investigation of anti-CD123-CAR-NK cells to target AML, and (3) points towards potential strategies to further improve CAR-NK anti-AML activity.

Characterization of Plasmacytoid Dendritic Cells, Microbial Sequences, and Identification of a Candidate Public T-Cell Clone in Kikuchi-Fujimoto Disease.

OBJECTIVES: Kikuchi-Fujimoto disease (KFD) is a self-limited lymphadenitis of unclear etiology. We aimed to further characterize this disease in pediatric patients, including evaluation of the CD123 immunohistochemical (IHC) staining and investigation of potential immunologic and infectious causes. METHODS: Seventeen KFD cases and 12 controls were retrospectively identified, and the histologic and clinical features were evaluated. CD123 IHC staining was quantified by digital image analysis. Next generation sequencing was employed for comparative microbial analysis via RNAseq (5 KFD cases) and to evaluate the immune repertoire (9 KFD cases). RESULTS: In cases of lymphadenitis with necrosis, >0.85% CD123+ cells by IHC was found to be six times more likely in cases with a final diagnosis of KFD (sensitivity 75%, specificity 87.5%). RNAseq based comparative microbial analysis did not detect novel or known pathogen sequences in KFD. A shared complementarity determining region 3 (CDR3) sequence and use of the same T-cell receptor beta variable region family was identified in KFD LNs but not controls, and was not identified in available databases. CONCLUSIONS: Digital quantification of CD123 IHC can distinguish KFD from other necrotizing lymphadenitides. The presence of a unique shared CDR3 sequence suggests that a shared antigen underlies KFD pathogenesis.

Combining phage display with SMRTbell next-generation sequencing for the rapid discovery of functional scFv fragments.

Phage display technology in combination with next-generation sequencing (NGS) currently is a state-of-the-art method for the enrichment and isolation of monoclonal antibodies from diverse libraries. However, the current NGS methods employed for sequencing phage display libraries are limited by the short contiguous read lengths associated with second-generation sequencing platforms. Consequently, the identification of antibody sequences has conventionally been restricted to individual antibody domains or to the analysis of single domain binding moieties such as camelid VHH or cartilaginous fish IgNAR antibodies. In this study, we report the application of third-generation sequencing to address this limitation. We used single molecule real time (SMRT) sequencing coupled with hairpin adaptor loop ligation to facilitate the accurate interrogation of full-length single-chain Fv (scFv) libraries. Our method facilitated the rapid isolation and testing of scFv antibodies enriched from phage display libraries within days following panning. Two libraries against CD160 and CD123 were panned and monitored by NGS. Analysis of NGS antibody data sets led to the isolation of several functional scFv antibodies that were not identified by conventional panning and screening strategies. Our approach, which combines phage display selection of immune libraries with the full-length interrogation of scFv fragments, is an easy method to discover functional antibodies, with a range of affinities and biophysical characteristics.

A CRISPR Screen Reveals Resistance Mechanisms to CD3-Bispecific Antibody Therapy.

CD3-bispecific antibodies represent an important therapeutic strategy in oncology. These molecules work by redirecting cytotoxic T cells to antigen-bearing tumor cells. Although CD3-bispecific antibodies have been developed for several clinical indications, cases of cancer-derived resistance are an emerging limitation to the more generalized application of these molecules. Here, we devised whole-genome CRISPR screens to identify cancer resistance mechanisms to CD3-bispecific antibodies across multiple targets and cancer types. By validating the screen hits, we found that deficiency in IFNγ signaling has a prominent role in cancer resistance. IFNγ functioned by stimulating the expression of T-cell killing-related molecules in a cell type-specific manner. By assessing resistance to the clinical CD3-bispecific antibody flotetuzumab, we identified core fucosylation as a critical pathway to regulate flotetuzumab binding to the CD123 antigen. Disruption of this pathway resulted in significant resistance to flotetuzumab treatment. Proper fucosylation of CD123 was required for its normal biological functions. In order to treat the resistance associated with fucosylation loss, flotetuzumab in combination with an alternative targeting CD3-bispecific antibody demonstrated superior efficacy. Together, our study reveals multiple mechanisms that can be targeted to enhance the clinical potential of current and future T-cell-engaging CD3-bispecific antibody therapies.

