Surface Antigen CD20 Research Articles

Direct Comparison of Anti-CD33 and Anti-CD123 Chimeric Antigen Receptor T Cells with CD28 or 41BB Co-Stimulatory Domains for Treatment of Acute Myeloid Leukemia

Introduction: Acute Myeloid Leukemia (AML) is an aggressive malignancy with very poor prognosis and limited treatment options. Chimeric antigen receptor expressing T (CART) cell therapy has been highly successful against refractory B cell acute lymphoblastic leukemia and lymphoma. Effective use of CART cells in AML has not yet been well established. Differences in efficacy and potency of CD28 or 41BB co-stimulatory domains of the CART cells have not been thoroughly studied particularly in the context of AML. Here, we systematically compare potency and efficacy of anti-CD123 and anti-CD33 CART cells with CD28 or 41BB co-stimulatory domains against AML both in vitro and in vivo .Methods: anti-CD33 and anti-CD123 CART cells were developed using two different anti-CD33 and a single anti-CD123 single chain fragment variable sequences and cloning into a third-generation lentiviral plasmid containing a CD8 transmembrane domain, a co-stimulatory signaling domain (either CD28 or 41BB) and a CD3zeta domain. CART surface expression on T cell surface was measured by flow cytometry. Efficacy of CAR expressing T cells was tested against a panel of AML cell lines with relatively uniform expression of CD33 and variable expression of CD123 including MOLM14, MV4;11, THP1 and U937. Enzyme-linked immunosorbent assay kits for interleukin-2 (IL2) and interferon-gamma (IFN-gamma) were used to assess in vitro CART functionality, 105 tumor cells were co-incubated with 105 transduced CART cells or mock T cells for 16 hours in 96-well plate. In vitro killing of tumor by CART cells was evaluated by measuring green and red fluorescence by IncuCyte ZOOM, 105 tumor cells that were co-incubated with 105 transduced CART cells or mock T cells for 40 hours in 96-well plates. In vivo CART functionality was assessed by monitoring leukemia burden through bioluminescent imaging using mice engrafted with AML cell lines that received anti-CD33 and anti-CD123 CART cell therapy (tumor challenge of 1x106 cells was given and CART cell therapy dose of 7x106 transduced cells was given). Bone marrow and spleen of mice were assessed by flow cytometry for CART cell and leukemia presence.Results: In vitro cytokine production of IL2 and IFN-gamma when anti-CD123 and anti-CD33 CART cells are co-incubated with various AML cell lines indicate greater production of cytokine in CART cells with a CD28 co-stimulatory domain. In vitro killing assay indicates no significant difference in killing capacity of anti-CD33 and anti-CD123 with CD28 or 41BB co-stimulatory domains, however there is some evidence that the CD28 co-stimulatory domain may yield more killing at earlier time points than 41BB co-stimulatory domain. In vivo activity of anti-CD33 and anti-CD123 CART cells against MOLM14, THP1 and U937 shows that clearance of AML occurs more potently and at earlier time points with the CD28 co-stimulatory domain when compared to the 41BB co-stimulatory domain. Bone marrow and spleen analyses show the lack of presence of leukemia but presence of CART cells with CD28 co-stimulatory domain.Conclusions: CART cells targeting surface antigen CD33 and CD123 in AML demonstrate in vitro and in vivo activity against AML, showing clearance of leukemia and presence of CART cells. Additionally, in vitro and in vivo comparison of CD28 and 41BB co-stimulatory domains indicates that the CD28 may be more potent and efficacious in clearing AML. Further experiments are ongoing to more fully evaluate relative potency of CAR T cells incorporating different co-stimulatory domains. DisclosuresNo relevant conflicts of interest to declare.

