Cell Surface CD44 Research Articles

Alternative splicing encodes functional intracellular CD59 isoforms that mediate insulin secretion and are down-regulated in diabetic islets

Human pancreatic islets highly express CD59, which is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein and is required for insulin secretion. How cell-surface CD59 could interact with intracellular exocytotic machinery has so far not been described. We now demonstrate the existence of CD59 splice variants in human pancreatic islets, which have unique C-terminal domains replacing the GPI-anchoring signal sequence. These isoforms are found in the cytosol of β-cells, interact with SNARE proteins VAMP2 and SNAP25, colocalize with insulin granules, and rescue insulin secretion in CD59-knockout (KO) cells. We therefore named these isoforms IRIS-1 and IRIS-2 (Isoforms Rescuing Insulin Secretion 1 and 2). Antibodies raised against each isoform revealed that expression of both IRIS-1 and IRIS-2 is significantly lower in islets isolated from human type 2 diabetes (T2D) patients, as compared to healthy controls. Further, glucotoxicity induced in primary, healthy human islets led to a significant decrease of IRIS-1 expression, suggesting that hyperglycemia (raised glucose levels) and subsequent decreased IRIS-1 expression may contribute to relative insulin deficiency in T2D patients. Similar isoforms were also identified in the mouse CD59B gene, and targeted CRISPR/Cas9-mediated knockout showed that these intracellular isoforms, but not canonical CD59B, are involved in insulin secretion from mouse β-cells. Mouse IRIS-2 is also down-regulated in diabetic db/db mouse islets. These findings establish the endogenous existence of previously undescribed non–GPI-anchored intracellular isoforms of human CD59 and mouse CD59B, which are required for normal insulin secretion.

Enhancement of the dissolution and in-vitro activity of a new antineoplastic agent

The cell-surface molecule CD44 plays a major role in the regulation of cancer stem cells. The CD44 inhibitor compound N′-(1-dimethylaminomethyl-2-oxoindolin-3-ylidene)-2-(benzyloxy)benzohydrazide (OYB), anticancer agent is practically insoluble in water. Hence, the solid dispersion (SD) technique was used for enhancing the dissolution of OYB. The SD of OYB was achieved using OYB:poloxamer 188 (1:7) via the fusion method. The anticancer activities of the free-OYB solution and the SD formulation (OYB-SD) were investigated in-vitro. The dissolution rate of OYB-SD (1:7) increased by 2-fold compared with the untreated drug (51.52–100% at pH 1.2 and 8.25–19.15% at pH 7 buffer). In addition, OYB-SD afforded 3-folds cytotoxic effect, against LoVo cells, compared to the untreated compound (IC50 4.72 ± 0.57 and 13.97 ± 0.90 µg/ml, respectively) and against HepG2 (∼3-fold) (4.98 ± 0.368 and 13.85 ± 1.82 µg/ml, respectively) and MCF-7 (1.4-fold) cells (15.20 ± 0.20 and 21.12 ± 0.51 µg/ml, respectively), and enhanced the apoptotic potential in LoVo cells compared with free-OYB. The improved cytotoxic activity of the drug might be attributable to the enhanced dissolution of OYB.

Development of CD44E/s dual-targeting DNA aptamer as nanoprobe to deliver treatment in hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) is the predominant subtype of liver cancer with an extraordinary high mortality. Resistance to systemic therapy is a major cause of inferior clinical outcome in most patients with HCC. CD44 is a transmembrane cell-surface glycoprotein that is characterized by its variants displaying differential overexpression in human cancers. Aptamers, also known as chemical antibodies, can target cell-surface molecules with high affinity and specificity via structural recognition. Aptamer-mediated drug delivery hence is of high potentials in guiding therapy to improve efficacy.Methods: Variants CD44E and CD44s were studied for HCC relevance by investigating their expressions in primary HCC tumors, adjacent cirrhotic/fibrotic livers and normal livers using junction specific primers in qPCR assay. CD44E/s dual-targeted aptamers were uncovered by integrating loss-gain cell-SELEX and next generation sequencing. Selected aptamers were characterized for binding affinity and specificity, biostability, in vivo and in vitro cytotoxicity, in vivo homing and biodistribution, and ability to deliver 5-FU into targeted cells in vitro.Results: Both CD44E and CD44s isoforms showed significant upregulations in HCC tumors with CD44E/s activities promoting cell proliferation and migration. Loss-gain cell-SELEX uncover a CD44E/s dual-targeting aptamer, termed CD44-Apt1. Strong binding of CD44-Apt1 to cell-surface CD44 positive cells but not CD44-negative cells was demonstrated by flow-cytometry. CD44-Apt1 displayed strong affinity to CD44E and CD44s with KD as low as 1 nM but not the hyaluronic acid binding domain of CD44. Confocal imaging of CD44-positive cells stained with fluorescent-labeled CD44-Apt1 showed profound cytoplasmic localization, suggesting efficient cell-penetrating ability. Meanwhile, no apparent staining was observed in CD44-negative cells. CD44-Apt1 when conjugated with inhibitor 5-FU showed efficient guidance of 5-FU into HCC cells that significantly enhanced drug toxicity by more than thousands-fold. Both in vitro cell treatment and in vivo animal biodistribution indicated that CD44-Apt1 is non-toxic. In HCC xenograft model, CD44-Apt1 efficiently homed to tumor xenografts in a CD44 expression-dependent manner.Conclusion: Novel discovery of aptamer CD44-Apt1 that can bind both CD44E and CD44s illustrates high potential as nanoprobe to deliver anti-cancer therapeutics.

