Inhibitor of bromodomain and extraterminal domain proteins decreases transcription of Cd33 in the brain of mice subjected to systemic inflammation; a promising strategy for neuroprotection.

The neuroinflammation is a crucial component of virtually all neurodegenerative disorders, including Alzheimer's disease (AD). The bacterial lipopolysaccharide (LPS), a potent activator of the innate immune system, was suggested to influence or even trigger the neuropathological alterations in AD. LPS-induced neuroinflammation involves changes in transcription of several genes, thus controlling these molecular processes may be a potentially efficient strategy to attenuate the progression of AD. Since genome-wide association studies showed that the majority of AD-related genetic risk factors (AD-GRF) are connected to the immune system, our aim was to identify AD-GRF affected in the hippocampus by LPS-induced systemic inflammatory response (SIR). Moreover, we analysed the role of bromodomain and extraterminal domain (BET) proteins, the readers of the acetylation code, in controlling the transcription of selected AD-GRF in the brain during neuroinflammation. In our study, we used a mouse model of LPS-induced SIR and mouse microglial BV2 cells. JQ1 was used as an inhibitor of BET proteins. The level of mRNA was analysed using microarrays and qPCR. Our data demonstrated that among the established AD-GRF, only the expression of Cd33 was significantly upregulated in the hippocampus during SIR. In parallel, we observed an increase in the expression of Brd4, a BET family member. JQ1 prevented an LPS-evoked increase in Cd33 expression in the hippocampus of mice. Moreover, JQ1 reduced Cd33 expression in BV2 microglial cells stimulated with blood serum from LPS-treated mice. Our study suggests that LPS-evoked SIR may increase Cd33 gene expression in the brain, and inhibition of BET proteins through suppression of Cd33 expression could be a promising strategy in prevention or in slowing down the progression of neuroinflammation and may potentially affect the pathomechanism of AD.

Causal role of immune cells in prostate cancer: a bidirectional Mendelian-randomization analyses.

BACKGROUND: Immune cell signatures have been implicated in cancer progression and response to treatment. However, the causal relationship between immune cell signatures and prostate cancer (PCa) is still unclear. This study aimed to investigate the potential causal associations between immune cell signatures and PCa using Mendelian randomization (MR). METHOD: This study utilized genome-wide association studies (GWAS) summary statistics for PCa and immune cell signatures from publicly available datasets. MR analyses, including IVW, MR-Egger, and weighted median methods, were performed to evaluate the causal associations between immune cell signatures and PCa. Multiple sensitivity analysis methods have been adopted to test the robustness of our results. RESULTS: After FDR correction, our findings suggested that specific immune cell signatures, such as HLA DR on CD33+ HLA DR+ CD14dim (odds ratio [OR] = 1.47, 95% confidence interval [CI] = 1.12-1.92, p = 0.006), HLA DR on CD33+ HLA DR+ CD14- (OR = 1.32, 95% CI = 1.05-1.67, p = 0.018), and HLA DR on monocyte (OR = 1.23, 95% CI = 1.03-1.47, p = 0.021), were significantly associated with PCa. PCa had no statistically significant effect on immunophenotypes. These results remained robust in sensitivity analyses, supporting the validity of the causal associations. CONCLUSIONS: This study provides evidence of a potential causal relationship between certain immune cell signatures and PCa. We observed that immune cell signatures involving HLA DR expression on specific cell types are associated with an increased risk of PCa.

Safety and tolerability of AMG 330 in adults with relapsed/refractory AML: a phase 1a dose-escalation study.

AMG 330, a bispecific T-cell engager (BiTE®) that binds CD33 and CD3 on T cells facilitates T-cell-mediated cytotoxicity against CD33+ cells. This first-in-human, open-label, dose-escalation study evaluated the safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of AMG 330 in adults with relapsed/refractory acute myeloid leukemia (R/R AML). Amongst 77 patients treated with AMG 330 (0.5 µg/day-1.6 mg/day) on 14-day or 28-day cycles, maximum tolerated dose was not reached; median duration of treatment was 29 days. The most frequent treatment-related adverse events were cytokine release syndrome (CRS; 78%) and rash (30%); 10% of patients experienced grade 3/4 CRS. CRS was mitigated with stepwise dosing of AMG 330, prophylactic dexamethasone, and early treatment with tocilizumab. Among 60 evaluable patients, eight achieved complete remission or morphologic leukemia-free state; of the 52 non-responders, 37% had ≥50% reduction in AML bone marrow blasts. AMG 330 is a promising CD33-targeted therapeutic strategy for R/R AML.