CLL Cell-Derived Exosomes Promote Bone Marrow Fibrosis and Inhibit Normal Hematopoietic Colony Proliferation

CLL Cell-Derived Exosomes Promote Bone Marrow Fibrosis and Inhibit Normal Hematopoietic Colony Proliferation

MO316RITUXIMAB IN GLOMERULAR DISEASES: A COHORT ANALYSIS AND A LITERATURE REVIEW

Abstract Background and Aims Glomerular diseases (GD) account for about 20% of chronic kidney disease causes. They are a heterogeneous group of diseases and current treatment is still inadequate. Rituximab (RTX) is a chimeric antibody which binds specifically to the B-cell surface antigen CD20 and has been applied in the treatment of different GD. The authors present the single center experience of the use of RTX for the treatment of GD as well as a literature review. Method We performed a retrospective analysis of all patients with biopsy-proven GD treated with RTX as first or second-line therapy between January 2010 and March 2020. The protocol used was RTX 375 mg/m2 once a week for 4 weeks. Infusions were preceded by adequate premedication. Results Nineteen patients with biopsy-proven GD received RTX therapy. Seven patients had membranous nephropathy (MN) (36.8%), 4 patients had focal segmental glomerulosclerosis (FSGS), 4 patients had lupus nephritis (LN) and 4 patients had vasculitis (25.0% each). Five patients (26.3%) received RTX as first-line therapy and 14 patients (73.7%) as second-line therapy, namely long-term prednisolone (Pd) (n=15, 78.9%), calcineurin inhibitors (CNI) (n=6, 31.6%), cyclophosphamide (Cp) (n=7, 36.8%), mycophenolate mofetil (MMF) (n=5, 26.3%) and methotrexate (MTX) (n=1, 5.3%). Serum creatinine at diagnosis was 1.5 ± 1.7mg/dL and the 24-h urine protein at diagnosis was 4.9 ± 4.5g. Mean follow-up time was 7.7 ± 7.2 years. In MN, 2 patients (28.6%) had CR, 2 patients (28.6%) had PR and 3 patients (42.9%) had no response. In FSGS, 2 patients (50.0%) presented CR, 1 patient (25.0%) had no response and the other one had renal deterioration. Two patients (50.0%) had a LN class IV with a CR after RTX, 1 patient with LN class IIIC/V had no response and 1 patient with LN class II had renal deterioration. In vasculitis, 3 patients (75.0%) presented CR and 1 patient had PR. Mean serum creatinine after RTX was 1.6 ± 1.4mg/dL and the mean 24-h urine protein was 1.9 ± 3.1g. Two patients (10.5%) presented infusion reactions and one patient had multiple respiratory infections. This patient had received previous immunosuppression with cyclophosphamide. Conclusions A total of 19 patients with biopsy-proven GD received RTX therapy and 12 patients (63.2%) presented a complete or partial remission suggesting the efficacy of RTX in different types of GD. In MN, our response rate was similar to studies such as GEMRITUX and MENTOR trial, which have shown remission in 60–70% of patients. In FSGS, 50% of patients had a stable and complete remission with RTX therapy similar with a meta-analysis of five studies with 51 patients with FSGS. Two patients had refractory LN class IV and both had complete remission (50% of patients with LN). This is in line with a systematic review of case reports and case series that reported sustained complete and partial response rates of 67% in LN class IV. In our cohort, patients with vasculitis received RTX as first-line therapy and complete remission was achieved in 75% of cases, which is in line with the results found in RITUXVAS and RAVE trial. Rituximab was safe and effective in achieving remission in different types of immune-mediated glomerular diseases.

Promotion of Differentiating Bone Marrow Mesenchymal Stromal Cells (BMSCs) into Cardiomyocytes via HCN2 and HCN4 Cotransfection.

Aim Investigation of the influences HCN2 and HCN4 has on bone marrow mesenchymal stromal cells (BMSCs) on cardiomyocyte differentiation. Methods Miniature adult pigs were used for bone marrow extraction and isolation of BMSCs. The identification of these BMSCs was done by using flow cytometry for the detection of expressed surface antigens CD45, CD11B, CD44, and CD90. Using HCN2 and HCN4 genes cotransfected into BMSCs as group HCN2+HCN4 while myocardial induction solution was used to induced BMSC differentiation in the BMSC induction group. Myocardial marker proteins α-actin and cTnT were detected by immunofluorescence staining, while α-actin, cTnT, and Desmin myocardial marker proteins expressed were detected by Western blot. The whole-cell patch-clamp technique was used to identify and detect cellular HCN2 channels, HCN4 channel current activation curve, and the inhibitory effect of CsCl on heterologous expression currents. Results Flow cytometry results showed that CD45 and CD11B were expressed negatively while CD90 and CD44 were positive. Post HCN2 and HCN4 gene transfection, immunofluorescence staining, and Western blot showed significantly increased HCN2, HCN4, α-actin, and cTnT expressed in group HCN2+HCN4 were, which could be compared to the expression levels in the BMSC-induced group. The HCN2+HCN4 group was able to document cell membrane channel ion currents that were similar to If properties. Conclusion HCN2 and HCN4 overexpression can considerably enhance the MSC ability to differentiate into cardiomyocytes in vitro and restore the ionic current.