Genetic Control of Splicing at SIRPG Modulates Risk of Type 1 Diabetes.

Signal regulatory protein SIRPγ (CD172G) is expressed on the surface of lymphocytes, where it acts by engaging its ligand, CD47. SIRPG, which encodes SIRPγ, contains a nonsynonymous coding variant, rs6043409, which is significantly associated with risk for type 1 diabetes. SIRPG produces multiple transcript isoforms via alternative splicing, all encoding potentially functional proteins. We show that rs6043409 alters a predicted exonic splicing enhancer, resulting in significant shifts in the distribution of SIRPG transcript isoforms. All of these transcript isoforms produced protein upon transient expression in vitro. However, CRISPR/Cas9 targeting of one of the alternatively spliced exons in SIRPG eliminated all SIRPγ expression in Jurkat T cells. These targeted cells formed fewer cell-cell conjugates with each other than with wild-type Jurkat cells, expressed reduced levels of genes associated with CD47 signaling, and had significantly increased levels of cell-surface CD47. In primary CD4+ and CD8+ T cells, cell-surface SIRPγ levels in response to anti-CD3 stimulation varied quantitatively by rs6043409 genotype. Our results suggest that SIRPG is the most likely causative gene for type 1 diabetes risk in the 20p13 region and highlight the role of alternative splicing in lymphocytes in mediating the genetic risk for autoimmunity.