Alzheimer's disease associated isoforms of human CD33 distinctively modulate microglial cell responses in 5XFAD mice.

Microglia play diverse pathophysiological roles in Alzheimer's disease (AD), with genetic susceptibility factors skewing microglial cell function to influence AD risk. CD33 is an immunomodulatory receptor associated with AD susceptibility through a single nucleotide polymorphism that modulates mRNA splicing, skewing protein expression from a long protein isoform (CD33M) to a short isoform (CD33m). Understanding how human CD33 isoforms differentially impact microglial cell function in vivo has been challenging due to functional divergence of CD33 between mice and humans. We address this challenge by studying transgenic mice expressing either of the human CD33 isoforms crossed with the 5XFAD mouse model of amyloidosis and find that human CD33 isoforms have opposing effects on the response of microglia to amyloid-ß (Aß) deposition. Mice expressing CD33M have increased Aß levels, more diffuse plaques, fewer disease-associated microglia, and more dystrophic neurites compared to 5XFAD control mice. Conversely, CD33m promotes plaque compaction and microglia-plaque contacts, and minimizes neuritic plaque pathology, highlighting an AD protective role for this isoform. Protective phenotypes driven by CD33m are detected at an earlier timepoint compared to the more aggressive pathology in CD33M mice that appears at a later timepoint, suggesting that CD33m has a more prominent impact on microglia cell function at earlier stages of disease progression. In addition to divergent roles in modulating phagocytosis, scRNAseq and proteomics analyses demonstrate that CD33m+ microglia upregulate nestin, an intermediate filament involved in cell migration, at plaque contact sites. Overall, our work provides new functional insights into how CD33, as a top genetic susceptibility factor for AD, modulates microglial cell function.

Developing a membrane-proximal CD33-targeting CAR T cell.

BACKGROUND: CD33 is a tractable target in acute myeloid leukemia (AML) for chimeric antigen receptor (CAR) T cell therapy, but clinical success is lacking. METHODS: We developed 3P14HLh28Z, a novel CD33-directed CD28/CD3Z-based CAR T cell derived from a high-affinity binder obtained through membrane-proximal fragment immunization in humanized mice. RESULTS: We found that immunization exclusively with the membrane-proximal domain of CD33 is necessary for identification of membrane-proximal binders in humanized mice. Compared with clinically validated lintuzumab-based CAR T cells targeting distal CD33 epitopes, 3P14HLh28Z showed enhanced in vitro functionality as well as superior tumor control and increased overall survival in both low antigen density and clinically relevant patient-derived xenograft models. Increased activation and enhanced polyfunctionality led to enhanced efficacy. CONCLUSIONS: Showing for the first time that a membrane-proximal CAR is superior to a membrane-distal one in the setting of CD33 targeting, our results demonstrate the rationale for targeting membrane-proximal epitopes with high-affinity binders. We also demonstrate the importance of optimizing CAR T cells for functionality in settings of both low antigen density and clinically relevant patient-derived models.

Targeting CD33+ Acute Myeloid Leukemia with GLK-33, a Lintuzumab-Auristatin Conjugate with a Wide Therapeutic Window.

CD33 (Siglec-3) is a cell surface receptor expressed in approximately 90% of acute myeloid leukemia (AML) blasts, making it an attractive target for therapy of AML. Although previous CD33-targeting antibody-drug conjugates (ADC) like gemtuzumab ozogamicin (GO, Mylotarg) have shown efficacy in AML treatment, they have suffered from toxicity and narrow therapeutic window. This study aimed to develop a novelADCwith improved tolerability and a wider therapeutic window. GLK-33 consists of the anti-CD33 antibody lintuzumab and eight mavg-MMAU auristatin linkerpayloads per antibody. The experimental methods included testing in cell cultures, patient-derived samples, mouse xenograft models, and rat toxicology studies. GLK-33 exhibited remarkable efficacy in reducing cell viability within CD33-positive leukemia cell lines and primary AML samples. Notably, GLK-33 demonstrated antitumor activity at single dose as low as 300 mg/kg in mice, while maintaining tolerability at single dose of 20 to 30 mg/kg in rats. In contrast with both GO and lintuzumab vedotin, GLK-33 exhibited a wide therapeutic window and activity against multidrug-resistant cells. The development of GLK-33 addresses the limitations of previous ADCs, offering a wider therapeutic window, improved tolerability, and activity against drug-resistant leukemia cells. These findings encourage further exploration of GLK-33 in AML through clinical trials.