Inhibition of PIM Kinases in Diffuse Large B-Cell Lymphoma Cells Targets MYC-Dependent Transcriptional Program, Increases CD20 Expression and Augments the Efficacy of Anti-CD20 Antibodies

R-CHOP immunochemotherapy remains standard frontline therapy for newly diagnosed diffuse large B-cell lymphoma (DLBCL) patients. However, this therapy is ineffective in approximately 1/3 of patients, underscoring the need for better treatment modalities. Targeting DLBCL oncogenic drivers is a promising strategy to improve the treatment efficacy and outcome. Although MYC transcription factor is one of the key oncogenes in DLBCL development, direct MYC targeting strategies have been largely ineffective, highlighting the need for other, indirect approaches. For example, MYC expression is stabilized by PIM serine-threonine kinases, indicating that PIM inhibition might be a rational approach to indirectly target MYC. In this study, we assessed the PIM-MYC relationship and the consequences of PIM inhibition in DLBCL. We first evaluated the expression of PIM1-3 and MYC proteins in 57 DLBCL diagnostic sections by immunohistochemistry. In this series, 70.17% of specimens were positive for at least one PIM isoform and 84.22% cases were MYC-positive. 100% of cases with high MYC expression (MYC present in ≥30% of the cells, n=35) were PIM-positive, whereas 86,36% of cases with undetectable or low MYC expression (MYC detected in ≤20% of cells, n= 22) were PIM-negative (Fisher's exact test, p<0.0001). Since the coexpression of MYC and PIMs highlights the functional link between these proteins in DLBCLs, we evaluated the expression of PIM kinases in cell lines following siRNA-mediated MYC knockdown or treatment with MYC-MAX dimerization inhibitor, 10058F4. The genetic or chemical MYC inhibition markedly decreased PIM1-3 expression in six GCB and ABC cell lines. Likewise, knockdown of all three PIM isoforms decreased MYC levels, attenuated proliferation and induced apoptosis. Similarly, PIM blockade with SEL24/MEN1703, a novel pan-PIM/FLT3 inhibitor tested currently in clinical trial in AML patients and exhibiting favorable safety profile, decreased the expression of multiple MYC-dependent genes. To assess the MYC role in PIM inhibitor-mediated toxicity, we generated DHL4 cells expressing degradation-resistant MYC_T58A mutant. MYC_T58A expression partially protected cells from PIM inhibitor-induced proliferation arrest and apoptosis, indicating that the inhibitor's toxicity is at least partially mediated by MYC depletion. The MS4A1 gene, encoding CD20 surface antigen and rituximab target, is regulated by an upstream promoter containing potential MYC-binding sites (E-boxes). MYC association to these regions was confirmed in chromatin immunoprecipitation assays. As expected, in SEL24/MEN1703-treated cells, MYC occupancy at the MS4A1 promoter markedly decreased. To determine the consequences of MYC binding to the MS4A1 promoter, we assessed CD20 levels in a lymphoblastoid cell line carrying tetracycline-regulated (tet-off) MYC. MYC repression markedly elevated transcript and surface CD20 levels in a time-dependent manner, reaching 17.3-fold (transcript) and 3.82-fold (surface) inductions at 96 h. Consistently, the pan-PIM inhibitor decreased MYC expression in DHL4 and RAJI cells and resulted in increased surface CD20 levels up to 3.72-fold of baseline. In cells expressing the MYC_T58A mutant, PIM inhibition did not increase CD20 level, indicating that PIM kinases modulate CD20 surface expression via MYC. Importantly, PIM inhibitors increased CD20 levels also in primary, patient-derived DLBCL cells. These data suggest that indirect MYC targeting via PIM inhibition would lead to increased rituximab activity. Indeed, in PIM inhibitor-treated DHL4 and RAJI cells, rituximab triggered higher complement-dependent toxicity. Likewise, PIM inhibitor potentiated rituximab-dependent uptake of DHL4 and DHL6 cells by human monocyte-derived macrophages in antibody-dependent cellular phagocytosis assay. Taken together, we characterize a PIM-MYC regulatory circuit promoting DLBCL growth and resistance to anti-CD20 antibody. We also demonstrate that PIM inhibition exhibits pleiotropic effects that combine direct cytotoxicity with increased surface CD20 levels and increased susceptibility to anti-CD20 antibody-based therapies. Study supported by Foundation for Polish Science (POIR.04.04.00-00-5C84/17-00), Polish National Science Centre (2016/22/M/NZ5/00668 and 2017/26/D/NZ5/00561) and Ministry of Science and Higher Education in Poland (iONCO) grants. Disclosures Golas: Ryvu Therapeutics: Current Employment. Green:KDAc Therapeutics: Current equity holder in private company. Tomirotti:Menarini Ricerche: Current Employment. Brzózka:Ryvu Therapeutics: Current Employment. Juszczynski:Ryvu Therapeutics: Other: member of advisory board.