Suppression of Extracellular Vesicle CD47 Induces Systemic Anti-DLBCL Immunity

Purpose:Tumor cells evade the immune surveillance by up-regulating surface expression of CD47, which interacts with SIRPa on macrophages to elicit the immune checkpoint response. Anti-CD47 antibody (IBI188) has shown promise in treating tumors, including Diffuse large B cell lymphoma (DLBCL). The objective response rate among DLBCL was 73% (ClinicalTrials.gov number, NCT02953509.). The basis of differential therapeutic success between patients remains unknown. Extracellular vesicles (EVs) carry bioactive molecules that influence the immune system. CD47 has be found on surface of EVs. The purpose of this study is to explore whether EV CD47 contributes to immunosuppression and is associated with anti-CD47 response in DLBCL.Methods: 1.Kaplan-Meier curves and Cox regression models were used to analyze PFS and OS. The area under the ROC curve (AUC) was used to assess the predicted validity of the National Comprehensive Cancer Network-International Prognostic Index (NCCN-IPI) model and the NCCN-IPI+ CD47 model.2.Immunofluorescence experiment was used to detect the infiltration of M1 and M2 subtype macrophages in DLBCL tumor tissue. M1 and M2 macrophages were generated from peripheral blood mononuclear cells obtained from healthy donors. Immunoelectron microscopy, flow cytometry and western blot were performed to detect CD47 on the surface of the EVs derived from DLBCL cells. The activity of macrophage phagocytosis of DLBCL cells was detected by confocal-based phagocytosis assay and flow cytometry-based phagocytosis assay.3.In DLBCL xenografts, the IBI188 antibody was a macrophage immune checkpoint inhibitor blocking CD47 that induces tumor-cell phagocytosis.4.Peripheral blood mononuclear cells from DLBCL patients and healthy donors was collected for EVs purification and subsequent detection of human CD47 proteins by ELISA.Results 1.The elevated expression of CD47 in DLBCL is significantly correlated with poor PFS and OS in a univariate analysis, and is statistically significant after adjusting for the NCCN-IPI in the univariate and multivariate analysis (Figure 1A, B). AUC analysis with cross-validation showed that the combination of the CD47 signature and NCCN-IPI had a better prediction of PFS and OS than without the CD47 signature (Figure 1C). Thus, CD47 in DLBCL can be used as a biomarker of prognosis and developed the prognostic stratification of DLBCL patients.2.EV CD47 derived from DLBCL cells has the same membrane topology as the cell surface CD47, with its extracellular domain exposed on the surface of the EVs (Figure 1D-G). Moreover, EV CD47 could function similarly to cell-surface CD47 in the suppression of macrophage phagocytosis of DLBCL cells (Figure 1H, I). DLBCL cells that had higher levels of cellular CD47 protein packaged greater amounts of CD47 into EVs, and these EVs displayed an increased binding to SIRPa of macrophages, thus inhibiting macrophage-mediated phagocytosis of DLBCL cells (Figure 1J-O).3.Antibodies against human CD47 specifically identified human CD47 on the circulating exosomes from mice bearing DLBCL xenografts but not the control mice. The level of circulating EV CD47 positively correlated with tumor size. Injection of EVs derived from DLBCL cells promoted the growth of tumors, whereas pre-treatment of the EVs with anti-CD47 antibodies (IBI188), but not IgG isotype or CD63-blocking antibodies, inhibited the effect (Figure 1P, Q).4.The presence of CD47 in the EVs isolated from the plasma of DLBCL patients, and the level of CD47 on the circulating EVs was significantly higher in DLBCL patients than that in healthy donors (Figure 1R, S). The receiver ROC curve shows that the level of circulating EV CD47 could distinguish DLBCL patients from healthy donors.ConclusionOur studies suggest that the EV CD47-SIRPa interaction may represent a critical mechanism by which DLBCL cells escape immune-mediated clearance. Our study also raises the possibility that disrupting the interaction between the EV CD47 and macrophage SIRPa is a mechanism in the CD47-SIRPa blockade-based therapies. Moreover, high levels of circulating EV CD47 would follow and correlate positively with the phagocytic activity of macrophages, and reflect the presence of a successful anti-tumor immunity elicited by the anti-CD47 therapy. Together, these findings show that EV CD47 represents an unexplored therapeutic target, which could overcome resistance to current CD47 antibody approaches. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Impact of CD19 Expression Combined with Measurable Residual Disease on Post-Remission Treatment and Outcome in Adult t(8;21) Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) with t(8;21) has considerable clinical heterogeneity. Only 50% of these patients receiving multiple cycles of high-dose cytarabine as post-remission treatments could achieve long term survival, and relapse occures in up to 40% of them. Therefore, further risk stratification is needed to guide appropriate post-remission treatment for t(8;21) AML patitents in first complete remission(CR1). Measurable residual disease (MRD) monitored by RUNX1-RUNX1T1 transcript levels after treatment is established as a powerful marker to predict relapse and guide treatment. Recent studies revealed cell-surface antigen CD19 negativity (CD19-) was a risk factor for relapse in t(8;21) AML. However, reports about the impact of CD19 expression combined with RUNX1-RUNX1T1 MRD on post-remission treatment remains absent to date.Methods: A total of 155 consecutive patients diagnosed with t(8;21) AML were enrolled in this study, including 69 patients who received chemotherapy (CMT) and 86 who received allogeneic stem cell transplantation (allo-SCT) as post remission treatment in CR1. MRD+ was defined as a < 3 log reduction of RUNX1/RUNX1T1 level after the second consolidation therapy.Results:With a median follow-up of 40 months (range, 4-133 months), the 3-year OS, LFS and CIR rates were 75.57% ,70.46% and 27.50% for the entire population, respectively. The 3-year CIR was dramatically decreased in allo-SCT than in CMT group (13.47% vs 49.19%, p<0.001). Allo-SCT demonstrated a significantly superior 3-year OS (85.78% vs 58.89%, p< 0.001) and LFS (85.64% vs 45.84%, p< 0.001) over CMT, respectively. The 3-year cumulative incidence of TRM was 9.27% and 7.04% in the CMT and allo-HSCT groups, respectively (p=0.890). KIT exon 17 mutation was a risk factor for OS in multivariate analysis, CD19 negativity was a risk factor for RFS in univariate analysis. Allo-SCT was a beneficial factor for LFS and OS in multivariate analysis when taking CMT as a reference. For CD19- patients (n=59), allo-HSCT demonstrated significantly lower CIR (17.20% vs 51.03%, p=0.006) , better OS (95.24% vs 51.55%, p < 0.001) and LFS (82.8% vs 39.71%, p = 0.001) than CMT. On the other hand, CMT had a comparable CIR (26.70% vs 11.40, p=0.064 ), OS (73.19% vs 86.01%, p=0.153) and LFS (71.58% vs 87.76%, p=0.067) with allo-SCT for CD19+ patients (n=80). Notably, when combined with RUNX1-RUNX1T1 transcripts after the second consolidation, allo-SCT preserved a significantly lower CIR (6.67% vs 66.67%, p=0.002), improved OS (100% vs 33.33%, p< 0.001) and LFS (93.33% vs 33.33%, p=0.001) than CMT in CD19-MRD+ group (n=22), respectively. In contranst, CMT revealed similar OS (80.0% vs 87.5%, p=0.677 ), LFS (60% vs 62.5%, p=0.663) and CIR (40.00% vs 37.50%, p=0.795 ) with allo-SCT in CD19-MRD- patients (n=13), respectively. For CD19+ patients, addition of MRD status had no impact on the outcome of allo-SCT and CMT. CMT preserved comparable OS (75% vs 84.66%, p =0.516), LFS (85.71% vs 96.30%, p =0.357 ) and CIR (12.50% vs 3.70%, p =0.367) with allo-SCT for CD19+MRD+ patients (n=36), and comparable OS (100% vs 92.31%, p =0.459), LFS (83.33% vs 85.29%, p =0.940 ) and CIR (16.67% vs 13.54%, p =0.998) with allo-SCT in CD19+MRD- patients (n=33).Conclusion: Combination of CD19 expression and RUNX1-RUNX1T1 MRD improved risk stratification and treatment guidance for t(8;21) AML in CR1. Allo-SCT might be recommended for the CD19-MRD+ patients, while CMT might be a reasnonable choice for CD19-MRD+ and CD19+ patients. Multicenter prospective studies are warranted to confirm the current results.Key words: t(8;21) AML, CD19, measurable residual disease, post-remission treatment, allo-SCT, chematherapy DisclosuresNo relevant conflicts of interest to declare.

Characterization of Aleta-001, a CAR T Engager Designed to Optimize Responses and Prevent Relapses from CAR-19 Therapy