Development of Highly Optimized Antibody-Drug Conjugates against CD33 and CD123 for Acute Myeloid Leukemia.

PURPOSE: Mortality due to acute myeloid leukemia (AML) remains high, and the management of relapsed or refractory AML continues to be therapeutically challenging. The reapproval of Mylotarg, an anti-CD33-calicheamicin antibody-drug conjugate (ADC), has provided a proof of concept for an ADC-based therapeutic for AML. Several other ADCs have since entered clinical development of AML, but have met with limited success. We sought to develop a next-generation ADC for AML with a wide therapeutic index (TI) that overcomes the shortcomings of previous generations of ADCs. EXPERIMENTAL DESIGN: We compared the TI of our novel CD33-targeted ADC platform with other currently available CD33-targeted ADCs in preclinical models of AML. Next, using this next-generation ADC platform, we performed a head-to-head comparison of two attractive AML antigens, CD33 and CD123. RESULTS: Our novel ADC platform offered improved safety and TI when compared with certain currently available ADC platforms in preclinical models of AML. Differentiation between the CD33- and CD123-targeted ADCs was observed in safety studies conducted in cynomolgus monkeys. The CD33-targeted ADC produced severe hematologic toxicity, whereas minimal hematologic toxicity was observed with the CD123-targeted ADC at the same doses and exposures. The improved toxicity profile of an ADC targeting CD123 over CD33 was consistent with the more restricted expression of CD123 in normal tissues. CONCLUSIONS: We optimized all components of ADC design (i.e., leukemia antigen, antibody, and linker-payload) to develop an ADC that has the potential to translate into an effective new therapy against AML.

CD123 bi-specific antibodies in development in AML: What do we know so far?

Bispecific antibodies are synthetic molecules designed to simultaneously bind two separate antigens. Given the recent success of blinatumomab in the treatment of acute lymphoblastic leukemia, there is growing interest in the use of bispecific antibodies as T-cell redirecting antibody for the treatment of cancer. In acute myeloid leukemia (AML), CD123 (also known as the interleukin receptor 3 alpha subunit) has emerged as a promising therapeutic target for bispecific antibodies. Prior attempts to target CD123 with unconjugated antibodies and antibody-drug conjugates have been mixed. However, available data from CD123-directed bispecific antibodies currently in clinical trials have been encouraging. In this review, we discuss the biology of CD123 and prior attempts to target this cell surface marker as part of anti-leukemic therapy. We then summarize and discuss the five CD123-directed bispecific antibodies currently in clinical trials for treatment of AML and provide practical insights regarding the use of these agents.

Expression of putative leukemia stem cell targets in genetically-defined acute myeloid leukemia subtypes.

Although most acute myeloid leukemia (AML) patients achieve complete remissions, the majority still eventually relapse and die of their disease. Rare primitive leukemia cells, so-called leukemia stem cells (LSCs), represent one potential type of resistant cell subpopulation responsible for this dissociation between response and cure. Several LSC targets have been described, but there is limited evidence about their relative utility or that targeting any can prevent relapse. LSCs not only appear to be biologically heterogeneous, but the classic immunocompromised mouse transplantation model also has serious shortcomings as an LSC assay. Out data suggest that the most immature cell phenotype that can be identified within a patient's leukemia may be clinically relevant and represent the de facto LSC. Moreover, although phenotypically heterogeneous, these putative LSCs show consistent phenotypes within individual genetically defined groups. Using this LSC definition, we studied several previously described putative LSC targets, CD25, CD26, CD47, CD96, CD123, and CLL-1, and all were expressed across heterogeneous LSC phenotypes. In addition, with the exception of CD47, there was at most low expression of these targets on normal hematopoietic stem cells (HSCs). CD123 and CLL-1 demonstrated the greatest expression differences between putative LSCs and normal HSCs. Importantly, CD123 monoclonal antibodies were cytotoxic in vitro to putative LSCs from all AML subtypes, while showing limited to no toxicity against normal HSCs and hematopoietic progenitors. Since minimal residual disease appears to be a more homogeneous population of cells responsible for relapse, targeting CD123 in this setting may be most effective.

Targeting CD123 in AML.