A Phase I Study of CD19-Targeted 19(T2)28z1xx CAR T Cells in Adult Patients with Relapsed or Refractory B-Cell Malignancies

Background: Autologous CAR T cell therapy targeting the B-cell specific surface antigen CD19 has demonstrated favorable clinical responses in relapsed or refractory (R/B) B-cell lymphomas (BCL). However, despite 40-60% initial complete response (CR) rates, only a subset of patients experience durable remissions, and there is a need to further improve the efficacy of CAR therapies by preventing relapse and attaining a deeper CR. We hypothesized that the redundancy of CD28 and CD3V signaling in a CAR design incorporating all 3 CD3Vimmunoreceptor tyrosine-based activation motifs (ITAMs) might foster counterproductive T cell differentiation and exhaustion, and therefore created a new CD19 CAR construct with calibrated CAR activation potential by mutating 2 of the 3 ITAMs, termed 1XX. In systemic ALL mouse models, 19-28z1XX CAR induced effective tumor eradication at low CAR T cell doses with improved survival compared to conventional 19-28z CAR. Further preclinical studies demonstrated that the enhanced therapeutic benefit resulted from the reduced strength of activation mediated by the 19-28z1XX CAR, achieving a favorable balance of effector and memory functions, thereby enhancing persistence of functional CAR T cells and promoting effective elimination of CD19+ leukemia at lower T cell doses than needed with 19-28z CAR T cells (Feucht J et al. Nat Med 2019). To further improve the persistence of functional CAR T cells, we screened different humanized CD19-directed scFv in the context of a 19-28z1XX CAR design and proved high specificity and functionality of 19-28z1XX CARs containing a novel humanized scFv T2 - termed 19(T2)28z1XX. Study Design and Methods: This study is a single center Phase I clinical trial of 19(T2)28z1XX in patients with R/R B-cell malignancies at Memorial Sloan Kettering Cancer Center (NCT04464200). Key disease eligibility criteria include R/R diffuse large B cell lymphoma (DLBCL), high grade BCL, primary mediastinal BCL, indolent BCL and chronic lymphocytic leukemia (CLL). Patients with prior CD19 CAR therapies are eligible as long as expression of CD19 is confirmed. Key exclusion criteria include ongoing immunosuppression such as systemic GvHD therapy and active CNS disease. The study uses a 3+3 dose-escalation design to identify the maximum tolerated dose for BCL. There are 5 planned flat-dose levels. Patients will receive conditioning chemotherapy consisting of 3 days of fludarabine and cyclophosphamide followed by a single infusion of 19(T2)28z1XX CAR T cells. In the dose-escalation phase, patients with DLBCL, high grade BCL, and primary mediastinal BCL are eligible to participate. Once the recommended phase 2 dose (RP2D) is determined, the study will open to dose expansion phase with two cohorts. Cohort 1 includes DLBCL, high grade BCL and primary mediastinal BCL (i.e. same eligibility criteria as the dose-escalation phase). Cohort 2 will include patients with indolent BCL, CLL, and Richter's transformation. The dose-expansion part of the trial is designed to further characterize the safety, efficacy, and pharmacokinetics of 19(T2)28z1XX CAR in multiple indications. The primary objective of the trial is to evaluate safety and tolerability and determine the recommended Phase 2 dose of 19(T2)28z1XX. Key secondary objectives include evaluation of 19(T2)28z1XX's efficacy and cellular kinetics. Exploratory objectives include assessment of B cell aplasia, and analysis of serum cytokines. The trial has begun enrollment in August 2020. The investigators are hopeful this study will lead to development of improved CD19 CAR T cell therapy with enhanced efficacy and favorable toxicity profiles with lower infused T cell dose. Disclosures Park: AstraZeneca: Consultancy; Servier: Consultancy, Research Funding; Autolus: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Novartis: Consultancy; Minverva: Consultancy; Artiva: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Research Funding; Kite: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Genentech/Roche: Research Funding; Juno Therapeutics: Research Funding; GSK: Consultancy; Intellia: Consultancy; Allogene: Consultancy. Riviere:Fate Therapeutics Inc.: Consultancy, Other: Ownership interest , Research Funding; FloDesign Sonics: Consultancy, Other: Ownership interest; Juno Therapeutics: Other: Ownership interest, Research Funding; Takeda: Research Funding; Atara: Research Funding. Palomba:Genentech: Research Funding; Juno Therapeutics, a Bristol-Meyers Squibb Company: Honoraria, Research Funding; Regeneron: Research Funding; Novartis: Honoraria; Merck: Honoraria; Celgene: Honoraria; Pharmacyclics: Honoraria. Brentjens:BMS: Research Funding; Gracell Therapeutics: Consultancy; Juno Therapeutics (a Bristol Myers Squibb company): Patents & Royalties. Sadelain:Atara: Patents & Royalties, Research Funding; Fate Therapeutics: Patents & Royalties, Research Funding; Minerva: Other: Biotechnologies , Patents & Royalties; Mnemo: Patents & Royalties; Takeda: Patents & Royalties, Research Funding. OffLabel Disclosure: Cyclophosphamide and fludarabine will be used as conditioning therapy prior to 19(T2)28z1XX CAR T cell administration.