Adoptive T cell therapies directed to the B cell malignancy antigen CD19 (CAR19 T cells) have transformed the care of otherwise refractory, last-line leukemia and lymphoma patients. The overall response rates achieved are very high, routinely above 60%, and a substantial number of responding patients have durable responses that can last many years. However, 50% of responders relapse within 6 months, and outcomes for these relapsed patients are poor. Relapses occur most often in patients whose initial CAR19 T cell expansion is suboptimal, and whose tumor cells reduce or lose expression of the target antigen, CD19.We have created a CAR T Engager protein that is designed to improve responses to anti-CD19 CAR T treatment and is further designed to prevent relapses. This modular protein contains an anti-CD20 llama VHH linked to an optimized CD19 protein and further linked to an anti-albumin llama VHH. Thus, this CAR T Engager, called Aleta-001, binds to CD20 on B cell tumor cells, displays CD19 on the tumor cell surface thereby activating anti-CD19 CAR T cells, and binds to albumin, providing for a long half-life upon injection. Aleta-001 increases CD19 antigen density and/or replaces lost CD19 expression by coating cell surface CD20 with CD19.Here we present studies that establish the efficacious dose to support the upcoming Phase 1/2 clinical trial to be run in collaboration with Cancer Research UK. Extensive in vitro modeling established that the Aleta-001 CAR T Engager specifically bound to CD20-positive/CD19 negative lymphoma cells, JeKo-1-CD19KO, that represent the phenotype of a lymphoma relapsing after losing CD19 expression. In the presence of CAR19 T cells, the Engager protein mediated cytotoxicity against the JeKo-1-CD19KO cells at sub-nM concentrations. The JeKo-1-CD19KO cells induced a rapid and lethal lymphoma when implanted into NSG mice. Administration of the Aleta-001 CAR T Engager and CAR19 T cells eliminated the JeKo-1-CD19KO lymphoma at administered protein concentrations as low as 0.5mg/kg.These results support the clinical development of the Aleta-001 Engager protein. The protein is designed to be administered to patients who have received CAR19 T cell therapy and who fail to achieve a complete response at the time of their first clinical evaluation, or who relapse from a complete response thereafter. Patients who are enrolled will be dosed with the Aleta-001 Engager protein every two weeks. Aleta-001 has entered GMP production and IND enabling studies and will then enter Phase 1 dose escalation clinical studies in the UK. [Display omitted] DisclosuresRennert: Aleta Biotherapeutics Inc.: Current Employment, Current holder of stock options in a privately-held company. Su: Aleta Biotherapeutics Inc.: Current Employment, Current holder of stock options in a privately-held company. Wu: Aleta Biotherapeutics Inc.: Current Employment, Current holder of stock options in a privately-held company. Lobb: Aleta Biotherapeutics Inc.: Consultancy, Current holder of stock options in a privately-held company. Ambrose: Aleta Biotherapeutics Inc.: Current Employment, Current holder of stock options in a privately-held company.

The Role of CD38 in Multiple Myeloma Cell Biology

Introduction. Anti-CD38 monoclonal antibodies, such as daratumumab and isatuximab, which exerts therapeutic effect against multiple myeloma (MM) cells through direct cell damage, antibody dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), has shown its high efficacy in clinical practice. However, the role of CD38 in MM cell biology is still unclear. CD38 is known as a major nicotinamide adenine dinucleotide (NAD +) glycohydrase (NADase) in mammalian tissues, which regulate cellular levels of NAD +. In the present study, we compared metabolic and proteomic profile between CD38 positive and negative MM cell lines to analyze the biological significance of CD38 in MM cells. Additionally, we performed CD38 enzyme activity inhibition on MM cells using 78c, a NADase enzyme inhibitor of CD38, in order to study the role of CD38 NADase activity in MM cell survival.Materials and methods. MM cell lines harboring CD38 positive and negative fractions (KMS-12BM, KMS-11) were sorted according to CD38 expression. Intracellular NAD+ and NADH concentrations between CD38 positive and negative cells were analyzed using NAD/NADH Assay kit. CD38 positive and negative MM cells were subjected to metabolome and proteome analysis using Shimadzu TQ8050 GC-MS/MS and TripleTOF 5600 respectively. Metabolites and proteins significantly enriched in CD38 negative MM cells were analyzed using MetaboAnalyst and Metascape. Cell cycle status between CD38 positive and negative cells were determined by flow cytometry after staining by BRDU and 7AAD. CD38 positive MM cell lines (NCI-H929 and KMS-12PE) and patient derived bone marrow cells were treated with 78c, a CD38 NADase inhibitor, in vitro. MM cell viability were determined by flow cytometry post Annexin V and PI staining. Differences in metabolites between 78c treated and control MM cell lines were also analyzed using Shimadzu TQ8050 GC-MS/MS.Results. Higher NAD+ and NAD+/NADH ratio was observed in CD38 negative fraction of MM cell lines compared to their CD38 positive counterparts, demonstrating that cell surface CD38 expression influences intracellular NAD+ concentration. Both metabolome and proteome analysis revealed that CD38 negative cells tend to have higher glycolytic activity compared to CD38 positive cells. Significant suppression of cell cycle, accompanying G0/G1 phase arrest, was observed in CD38 negative MM cells, indicating that metabolic shift in CD38 negative MM cells may lead to change in cell proliferation. Marked increase of NAD+/NADH ratio was observed in 78c treated MM cell lines compared to control, proving that CD38 NADase inhibiton indeed affects intracellular NAD+ concentration in MM cells. 78c was capable of inducing cell death in MM cell lines and patient derived MM cells, accompanying cell cycle arrest. Metabolites significantly upregulated in 78c treated MM cells compared to control were associated with glycolysis, demonstrating that CD38 NADase activity has a significant effect on MM cell metabolism.Conclusions. CD38 is the major NADase in mammalian tissues, involved in catabolism of NAD +. Although CD38 is highly expressed in normal plasma cells and MM cells, its role in MM cell biology has not been studied in detail. By comparing CD38 positive and negative cells and using CD38 NADase inhibitor, we showed for the first time that CD38 on MM cells decrease intracellular NAD+, reduces intracellular glycolysis and as a result, has an influence on cell cycle. The present study sheds light on the significance of CD38 enzyme activity in MM cell biology and may also contribute to understanding the mechanism of resistance to CD38 targeted therapy. DisclosuresKawano: Janssen Pharmaceuticals: Honoraria; Ono pharmaceutical: Honoraria; Sanofi: Honoraria; Bristol Myers Squibb: Honoraria; Takeda Pharmaceuticals: Honoraria.