The CD33xCD123xCD70 Multispecific CD3-Engaging DARPin MP0533 Induces Selective T Cell-Mediated Killing of AML Leukemic Stem Cells.

The prognosis of patients with acute myeloid leukemia (AML) is limited, especially for elderly or unfit patients not eligible for hematopoietic stem cell (HSC) transplantation. The disease is driven by leukemic stem cells (LSCs), which are characterized by clonal heterogeneity and resistance to conventional therapy. These cells are therefore believed to be a major cause of progression and relapse. We designed MP0533, a multispecific CD3-engaging designed ankyrin repeat protein (DARPin) that can simultaneously bind to three antigens on AML cells (CD33, CD123, and CD70), aiming to enable avidity-driven T cell-mediated killing of AML cells coexpressing at least two of the antigens. In vitro, MP0533 induced selective T cell-mediated killing of AML cell lines, as well as patient-derived AML blasts and LSCs, expressing two or more target antigens, while sparing healthy HSCs, blood, and endothelial cells. The higher selectivity also resulted in markedly lower levels of cytokine release in normal human blood compared to single antigen-targeting T-cell engagers. In xenograft AML mice models, MP0533 induced tumor-localized T-cell activation and cytokine release, leading to complete eradication of the tumors while having no systemic adverse effects. These studies show that the multispecific-targeting strategy used with MP0533 holds promise for improved selectivity toward LSCs and efficacy against clonal heterogeneity, potentially bringing a new therapeutic option to this group of patients with a high unmet need. MP0533 is currently being evaluated in a dose-escalation phase 1 study in patients with relapsed or refractory AML (NCT05673057).

Targeting inhibitory Siglec-3 to suppress IgE-mediated human basophil degranulation.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin-3 (Siglec-3 [CD33]) is a major Siglec expressed on human mast cells and basophils; engagement of CD33 leads to inhibition of cellular signaling via immunoreceptor tyrosine-based inhibitory motifs. OBJECTIVE: We sought to inhibit human basophil degranulation by simultaneously recruiting inhibitory CD33 to the IgE-FcεRI complex by using monoclonal anti-IgE directly conjugated to CD33 ligand (CD33L). METHODS: Direct and indirect basophil activation tests (BATs) were used to assess both antigen-specific (peanut) and antigen-nonspecific (polyclonal anti-IgE) stimulation. Whole blood from donors with allergy was used for direct BAT, whereas blood from donors with nonfood allergy was passively sensitized with plasma from donors with peanut allergy in the indirect BAT. Blood was incubated with anti-IgE-CD33L or controls for 1 hour or overnight and then stimulated with peanut, polyclonal anti-IgE, or N-formylmethionyl-leucyl-phenylalanine for 30 minutes. Degranulation was determined by measuring CD63 expression on the basophil surface by flow cytometry. RESULTS: Incubation for 1 hour with anti-IgE-CD33L significantly reduced basophil degranulation after both allergen-induced (peanut) and polyclonal anti-IgE stimulation, with further suppression after overnight incubation with anti-IgE-CD33L. As expected, anti-IgE-CD33L did not block basophil degranulation due to N-formylmethionyl-leucyl-phenylalanine, providing evidence that this inhibition is IgE pathway-specific. Finally, CD33L is necessary for this suppression, as monoclonal anti-IgE without CD33L was unable to reduce basophil degranulation. CONCLUSIONS: Pretreating human basophils with anti-IgE-CD33L significantly suppressed basophil degranulation through the IgE-FcεRI complex. The ability to abrogate IgE-mediated basophil degranulation is of particular interest, as treatment with anti-IgE-CD33L before antigen exposure could have broad implications for the treatment of food, drug, and environmental allergies.

Drug-regulated CD33-targeted CAR T cells control AML using clinically optimized rapamycin dosing.