Significance of hematopoietic surface antigen CD34 in neuroblastoma prognosis and the genetic landscape of CD34-expressing neuroblastoma CSCs.

High-risk neuroblastoma (HR-NB) is branded with hematogenous metastasis, relapses, and dismal long-term survival. Intensification of consolidation therapy with tandem/triple autologous stem cell (SC) rescue (with bone marrow [BM]/peripheral blood [PB] CD34+ selection) after myeloablative chemotherapy has improved long-term survival. However, the benefit is limited by the indication of NB cells in CD34+ PBSCs, CD34 expression in NB cells, and the risk of reinfusing NB cancer stem cells (NB CSCs) that could lead to post-transplant relapse. We investigated the association of CD34 surface expression (92 patients) with NB evolution/clinical outcomes. CD34 gene-level status in NB was assessed through RNA-Seq data mining (18 cohorts, n, 3324). Genetic landscape of CD34-expressing NB CSCs (CD133+CD34+) was compared with CD34- CSCs (CD133+CD34-). RNA-seq data revealed equivocal association patterns of CD34 expression with patient survival. Our immunohistochemistry data revealed definite, but rare (mean, 0.73%; range 0.00-7.87%; median, 0.20%) CD34 positivity in NB. CD34+ significantly associated with MYCN amplification (p, 0.003), advanced disease stage (p, 0.016), and progressive disease (PD, p < 0.0009) after clinical therapy. A general high-is-worse tendency was observed in patients with relapsed disease. High CD34+ correlated with poor survival in patients with N-MYC-amplified HR-NB. Gene expression analysis of CD34+-NB CSCs identified significant up (4631) and downmodulation (4678) of genes compared with NB CSCs that lack CD34. IPA recognized the modulation of crucial signaling elements (EMT, stemness maintenance, differentiation, inflammation, clonal expansion, drug resistance, metastasis) that orchestrate NB disease evolution in CD34+CSCs compared with CD34- CSCs. While the function of CD34 in NB evolution requires further in-depth investigation, careful consideration should be exercised for autologous stem cell rescue with CD34+ selection in NB patients. Graphical abstract.