375 Development of thrombospondin-1 as a clinical pharmacodynamic biomarker for VT1021, a first-in-class therapeutic agent that reprograms the tumor microenvironment

BackgroundVT1021 is a first-in-class therapeutic agent in Phase I clinical studies in solid tumors. In vivo preclinical studies demonstrated that VT1021 inhibited tumor growth via stimulation of p53 and Thrombospondin-1 (Tsp-1) in MDSCs. Moreover, induction of Tsp-1 reprogrammed the tumor microenvironment and induced apoptosis in tumor cells via its cell surface receptors CD36 and CD47. Here we report on the utility of Tsp-1 as a pharmacodynamic biomarker and its correlation with clinical response.MethodsTsp-1 protein levels in PBMCs, platelets and plasma were assessed by ELISA following SepMate-based fractionation of subject blood samples. Tsp-1 mRNA levels in peripheral blood cells were analyzed by quantitative RT-PCR following extraction of total RNA from subject whole blood samples via PAXgene Blood RNA kit. Tsp-1 levels in subject biopsy samples were analyzed by immunohistochemistry as described previously.ResultsUp-regulation of Tsp-1 protein was observed in PBMCs, platelets and plasma in all evaluable subjects following treatment with VT1021 across multiple indications in a phase 1 clinical study, indicating that Tsp-1 induction is a pharmacodynamic biomarker for VT1021 (figure 1). Induction of Tsp-1 by VT1021 was also shown at the transcriptional level via RT-PCR measurement of whole blood samples. Strikingly, maximum PBMC Tsp-1 levels induced by VT1021 were higher in subjects with glioblastoma (GBM) that had objective responses (complete or partial response) compared to subjects with stable disease (SD) or progressive disease (PD). Of note, pre-dose levels of Tsp-1 were predictive of response, as subjects with objective responses had higher basal levels of Tsp-1 than those that had stable or progressive disease. For subjects with pancreatic cancer, Tsp-1 induction was higher in SD subjects compared to PD subjects, suggesting that Tsp-1 induction in PBMCs can be a potential prognostic biomarker. In tumor biopsy samples from subjects with pancreatic cancer, increased colocalization of Tsp-1 and CD11b was observed in on-study samples, supporting a role of Tsp-1 in reprogramming the tumor microenvironment (figure 2).Abstract 375 Figure 1Up-regulation of Tsp-1 protein levels have been observed in PBMCs(A), platelets (B) and plasma (C) in all evaluable subjects post-dosing with VT1021 across multiple inductions in a phase 1 clinical study.Abstract 375 Figure 2Increased colocalization of Tsp-1 and CD11b was observed in tumor microenvironment in tumor biopsy samples from a subject with pancreatic cancer post-dosing with VT1021ConclusionsBased on both protein and mRNA levels, Tsp-1 induction has the potential to be a useful prognostic pharmacodynamic biomarker for VT1021 in various tumor types. In subjects with GBM, both basal and induced Tsp-1 levels in PBMCs are potential predictive and prognostic biomarkers, respectively. For subjects with pancreatic cancer, Tsp-1 protein induction in PBMCs is a potential prognostic biomarker. The predictive/prognostic utility coupled with the ability to measure levels in peripheral blood makes Tsp-1 a powerful biomarker to assess and predict clinical response to VT1021.

Upregulation of CD22 by Chidamide promotes CAR T cells functionality

Treatment failure or relapse due to tumor escape caused by reduction in target antigen expression has become a challenge in the field of CART therapy. Target antigen density is closely related to the effectiveness of CART therapy, and reduced or lost target antigen expression limits the efficacy of CART therapy and hinders the durability of CAR T cells. Epigenetic drugs can regulate histones for molecular modifications to regulate the transcriptional, translational and post-translational modification processes of target agents, and we demonstrated for the first time the role in regulating CD22 expression and its effect on the efficacy of CD22 CART. In this paper, we found that Chidamide promoted the expression of CD22 on the surface of B-cell tumor cells in vitro and in vivo, and enhanced the function of CD22 CART. As for mechanisms, we demonstrated that Chidamide did not affect CD22 mRNA transcription, but significantly increased the expression of total CD22 protein, indicating that Chidamide may upregulate cell surface CD22 expression by affecting the distribution of CD22 protein. In summary, our results suggest that Chidamide may enhance the efficacy of CD22 CART by inhibiting histone deacetylases to regulate post-transcriptional modifications that affect protein distribution to increase the expression of CD22 on the cell surface.

Comparative Analysis of Human Adipose-Derived Stromal/Stem Cells and Dermal Fibroblasts.