Chimeric antigen receptor (CAR) designs that incorporate pharmacologic control are desirable; however, designs suitable for clinical translation are needed. We designed a fully human, rapamycin-regulated drug product for targeting CD33+ tumors called dimerizaing agent-regulated immunoreceptor complex (DARIC33). T cell products demonstrated target-specific and rapamycin-dependent cytokine release, transcriptional responses, cytotoxicity, and in vivo antileukemic activity in the presence of as little as 1 nM rapamycin. Rapamycin withdrawal paused DARIC33-stimulated T cell effector functions, which were restored following reexposure to rapamycin, demonstrating reversible effector function control. While rapamycin-regulated DARIC33 T cells were highly sensitive to target antigen, CD34+ stem cell colony-forming capacity was not impacted. We benchmarked DARIC33 potency relative to CD19 CAR T cells to estimate a T cell dose for clinical testing. In addition, we integrated in vitro and preclinical in vivo drug concentration thresholds for off-on state transitions, as well as murine and human rapamycin pharmacokinetics, to estimate a clinically applicable rapamycin dosing schedule. A phase I DARIC33 trial has been initiated (PLAT-08, NCT05105152), with initial evidence of rapamycin-regulated T cell activation and antitumor impact. Our findings provide evidence that the DARIC platform exhibits sensitive regulation and potency needed for clinical application to other important immunotherapy targets.

HuM195 and its single-chain variable fragment increase Aß phagocytosis in microglia via elimination of CD33 inhibitory signaling.

CD33 is a transmembrane receptor expressed on cells of myeloid lineage and regulates innate immunity. CD33 is a risk factor for Alzheimer's disease (AD) and targeting CD33 has been a promising strategy drug development. However, the mechanism of CD33's action is poorly understood. Here we investigate the mechanism of anti-CD33 antibody HuM195 (Lintuzumab) and its single-chain variable fragment (scFv) and examine their therapeutic potential. Treatment with HuM195 full-length antibody or its scFv increased phagocytosis of ß-amyloid 42 (Aß42) in human microglia and monocytes. This activation of phagocytosis was driven by internalization and degradation of CD33, thereby downregulating its inhibitory signal. HumM195 transiently induced CD33 phosphorylation and its signaling via receptor dimerization. However, this signaling decayed with degradation of CD33. scFv binding to CD33 leads to a degradation of CD33 without detection of the CD33 dimerization and signaling. Moreover, we found that treatments with either HuM195 or scFv promotes the secretion of IL33, a cytokine implicated in microglia reprogramming. Importantly, recombinant IL33 potentiates the uptake of Aß42 in monocytes. Collectively, our findings provide unanticipated mechanistic insight into the role of CD33 signaling in both monocytes and microglia and define a molecular basis for the development of CD33-based therapy of AD.

Multi-omics analysis of Siglec family genes in cutaneous melanoma.

Background: Melanoma is widely recognized as the most aggressive and fatal type of skin cancer; however, effective prognostic markers are lacking. The sialic acid-binding immunoglobulin-type lectin (Siglec) gene family plays an important role in the development of tumors and immune escape, but its prognostic role in melanoma remains unknown. Results: Siglec genes have a high mutation frequency, with up to 8% in SIGLEC7. High expression levels of Siglecs in tumor bulk suggests a better prognosis. Siglecs also show a high degree of synergistic expression. Immunohistochemistry was used to analyze the expression of SIGLEC9 in tumor tissue microarray. The expression of SIGLEC9 in tumor tissue without metastasis was higher than that in tumor tissue with metastasis. We used unsupervised clustering to create a high expression of Siglec (HES) cluster and a low expression of Siglec (LES) cluster. The HES cluster correlated with high overall survival and increased expression levels of Siglec genes. The HES cluster also showed significant immune cell infiltration and activation of immune signaling pathways. We used least absolute shrinkage and selection operator (LASSO) regression analysis to reduce the dimensionality of Siglec cluster-related genes and constructed a prognostic model composed of SRGN and GBP4, which can risk-stratify patients in both the training and test datasets. Conclusion: We conducted a multi-omics analysis of the Siglec family genes in melanoma and found that Siglecs play an important role in the occurrence and development of melanoma. Typing constructed using Siglecs can show risk stratification and derived prognostic models can predict a patient's risk score. In summary, Siglec family genes are potential targets for melanoma treatment as well as prognostic markers that can direct individualized treatments and improve overall survival.

CD33 BiTE® molecule-mediated immune synapse formation and subsequent T-cell activation is determined by the expression profile of activating and inhibitory checkpoint molecules on AML cells.