Abstract 1921: The DDB2 intronic variation rs830083 is a strong predictor of gemtuzumab ozogamicin response in AML patients: A calicheamicin pathway-directed analysis from the COG-AAML0531 trial

Abstract Polymorphisms in DNA damage repair (DDR) genes could serve as predictors of response to gemtuzumab ozogamicin (GO), a CD33 targeting antibody conjugated to the DNA damaging agent calicheamicin approved for use in immunotherapy for acute myeloid leukemia (AML). GO is internalized upon binding to the myeloid cell surface antigen CD33, allowing calicheamicin-mediated DNA damage and subsequent apoptosis. Following concerns over safety, GO was withdrawn in 2010 but was re-approved in 2017 with the emergence of new evidence of its clinical efficacy and safety. In clinical trials, significantly improved event free survival (EFS), disease free survival (DFS), and relapse risk (RR) were observed in a subset of AML patients with the addition of GO to conventional AML chemotherapy. Combined with low overall survival (OS) and high RR with the relatively unchanged conventional AML chemotherapy, GO usage is anticipated to increase. We used samples from the Children's Oncology Group (COG) trial AAML0531 to determine the association between polymorphism in DDR genes and OS, EFS, DFS, and RR with GO treatment as the primary end points. In this trial, newly diagnosed AML patients were randomized to receive either the standard 5 course chemotherapy alone (Arm A) or with two doses of GO (3mg/m2/dose) during induction 1 and intensification 2 (Arm B). Briefly, genomic DNA samples from a total of 947 patients from Arm A and Arm B were genotyped for 131 SNPs across 43 key genes involved in calicheamicin-mediated DDR. Overall, 28 SNPs in 21 genes were significantly associated with OS, EFS, DFS, and RR. Of these, 21 SNPs in 16 genes were associated with response to GO treatment (Arm B; p ≤ 0.05) and not with conventional AML chemotherapy (Arm A; p ≥ 0.05). The top 5 SNPs occurred within DDB2, XPA, XPC, and XRCC1 – genes involved in the nucleotide excision repair (NER) DDR pathway. The intronic variation rs830083 (G&amp;gt;C) in DDB2 had the poorest outcome (OS Cox p = 0.01; EFS cox p = 0.004; DFS p = 0.000; RR reg p = 0.0006). Furthermore, rs830083 was also most significantly associated with standard risk patients (DFS p = 0.003), EFS (p=0.01) and RR (p = 0.0006). DDB2 has been reported to play a critical role in apoptotic processes post-DNA damage induction by regulating p53, lending further importance to the association between rs830083 and poor response to treatment. In conclusion, the identification of several variants associated with treatment outcomes in our study underscores the importance of a pathway-directed SNP-based candidate gene approach to identify personalized approaches for AML treatment. Specifically, screening DDB2 SNPs could be applied in patient selection and predicting response to GO treatment. Citation Format: Vivek M. Shastri, Lata Chauhan, Todd A. Alonzo, Yi-Cheng Wang, Richard Aplenc, Betsy A. Hirsch, Alan S. Gamis, Soheil Meshinchi, Jatinder K. Lamba. The DDB2 intronic variation rs830083 is a strong predictor of gemtuzumab ozogamicin response in AML patients: A calicheamicin pathway-directed analysis from the COG-AAML0531 trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1921.