Dermal fibroblasts (DFs) share several qualities with mesenchymal stem cell/multipotent stromal cells (MSCs) derived from various tissues, including adipose-derived stromal/stem cells (ASCs). ASCs and DFs are morphologically comparable and both cell types can be culture expanded through the utilization of their plastic-adherence properties. Despite these similar characteristics, numerous studies indicate that ASC and DF display distinct therapeutic benefits in clinical applications. To more accurately distinguish between these cell types, human DFs and ASCs isolated from three individual donors were analyzed for multipotency and cell surface marker expressions. The detection of cell surface markers, CD29, CD34, CD44, CD73, CD90, and CD105, were used for phenotypic characterization of the DFs and ASCs. Furthermore, both cell types underwent lineage differentiation based on histochemical staining and the expression of adipogenic related genes, CCAAT/Enhancer-Binding Protein alpha (CEBPα), Peroxisome proliferator-activated receptor gamma (PPARγ), UCP1, Leptin (LEP), and Adiponectin (ADIPOQ); and osteogenic related genes, Runt related transcription factor 2 (Runx2), Alkaline phosphatase (ALPL), Osteocalcin (OCN), and Osteopontin (OPN). Evidence provided by this study demonstrates similarities between donor-matched ASC and DF with respect to morphology, surface marker expression, differentiation potential, and gene expression, although appearance of enhanced adipogenesis in the ASC based solely on spectrophotometric analyses with no significant difference in real-time polymerase chain reaction detection of adipogenic biomarkers. Thus, there is substantial overlap between the ASC and DF phenotypes based on biochemical and differentiation metrics.

Significance of CD8+ T cell infiltration-related biomarkers and the corresponding prediction model for the prognosis of kidney renal clear cell carcinoma.

Cytotoxic T cells expressing cell surface CD8 played a key role in anti-cancer immunotherapy, including kidney renal clear cell carcinoma (KIRC). Here we set out to comprehensively analyze and evaluate the significance of CD8+ T cell-related markers for patients with KIRC. We checked immune cell response in KIRC and identified cell type-specific markers and related pathways in the tumor-infiltrating CD8+ T (TIL-CD8T) cells. We used these markers to explore their prognostic signatures in TIL-CD8+ T by evaluating their prognostic efficacy and group differences at various levels. Through pan-cancer analysis, 12 of 63 up-regulated and 162 of 396 down-regulated genes in CD8+ T cells were found to be significantly correlated with the survival prognosis. Based on our highly integrated multi-platform analyses across multiple datasets, we constructed a 6-gene risk scoring model specific to TIL-CD8T. In this model, high TIL-CD8 sig score was corresponding to a higher incidence frequency of copy number variation and drug sensitivity to sorafenib. Moreover, the prognosis of patients with the same or similar immune checkpoint gene levels could be distinguished from each other by TIL-CD8 sig score.

A Novel Immunotherapy for Relapsed or Refractory Myeloma

Bispecific therapeutic antibodies are active in relapsed B-cell malignancies by dual engagement of a tumor cell surface antigen and CD3 on T-cells,

African Trypanosomiasis: Extracellular Vesicles Shed by Trypanosoma brucei brucei Manipulate Host Mononuclear Cells.

African trypanosomiasis or sleeping sickness is a zoonotic disease caused by Trypanosoma brucei, a protozoan parasite transmitted by Glossina spp. (tsetse fly). Parasite introduction into mammal hosts triggers a succession of events, involving both innate and adaptive immunity. Macrophages (MΦ) have a key role in innate defence since they are antigen-presenting cells and have a microbicidal function essential for trypanosome clearance. Adaptive immune defence is carried out by lymphocytes, especially by T cells that promote an integrated immune response. Like mammal cells, T. b. brucei parasites release extracellular vesicles (TbEVs), which carry macromolecules that can be transferred to host cells, transmitting biological information able to manipulate cell immune response. However, the exact role of TbEVs in host immune response remains poorly understood. Thus, the current study examined the effect elicited by TbEVs on MΦ and T lymphocytes. A combined approach of microscopy, nanoparticle tracking analysis, multiparametric flow cytometry, colourimetric assays and detailed statistical analyses were used to evaluate the influence of TbEVs in mouse mononuclear cells. It was shown that TbEVs can establish direct communication with cells of innate and adaptative immunity. TbEVs induce the differentiation of both M1- and M2-MΦ and elicit the expansion of MHCI+, MHCII+ and MHCI+MHCII+ MΦ subpopulations. In T lymphocytes, TbEVs drive the overexpression of cell-surface CD3 and the nuclear factor FoxP3, which lead to the differentiation of regulatory CD4+ and CD8+ T cells. Moreover, this study indicates that T. b. brucei and TbEVs seem to display opposite but complementary effects in the host, establishing a balance between parasite growth and controlled immune response, at least during the early phase of infection.

Neutrophils promote T-cell activation through the regulated release of CD44-bound Galectin-9 from the cell surface during HIV infection.

The interaction of neutrophils with T cells has been the subject of debate and controversies. Previous studies have suggested that neutrophils may suppress or activate T cells. Despite these studies, the interaction between neutrophils and T cells has remained a largely unexplored field. Here, based on our RNA sequencing (RNA-seq) analysis, we found that neutrophils have differential transcriptional and functional profiling depending on the CD4 T-cell count of the HIV-infected individual. In particular, we identified that neutrophils in healthy individuals express surface Galectin-9 (Gal-9), which is down-regulated upon activation, and is consistently down-regulated in HIV-infected individuals. However, down-regulation of Gal-9 was associated with CD4 T-cell count of patients. Unstimulated neutrophils express high levels of surface Gal-9 that is bound to CD44, and, upon stimulation, neutrophils depalmitoylate CD44 and induce its movement out of the lipid raft. This process causes the release of Gal-9 from the surface of neutrophils. In addition, we found that neutrophil-derived exogenous Gal-9 binds to cell surface CD44 on T cells, which promotes LCK activation and subsequently enhances T-cell activation. Furthermore, this process was regulated by glycolysis and can be inhibited by interleukin (IL)-10. Together, our data reveal a novel mechanism of Gal-9 shedding from the surface of neutrophils. This could explain elevated plasma Gal-9 levels in HIV-infected individuals as an underlying mechanism of the well-characterized chronic immune activation in HIV infection. This study provides a novel role for the Gal-9 shedding from neutrophils. We anticipate that our results will spark renewed investigation into the role of neutrophils in T-cell activation in other acute and chronic conditions, as well as improved strategies for modulating Gal-9 shedding.