Bispecific T-cell engager (BiTE®) molecules recruit T cells to cancer cells through CD3ε binding, independently of T-cell receptor (TCR) specificity. Whereas physiological T-cell activation is dependent on signal 1 (TCR engagement) and signal 2 (co-stimulation), BiTE molecule-mediated T-cell activation occurs without additional co-stimulation. As co-stimulatory and inhibitory molecules modulate the strength and nature of T-cell responses, we studied the impact of the expression profile of those molecules on target cells for BiTE molecule-mediated T-cell activation in the context of acute myeloid leukemia (AML). Accordingly, we created a novel in vitro model system using murine Ba/F3 cells transduced with human CD33 ± CD86 ± PD-L1. T-cell fitness was assessed by T-cell function assays in co-cultures and immune synapse formation by applying a CD33 BiTE molecule (AMG 330). Using our cell-based model platform, we found that the expression of positive co-stimulatory molecules on target cells markedly enhanced BiTE molecule-mediated T-cell activation. The initiation and stability of the immune synapse between T cells and target cells were significantly increased through the expression of CD86 on target cells. By contrast, the co-inhibitory molecule PD-L1 impaired the stability of BiTE molecule-induced immune synapses and subsequent T-cell responses. We validated our findings in primary T-cell-AML co-cultures, demonstrating a PD-L1-mediated reduction in redirected T-cell activation. The addition of the immunomodulatory drug (IMiD) lenalidomide to co-cultures led to stabilization of immune synapses and improved subsequent T-cell responses. We conclude that target cells modulate CD33 BiTE molecule-dependent T-cell activation and hence, combinatorial strategies might contribute to enhanced efficacy.

[Expression level and clinical significance of CD44 and CD33 in 77 patients with benign lymphoadenosis of oral mucosa].

PURPOSE: To investigate the expression and clinical significance of CD44 and CD33 in benign lymphoadenosis of oral mucosa(BLOM). METHODS: From January 2017 to March 2020, seventy-seven BLOM wax blocks from the Department of Pathology of Qingdao Traditional Chinese Medicine Hospital were selected as the experimental group, and 63 cases of normal oral mucosal tissue wax blocks during the same period were selected as the control group. Immunohistochemical method was used to detect the positive expression of CD44 and CD33 in the two groups.Spearman correlation analysis was used to analyze the correlation between the positive expression of CD33 and the positive expression of CD44 in the diseased tissues of BLOM patients.The general information about patients were collected.The relationship between the expression of CD33 and CD44 in the diseased tissues of BLOM patients and the clinicopathological characteristics of BLOM patients were analyzed. SPSS 21.0 software package was used for statistical analysis of the data. RESULTS: The positive expression rates of CD33 in the control group and the experimental group were 95.24% and 63.64%, respectively, and the difference was statistically significant(P＜0.05). The positive expression rates of CD44 in the control group and the experimental group were 93.65% and 67.53%, respectively, and the difference was statistically significant(P＜0.05). The results of Spearman correlation analysis showed that the positive expression of CD33 in the diseased tissues of BLOM patients was positively correlated with the positive expression of CD44 (r=0.834, P=0.002). The expression of CD33 and CD44 in the diseased tissues of patients with BLOM were related to clinical type, degree of inflammation, presence or absence of lymphoid follicles, and lymphocyte infiltration(P＜0.05), but not related to age, gender, course of disease, location, and epithelial surface keratinization(P＞0.05). CONCLUSIONS: The positive expression rate of CD33 and CD44 in the BLOM tissues decreased, which was closely related to the clinical type, degree of inflammation, presence or absence of lymphoid follicles, and lymphocyte infiltration.

Alzheimer's Disease-Associated Alternative Splicing of CD33 Is Regulated by the HNRNPA Family Proteins.

Genetic variations of CD33 have been implicated as a susceptibility factor of Alzheimer's disease (AD). A polymorphism on exon 2 of CD33, rs12459419, affects the alternative splicing of this exon. The minor allele is associated with a reduced risk of AD and promotes the skipping of exon 2 to produce a shorter CD33 isoform lacking the extracellular ligand-binding domain, leading to decreased suppressive signaling on microglial activity. Therefore, factors that regulate the splicing of exon 2 may alter the disease-associated properties of CD33. Herein, we sought to identify the regulatory proteins of CD33 splicing. Using a panel of RNA-binding proteins and a human CD33 minigene, we found that exon 2 skipping of CD33 was promoted by HNRNPA1. Although the knockdown of HNRNPA1 alone did not reduce exon 2 skipping, simultaneous knockdown of HNRNPA1 together with that of HNRNPA2B1 and HNRNPA3 promoted exon 2 inclusion, suggesting functional redundancy among HNRNPA proteins. Similar redundant regulation by HNRNPA proteins was observed in endogenous CD33 of THP-1 and human microglia-like cells. Although mouse Cd33 showed a unique splicing pattern of exon 2, we confirmed that HNRNPA1 promoted the skipping of this exon. Collectively, our results revealed novel regulatory relationships between CD33 and HNRNPA proteins.