Structure-guided engineering of the affinity and specificity of CARs against Tn-glycopeptides

The potency of adoptive T cell therapies targeting the cell surface antigen CD19 has been demonstrated in hematopoietic cancers. It has been difficult to identify appropriate targets in nonhematopoietic tumors, but one class of antigens that have shown promise is aberrant O-glycoprotein epitopes. It has long been known that dysregulated synthesis of O-linked (threonine or serine) sugars occurs in many cancers, and that this can lead to the expression of cell surface proteins containing O-glycans comprised of a single N-acetylgalactosamine (GalNAc, known as Tn antigen) rather than the normally extended carbohydrate. Previously, we used the scFv fragment of antibody 237 as a chimeric antigen receptor (CAR) to mediate recognition of mouse tumor cells that bear its cognate Tn-glycopeptide epitope in podoplanin, also called OTS8. Guided by the structure of the 237 Fab:Tn-OTS8-glycopeptide complex, here we conducted a deep mutational scan showing that residues flanking the Tn-glycan contributed significant binding energy to the interaction. Design of 237-scFv libraries in the yeast display system allowed us to isolate scFv variants with higher affinity for Tn-OTS8. Selection with a noncognate human antigen, Tn-MUC1, yielded scFv variants that were broadly reactive with multiple Tn-glycoproteins. When configured as CARs, engineered T cells expressing these scFv variants showed improved activity against mouse and human cancer cell lines defective in O-linked glycosylation. This strategy provides CARs with Tn-peptide specificities, all based on a single scFv scaffold, that allows the same CAR to be tested for toxicity in mice and efficacy against mouse and human tumors.

Bortezomib interferes with DNA repair and exerts synergistic anti-multiple myeloma activity with doxorubicin

目的探讨系统性免疫球蛋白轻链淀粉样变性（AL）初治患者的外周血免疫细胞表型特征及其与临床指标的相关性。方法采用流式细胞仪多参数免疫荧光分析技术，对36例AL初诊患者和28名健康供者的外周血单个核细胞的表面抗原CD3、CD56、CD4、CD8、CD25、CD45RA、CD28、CD57及核内抗原FOXP3进行检测和比较。根据梅奥2012分期对AL患者进行分期，比较Ⅰ~Ⅱ、Ⅲ~Ⅳ期患者的免疫细胞表型差异。分析λ轻链型AL患者T细胞亚群比例与多项临床指标的相关性。结果AL患者的外周血T（CD3+CD56−）和NKT（CD3+CD56+）细胞比例，T细胞中的CD4+CD8−、CD4−CD8+、Treg（CD4+CD25+FOXP3+）细胞比例与健康供者相比差异无统计学意义（P>0.05）。AL患者的CD4−CD8+细胞中，CD57+细胞的比例较健康供者显著降低（P<0.05），但CD45RA+和CD28+细胞的比例在AL和健康供者间差异无统计学意义。Ⅰ~Ⅱ期和Ⅲ~Ⅳ期AL患者T细胞及其亚群的比例差异无统计学意义（P>0.05）。在λ轻链型AL患者中，外周血CD4−CD8+细胞的比例与24 h尿蛋白和血肌酐呈正相关（P<0.05），与eGFR呈负相关（P<0.05），与其他临床指标无显著相关性。与此相反，CD4+CD8−细胞的比例与eGFR呈正相关，而与24 h尿蛋白和血肌酐呈负相关（P<0.05）。结论AL患者外周血的T细胞亚群与健康供者相比差异无统计学意义，但CD8+ T细胞的比例与肾脏损伤程度呈正相关，提示CD8+ T细胞的比例在评估AL患者肾脏预后中具有一定的价值。