An ideal cell surface target for therapy in leukemia would be: tumor-specific (not expressed on normal cells) or at least enriched on tumor cells, necessary for tumor but not for normal cell survival, internalized efficiently (if the surface-targeted agent is conjugated to chemotherapy or a toxin molecule), and recycled rapidly to the cell surface. While a single target that meets all of these criteria has not yet been discovered in AML, CD123 has emerged as an attractive candidate.1 The first-in-class CD123-targeting agent, tagraxofusp-erzs (SL-401) was approved in 2018 for patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN)2 and is currently in trials for several other hematologic malignancies, including AML. Several other CD123-targeted drugs are in development.

Blockade of VLA4 sensitizes leukemic and myeloma tumor cells to CD3 redirection in the bone marrow microenvironment.

Redirecting T cells to specifically kill malignant cells has been validated as an effective anti-cancer strategy in the clinic with the approval of blinatumomab for acute lymphoblastic leukemia. However, the immunosuppressive nature of the tumor microenvironment potentially poses a significant hurdle to T cell therapies. In hematological malignancies, the bone marrow (BM) niche is protective to leukemic stem cells and has minimized the efficacy of several anti-cancer drugs. In this study, we investigated the impact of the BM microenvironment on T cell redirection. Using bispecific antibodies targeting specific tumor antigens (CD123 and BCMA) and CD3, we observed that co-culture of acute myeloid leukemia or multiple myeloma cells with BM stromal cells protected tumor cells from bispecific antibody-T cell-mediated lysis in vitro and in vivo. Impaired CD3 redirection cytotoxicity was correlated with reduced T cell effector responses and cell-cell contact with stromal cells was implicated in reducing T cell activation and conferring protection of cancer cells. Finally, blocking the VLA4 adhesion pathway in combination with CD3 redirection reduced the stromal-mediated inhibition of cytotoxicity and T cell activation. Our results lend support to inhibiting VLA4 interactions along with administering CD3 redirection therapeutics as a novel combinatorial regimen for robust anti-cancer responses.

Different distribution patterns of plasmacytoid dendritic cells in discoid lupus erythematosus and lichen planopilaris demonstrated by CD123 immunostaining

Abstract Background: Clinical and histological features may overlap between lichen planopilaris-associated and discoid lupus erythematosus-associated scarring alopecia. Objectives: The aim of this study was to demonstrate the cutaneous infiltration of plasmacytoid dendritic cells and to compare their distribution pattern in discoid lupus erythematosus and lichen planopilaris. Methods: Twenty-four cases of discoid lupus erythematosus and 30 cases of lichen planopilaris were examined for immunostaining of the CD123 marker. The percentage and distribution pattern of plasmacytoid dendritic cells and the presence of the plasmacytoid dendritic cells clusters were evaluted in the samples. Results: The number of plasmacytoid dendritic cells was higher in the discoid lupus erythematosus specimens. Aggregations of 10 cells or more (large cluster) were observed in half of the discoid lupus erythematosus specimens and only 2 lichen planopilaris, with 50% sensitivity and 93% specificity for differentiating discoid lupus erythematosus from lichen planopilaris. Study limitations: Incidence and prevalence of discoid lupus erythematosus-associated scarring alopecia in the scalp are low, so the samples size of our study was small. Conclusions: We suggest that a plasmacytoid dendritic cells cluster of 10 cells or more is highly specific for distinguishing discoid lupus erythematosus from lichen planopilaris. It also appears that CD123 immunolabeling is valuable in both active and late stages of the disease.

Different distribution patterns of plasmacytoid dendritic cells in discoid lupus erythematosus and lichen planopilaris demonstrated by CD123 immunostaining.

BACKGROUND: Clinical and histological features may overlap between lichen planopilaris-associated and discoid lupus erythematosus-associated scarring alopecia. OBJECTIVES: The aim of this study was to demonstrate the cutaneous infiltration of plasmacytoid dendritic cells and to compare their distribution pattern in discoid lupus erythematosus and lichen planopilaris. METHODS: Twenty-four cases of discoid lupus erythematosus and 30 cases of lichen planopilaris were examined for immunostaining of the CD123 marker. The percentage and distribution pattern of plasmacytoid dendritic cells and the presence of the plasmacytoid dendritic cells clusters were evaluted in the samples. RESULTS: The number of plasmacytoid dendritic cells was higher in the discoid lupus erythematosus specimens. Aggregations of 10 cells or more (large cluster) were observed in half of the discoid lupus erythematosus specimens and only 2 lichen planopilaris, with 50% sensitivity and 93% specificity for differentiating discoid lupus erythematosus from lichen planopilaris. STUDY LIMITATIONS: Incidence and prevalence of discoid lupus erythematosus-associated scarring alopecia in the scalp are low, so the samples size of our study was small. CONCLUSIONS: We suggest that a plasmacytoid dendritic cells cluster of 10 cells or more is highly specific for distinguishing discoid lupus erythematosus from lichen planopilaris. It also appears that CD123 immunolabeling is valuable in both active and late stages of the disease.