Rituximab induces rapid blood repopulation by CLL cells mediated through their release from immune niches and complement exhaustion

The in vivo rituximab effects in B cell malignancies are only partially understood. Here we analyzed in a large chronic lymphocytic leukemia (CLL) cohort (n = 80) the inter-patient variability in CLL cell count reduction within the first 24 h of rituximab administration in vivo, and a phenomenon of blood repopulation by malignant cells after anti-CD20 antibody therapy. Larger CLL cell elimination after rituximab infusion was associated with lower pre-therapy CLL cell counts, higher CD20 levels, and the non-exhausted capacity of complement-dependent cytotoxicity (CDC). The absolute amount of cell-surface CD20 molecules (CD20 density x CLL lymphocytosis) was a predictor for complement exhaustion during therapy. We also describe that a highly variable decrease in CLL cell counts at 5 h (88 %–2%) following rituximab infusion is accompanied in most patients by peripheral blood repopulation with CLL cells at 24 h, and in ∼20 % of patients, this resulted in CLL counts higher than before therapy. We provide evidence that CLL cells recrudescence is linked with i) CDC exhaustion, which leads to the formation of an insufficient amount of membrane attack complexes, likely resulting in temporary retention of surviving rituximab-opsonized cells by the mononuclear-phagocyte system (followed by their release back to blood), and ii) CLL cells regression from immune niches (CXCR4dimCD5bright intraclonal subpopulation). Patients with major peripheral blood CLL cell repopulation exhibited a longer time-to-progression after chemoimmunotherapy compared to patients with lower or no repopulation, suggesting chemotherapy vulnerability of CLL cells that repopulate the blood.

SPPL3-dependent downregulation of the synthesis of (neo)lacto-series glycosphingolipid is required for the staining of cell surface CD59

CD59 is a small glycoprotein modified with a glycophosphatidylinositol (GPI) anchor that prevents the formation of the membrane attack complex, thereby protecting host cells from lysis. A previous study identified that cell surface CD59 staining required the intramembrane protease signal peptide peptidase-like 3 (SPPL3). However, the effect of SPPL3 on the staining of CD59 remains unknown. This study shows that SPPL3 is essential for the surface labeling of CD59 but not of major GPI-anchored proteins. Surface CD59 staining requires the intramembrane protease activity of SPPL3 and SPPL3-mediated suppression of the (neo)lacto-series glycosphingolipids (nsGSLs)—but not N-glycan—synthesis pathway. The abundance of nsGSLs may affect complement-dependent cytotoxicity by altering the abundance or accessibility of cell surface CD59.

Abstract 1877: IOA-237 - A potent anti-CD73 mAb demonstrates monotherapeutic and combination efficacy in solid tumor models

Abstract CD73, an ecto-5'-nucleotidase, is frequently overexpressed in the tumor microenvironment and can be found on tumor, stromal cells and immune cells. Its function is to catalyze the conversion of adenosine monophosphate (AMP) to adenosine and phosphate, both at the cell surface and as a soluble factor. CD73 has gained interest as a therapeutic target in cancer due to the immunosuppressive role of adenosine in the tumor microenvironment. IOA-237 is a fully human, phage derived monoclonal antibody fully cross-reactive to mouse and human and with high potency against soluble and cell surface CD73. This novel antibody demonstrates in vitro and in vivo characteristics that suggest a best-in-class potential. We demonstrate that IOA-237 is able to significantly reduce tumor outgrowth in multiple mouse models of solid tumors. In the low CD73 expressing CT26 mouse colorectal cancer model, monotherapeutic application of IOA-237 showed tumor outgrowth reduction compared with isotype control. Best-in-class potential was confirmed with head-to-head comparison against a clinical stage anti-CD73 mAb, MEDI9447. Furthermore, inhibition of CD73 by IOA-237 was combined with inhibitors of novel immuno-oncology targets to study complementary modes of action in mouse models of solid tumors. These data indicate the potential of IOA-237 in monotherapeutic and combination therapeutic approaches and warrants further development. Additional in vitro and in vivo efficacy studies are ongoing and non-clinical safety studies are being planned. Citation Format: Marcel A. Deken, Alan M. Carruthers, Karolina Niewola-Staszkowska, Gráinne Gernon, Hilary Sandig, Pritom Shah, Michael M. Lahn, Zoë Johnson. IOA-237 - A potent anti-CD73 mAb demonstrates monotherapeutic and combination efficacy in solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1877.