AI reveals insights into link between CD33 and cognitive impairment in Alzheimer's Disease.

Modeling biological mechanisms is a key for disease understanding and drug-target identification. However, formulating quantitative models in the field of Alzheimer's Disease is challenged by a lack of detailed knowledge of relevant biochemical processes. Additionally, fitting differential equation systems usually requires time resolved data and the possibility to perform intervention experiments, which is difficult in neurological disorders. This work addresses these challenges by employing the recently published Variational Autoencoder Modular Bayesian Networks (VAMBN) method, which we here trained on combined clinical and patient level gene expression data while incorporating a disease focused knowledge graph. Our approach, called iVAMBN, resulted in a quantitative model that allowed us to simulate a down-expression of the putative drug target CD33, including potential impact on cognitive impairment and brain pathophysiology. Experimental validation demonstrated a high overlap of molecular mechanism predicted to be altered by CD33 perturbation with cell line data. Altogether, our modeling approach may help to select promising drug targets.

A novel therapeutic bispecific format based on synthetic orthogonal heterodimers enables T cell activity against Acute myeloid leukemia.

Many therapeutic bispecific T-cell engagers (BiTEs) are in clinical trials. A modular and efficient process to create BiTEs would accelerate their development and clinical applicability. In this study, we present the design, production, and functional activity of a novel bispecific format utilizing synthetic orthogonal heterodimers to form a multichain modular design. Further addition of an immunoglobulin hinge region allowed a stable covalent linkage between the heterodimers. As proof-of-concept, we utilized CD33 and CD3 binding scFvs to engage leukemia cells and T-cells respectively. We provide evidence that this novel bispecific T-cell engager (termed IgGlue-BiTE) could bind both CD3+ and CD33+ cells and facilitates robust T-cell mediated cytotoxicity on AML cells in vitro. In a mouse model of minimal residual disease, we showed that the novel IgGlue-BiTE greatly extended survival, and mice of this treatment group were free of leukemia in the bone marrow. These findings suggest that the IgGlue-BiTE allows for robust simultaneous engagement with both antigens of interest in a manner conducive to T cell cytotoxicity against AML. These results suggest a compelling modular system for bispecific antibodies, as the CD3- and CD33-binding domains can be readily swapped with domains binding to other cancer- or immune cell-specific antigens.

Acute myeloid leukemia fusion genes can be found in CD33-negative cells.

INTRODUCTION: Targeted therapies and immunotherapies are emerging strategies for the treatment of leukemia. CD33 is a common and important therapeutic target for cellular immunotherapy or antibody immunotherapy. Drugs on targeting CD33 are also emerging. However, acute myeloid leukemia (AML) relapse still occurs after treatment with targeted CD33, for which the mechanism is unknown. METHODS: We used fluorescence in situ hybridization and real-time polymerase chain reaction to detect the expression of fusion genes in different populations of cells from AML patients. RESULT: Fusion gene can be express in CD33 negative cell proportions in newly diagnosed and relapsed AML patients. CONCLUSION: There are fusion genes in CD33-negative cells that are might not be cleared by CD33 targeting therapy. And this might be the source of relapse.

[Preparation of CD33 targeted bispecific- and trispecific-T cell engagers and their cytotoxicity on leukemia cells].