CD28/4-1BB CD123 CAR T cells in blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is associated with a remarkably poor prognosis and with no treatment consensus. The identification of relevant therapeutic targets is challenging. Here, we investigated the immune functions, antileukemia efficacy and safety of CD28/4-1BB CAR T cells targeting CD123 the interleukin (IL)-3 receptor alpha chain which is overexpressed on BPDCN. We demonstrated that both retroviral and lentiviral engineering CD28/4-1BB CD123 CAR T cells exhibit effector functions against BPDCN cells through CD123 antigen recognition and that they efficiently kill BPDCN cell lines and BPDCN-derived PDX cells. In vivo, CD28/4-1BB CD123 CAR T-cell therapy displayed strong efficacy by promoting a decrease of BPDCN blast burden. Furthermore we showed that T cells from BPDCN patient transduced with CD28/4-1BB CD123 CAR successfully eliminate autologous BPDCN blasts in vitro. Finally, we demonstrated in humanized mouse models that these effector CAR T cells exert low or no cytotoxicity against various subsets of normal cells with low CD123 expression, indicating a potentially low on-target/off-tumor toxicity effect. Collectively, our data support the further evaluation for clinical assessment of CD28/4-1BB CD123 CAR T cells in BPDCN neoplasm.

Daunorubicin-Loaded CdTe QDs Conjugated with Anti-CD123 mAbs: A Novel Delivery System for Myelodysplastic Syndromes Treatment.

INTRODUCTION: The myelodysplastic syndromes (MDS) are a very heterogeneous group of myeloid disorders characterized by peripheral blood cytopenias and increase risk of transformation to acute myeloid leukemia (AML). Daunorubicin (DNR) is an indispensable drug for the treatment of MDS and AML. However, its side effects including cardiac toxicity and bone marrow suppression severely limit clinical application. Many researches reported high expression of CD123 antigen on high-risk MDS cells, so we constructed a novel drug delivery system comprising daunorubicin-loaded CdTe QDs conjugated with anti-CD123 mAbs (DNR-CdTe-CD123) to develop targeted combination chemotherapy for MDS. METHODS: CdTe conjugated antiCD123 through amide bond, co-loaded with DNR with electrostatic bonding. Then, we determined characterization and release rate of DNR-CdTe-CD123. The therapeutic effect and side effect of drug delivery system were evaluated through in vitro and in vivo experiments. RESULTS: CdTe showed appropriate diameter and good dispersibility and DNR was loaded into CdTes with high encapsulation efficiency and drug loading. The maximum drug loading and encapsulation efficiency were 42.08 ± 0.64% and 74.52 ± 1.81%, respectively, at DNR concentration of 0.2mg/mL and anti-CD123 mAbs volume of 5ul (100ug/mL). Flow cytometry (FCM) showed that CD123 antigen was highly expressed on MUTZ-1 cells, and its expression rate was 72.89 ± 10.67%. In vitro experiments showed that the inhibition rate and apoptosis rate of MUTZ-1 cells treated with DNR-CdTe-CD123 were higher than those in the other groups (P<0.05). Compared with the other groups, the level of apoptosis-related protein (P53, cleaved caspase-9, Bax and cleaved caspase-3) were upregulated in DNR-CdTe-CD123 group (P<0.05). In vivo experiments, DNR-CdTe-CD123 can effectively inhibit the tumor growth of MDS-bearing nude mice and reduce the side effects of DNR on myocardial cells. CONCLUSION: The system of DNR-CdTe-CD123 enhances the therapeutic effects and reduce the side effects of DNR, thus providing a novel platform for MDS treatment.