Abstract LB171: The differentiated CD47 monoclonal antibody AO-176 exhibits significant in vivo activity against xenograft models of pediatric acute lymphoblastic leukemia (ALL)

Abstract Introduction: CD47 is a cell surface marker that interacts with signal regulatory protein alpha (SIRPα) on macrophages and dendritic cells to negatively regulate phagocytosis. This interaction elicits a “don't eat me” signal preventing phagocytosis by immune cells, enabling cell survival. CD47 is expressed on tumor cells, including acute leukemia cells. AO-176 is a highly differentiated monoclonal antibody that targets CD47, exerts potent in vivo activity in mouse xenograft models, has an established safety profile in cynomolgus monkeys, and is being evaluated in clinical trials. Given the broad expression of CD47 in malignant hematopoietic cells, it was of interest for the Pediatric Preclinical Testing Consortium to evaluate AO-176 in vivo against patient-derived xenograft (PDX) models of pediatric ALL. Methods: CD47 mRNA and protein expression were quantified by RNAseq (https://pedcbioportal.org) and flow cytometry (relative fluorescence intensity, RFI), respectively. AO-176 was tested in vivo against 3 T-ALL (ALL-8, ALL-30, ALL-121) and one early T-cell precursor (ETP) ALL (ETP-2) PDXs in NSG mice at 25 mg/kg intraperitoneally once per week for 4 weeks. Engraftment was evaluated by enumerating the proportion of human CD45+ cells in the peripheral blood (%huCD45+) at weekly intervals. Events were defined as the %huCD45+ ≥ 25%. At Day 28 post-treatment initiation or event (whichever occurred first), 3 mice/group were humanely killed to assess leukemia infiltration of hematolymphoid organs. Drug efficacy was assessed by event-free survival (EFS) of treated (T) and control (C) groups by T-C, T/C, and stringent objective response measures (Houghton et al, Pediatr Blood Cancer 49:928-40, 2007). Results: CD47 mRNA expression was significantly higher in a panel of T-lineage ALL PDXs (n=25; mean + SD FPKM, 149 + 57.9) compared with B-ALL PDXs (n=65; 65.3 + 18.7 FPKM) (P&amp;lt;0.0001, unpaired t-test). Cell surface CD47 expression in a subset of 8 PDXs (6 T-ALL, 2 ETP-ALL) was a median of 40.2 (RFI range 17.9-81.9). AO-176 was well tolerated in NSG mice and significantly (P&amp;lt;0.006) delayed the progression of 3/4 PDXs tested (3 T-ALL, one ETP-ALL) (T-C, 12.7-56.9 days; T/C 3.4-4.9) and one PDX elicited an objective response (Partial Response). AO-176 was least effective against the lowest CD47 expressing PDX. AO-176 significantly (P&amp;lt;0.005) decreased human leukemia infiltration of murine spleens, but not bone marrow, in 3/4 PDXs. Conclusion: Despite being tested in highly immune-deficient mice, the naked antibody AO-176 exhibited significant single-agent in vivo anti-leukemic activity against pediatric T-lineage ALL PDXs. The significant decrease in spleen infiltration elicited by AO-176 in 3/4 PDXs suggests on-target activity consistent with the known mechanism of action of CD47 targeting agents. (Supported by NCI Grants CA199000 and CA199922) Citation Format: Richard B. Lock, Kathryn Evans, Narimanne El-Zein, Eric J. Earley, Stephen W. Erickson, Beverly A. Teicher, Victoria Sung, Malcolm A. Smith. The differentiated CD47 monoclonal antibody AO-176 exhibits significant in vivo activity against xenograft models of pediatric acute lymphoblastic leukemia (ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB171.

Abstract LB174: Discovery and preclinical characterization of INCA00186, a humanized monoclonal antibody antagonist of CD73, as a cancer immunotherapy

Abstract In the tumor microenvironment, dying cells release ATP which gets converted to AMP by CD39. AMP is subsequently converted to immunosuppressive adenosine by the extracellular 5'-nucleotidase CD73. CD73 is linked to the cell surface by a GPI-anchor and can be released into the plasma as soluble CD73. In order to lower extracellular levels and reverse the immunosuppressive activity of adenosine in the tumor microenvironment, an antibody was researched and discovered that antagonizes CD73 function. INCA00186 is a humanized monoclonal antibody that binds and inhibits CD73 function. INCA00186 binds human and cynomolgus CD73 with sub-nanomolar affinity, and inhibits CD73 enzymatic activity in a manner that is non-competitive for AMP binding. In functional studies, INCA00186 restored effector T cell proliferation in the presence of high concentrations of AMP. In the human A375 melanoma tumor model in CD34+ humanized NSG mice, INCA00186 decreased levels of cell surface CD73, displayed high receptor occupancy on tumor cells, and decreased CD73 activity in ex vivo assays of tumor homogenates. In the human MDA-MB-231 breast tumor model in CD34+ humanized NSG mice, INCA00186 decreased intratumoral adenosine concentrations. In combination with the novel A2A/A2B adenosine receptor antagonist INCB106385, INCA00186 synergistically restored effector T cell activity as measured by interferon gamma (IFNγ) production in the presence of high concentrations of AMP. In the human MDA-MB-231 breast tumor model in CD34+ humanized NSG mice, combination treatment with INCA00186 and INCB106385 controlled tumor growth significantly better than monotherapies. In summary, the data presented in this study demonstrates that INCA00186 is a potent CD73 antagonist and effectively attenuates adenosine induced immunosuppression in the tumor microenvironment. INCA00186 demonstrated anti-tumor immunity alone and, to a greater extent, in combination with the A2A/A2B adenosine receptor antagonist INCB106385. Citation Format: Shaun Stewart, Rebecca Buonpane, Jing Zhou, Michael Hansbury, Michael Smith, Hui Wang, Lu, Bin Su, Rahel Awdew, Cheng-Yen Huang, Ashwini Kulkarni, Shane Harvey, Arpita Mondal, Steve Wang, Christina Stevens, Michael Pratta, Elham Behshad, Alexandra Fanuka, Xiaodi Ren, Holly Koblish, Horacio Nastri, Patrick Mayes. Discovery and preclinical characterization of INCA00186, a humanized monoclonal antibody antagonist of CD73, as a cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB174.