Objective: To investigate the effect of CD33-targeted bi-specific and tri-specific T-cell engagers on T-cell proliferation and explore their cytotoxicity on leukemia cells. Methods: The CD33-targeted bi-specific T-cell engager (CD33-BiTE) and tri-specific T-cell engager (CD33-TriTE) expression vectors were successfully constructed and expressed through a eukaryotic cell expression system. CD33-BiTE and CD33-TriTE were purified by affinity chromatography. The effects of CD33-BiTE and CD33-TriTE on T cells were analyzed through in vitro experiments. Results: ① CD33-BiTE and CD33-TriTE were successfully constructed and purified and could compete with flow cytometry antibodies for binding to the target cells. ② After 12 days of co-culture with CD33-BiTE and CD33-TriTE, the number of human T cells were expanded to 33.89±19.46 and 81.56±23.62 folds, respectively. CD33-TriTE induced a stronger proliferation of T cells than CD33-BiTE (P<0.05) . ③ Both CD33-BiTE and CD33-TriTE induced specific dose-dependent cytotoxicity on CD33(+) leukemia cells. ④ Compared to CD33-TriTE, leukemia cells were prone to express PD-L1 when co-cultured with T cells and CD33-BiTE. CD33-TriTE induced powerful cytotoxicity on leukemia cells with high PD-L1 expression. Conclusion: CD33-BiTE and CD33-TriTE expression vectors were constructed, and fusion proteins were expressed in eukaryotic cells. Our results support the proliferative and activating effects of BiTE and TriTE on T cells. Compared to that of CD33-BiTE, CD33-TriTE induced a stronger proliferative effect on T cells and a more powerful cytotoxicity on leukemia cells with high PD-L1 expression.

A ten-gene DNA-damage response pathway gene expression signature predicts gemtuzumab ozogamicin response in pediatric AML patients treated on COGAAML0531 and AAML03P1 trials.

Gemtuzumab ozogamicin (GO) is an anti-CD33 monoclonal antibody linked to calicheamicin, a DNA damaging agent, and is a well-established therapeutic for treating acute myeloid leukemia (AML). In this study, we used LASSO regression modeling to develop a 10-gene DNA damage response gene expression score (CalDDR-GEx10) predictive of clinical outcome in pediatric AML patients treated with treatment regimen containing GO from the AAML03P1 and AAML0531 trials (ADE + GO arm, N = 301). When treated with ADE + GO, patients with a high CalDDR-GEx10 score had lower complete remission rates (62.8% vs. 85.5%, P = 1.7 7 * 10-5) and worse event-free survival (28.7% vs. 56.5% P = 4.08 * 10-8) compared to those with a low CalDDR-GEx10 score. However, the CalDDR-GEx10 score was not associated with clinical outcome in patients treated with standard chemotherapy alone (ADE, N = 242), implying the specificity of the CalDDR-GEx10 score to calicheamicin-induced DNA damage response. In multivariable models adjusted for risk group, FLT3-status, white blood cell count, and age, the CalDDR-GEx10 score remained a significant predictor of outcome in patients treated with ADE + GO. Our findings present a potential tool that can specifically assess response to calicheamicin-induced DNA damage preemptively via assessing diagnostic leukemic cell gene expression and guide clinical decisions related to treatment using GO.

Human brain sialoglycan ligand for CD33, a microglial inhibitory Siglec implicated in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by accumulation of misfolded proteins. Genetic studies implicate microglia, brain-resident phagocytic immune cells, in AD pathogenesis. As positive effectors, microglia clear toxic proteins, whereas as negative effectors, they release proinflammatory mediators. An imbalance of these functions contributes to AD progression. Polymorphisms of human CD33, an inhibitory microglial receptor, are linked to AD susceptibility; higher CD33 expression correlates with increased AD risk. CD33, also called Siglec-3, is a member of the sialic acid-binding immunoglobulin-type lectin (Siglec) family of immune regulatory receptors. Siglec-mediated inhibition is initiated by binding to complementary sialoglycan ligands in the tissue environment. Here, we identify a single sialoglycoprotein in human cerebral cortex that binds CD33 as well as Siglec-8, the most abundant Siglec on human microglia. The ligand, which we term receptor protein tyrosine phosphatase zeta (RPTPζ)S3L, is composed of sialylated keratan sulfate chains carried on a minor isoform/glycoform of RPTPζ (phosphacan) and is found in the extracellular milieu of the human brain parenchyma. Brains from human AD donors had twofold higher levels of RPTPζS3L than age-matched control donors, raising the possibility that RPTPζS3L overexpression limits misfolded protein clearance contributing to AD pathology. Mice express the same structure, a sialylated keratan sulfate RPTPζ isoform, that binds mouse Siglec-F and crossreacts with human CD33 and Siglec-8. Brains from mice engineered to lack RPTPζ, the sialyltransferase St3gal4, or the keratan sulfate sulfotransferase Chst1 lacked Siglec binding, establishing the ligand structure. The unique CD33 and Siglec-8 ligand, RPTPζS3L, may contribute to AD progression.