Analiza fenotipa CD3+CD16+ limfocita periferne krvi svinja

The phenotype of porcine peripheral blood T cells and natural killer (NK) cells has been well-studied over the past three decades, though porcine peripheral blood lymphocytes with mixed T/NK-cell phenotype within perforin- and NKp46- positive CD3+ populations have also been identified. Despite the mixed phenotype, both populations showed in vitro NK cell-like major histocompatibility complex-unrestricted cytolysis. In this study, the peripheral blood lymphocytes of 15 crossbreed, 12-week-old pigs of both sexes, were analysed by flow cytometry for the expression of leukocyte surface antigens (cluster of differentiation, CD) that can be found on porcine T cells (CD3, TCR-γδ and CD4), NK cells (CD16) or on both cell populations (CD8α and SLA-DR). We found the presence of a minor population of CD3+CD16+ cells within peripheral blood lymphocytes (2.84%). Peripheral blood CD3+CD16+ lymphocytes consisted of all four subpopulations with respect to the expression of surface antigens CD4 and CD8α; most were CD4-CD8α+ (60.64%) and CD4-CD8α-(36.77%). While the proportion of SLA- DR+ cells within both subpopulations was similar (8.01% of CD3+CD16+CD4-CD8α+ lymphocytes and 7.41% of CD3+CD16+CD4- CD8α- lymphocytes), the proportion of TCR-γδ+ cells was noticeably higher within CD3+CD16+CD4-CD8α+ (43.48%) than CD3+CD16+CD4-CD8α- (16.55%) lymphocytes. When the expression of individual surface antigens was analysed on peripheral blood CD3+CD16+ lymphocytes, most were CD8α+ (62.44%), though some were also TCR- γδ+ (32.56%), SLA-DR+ (7.55%) or CD4+ (2.59%). Expression of CD8α on CD3+CD16+ lymphocytes was not related to co-expression of other surface antigens, though most CD3+CD16+TCR-γδ+ lymphocytes (81.04%) and most CD3+CD16+CD4+ lymphocytes (69.50%) expressed CD8α. Expression of SLA-DR was not related to the co-expression of TCR-γδ or CD8α, or to the co-expression of both antigens (TCR-γδ and CD8α) on CD3+CD16+CD4- lymphocytes. The results also showed the presence of peripheral blood lymphocytes with the combined phenotypeof T cells and NK cells in three-month old pigs. Though a functional analysis of the investigated cells was not performed in this study, future investigations should provide more insight about the functional properties of porcine peripheral blood CD3+CD16+ lymphocytes with distinct phenotypic characteristics, especially concerning antigen- specific responses and whether the results presented here are solely age-related.

Impact of Therapeutic Angiogenesis Using Autologous Bone Marrow-derived Mononuclear Cell Implantation in Patients with No-option Critical Limb Ischemia.

Recently, the limb salvage rate of patients with critical limb ischemia (CLI) has been improved due to the development of revascularization and wound care treatment. However, many patients with CLI are refractory to standard treatments, including revascularization such as endovascular treatment or surgical bypass. Establishment of a new cell therapy is required to improve the limb salvage rate and prognosis in patients with CLI. In 1997, endothelial progenitor cells were found to be derived from the bone marrow to circulate as CD34 surface antigen positive cells in peripheral blood and to affect therapeutic angiogenesis in ischemic tissues. Later, therapeutic angiogenesis using autologous bone marrow-derived mononuclear cell (BM-MNC) implantation was performed for patients with no-option CLI in clinical practice. Several reports showed the safety and efficacy of the BM-MNC implantation in patients with CLI caused by arteriosclerosis obliterans, thromboangiitis obliterans (TAO), and collagen diseases. In particular, in patients with CLI caused by TAO, limb salvage rate was significantly improved compared with standard treatments. The BM-MNC implantation may be feasible and safe in patients with no-option CLI. Here, we review the efficacy of BM-MNC implantation in no-option CLI, with a focus on therapeutic angiogenesis.

Post-rituximab immunoglobulin M (IgM) hypogammaglobulinemia.

Rituximab is a B cell depleting monoclonal antibody that targets the B cell-specific cell surface antigen CD20 and is currently used to treat several autoimmune diseases. The elimination of mature CD20-positive B lymphocytes committed to differentiate into autoantibody-producing plasma cells is considered to be the major effect of rituximab, that makes it a beneficial biological agent in treating autoimmune diseases. Hypogammaglobulinemia has been reported after rituximab therapy in patients with lymphoma and rheumatoid arthritis. Similar data are scarce for other autoimmune diseases. Low immunoglobulin G (IgG) or hypogammaglobulinemia has attracted the most attention because of its significant role in protective immunity. However, the incidence and clinical implications of low immunoglobulin M (IgM) or hypogammaglobulinemia have not been studied in detail. This review will focus on the frequency and the clinical concerns of low IgM levels that result as a consequence of the administration of rituximab. The etiopathogenic mechanisms underlying post-rituximab IgM hypogammaglobulinemia and its implications are presented. The long-term consequences, if any, are not known or documented. Multiple factors may be involved in whether IgG or IgM decreases secondary to rituximab therapy. It is possible that the autoimmune disease itself may be one of the important factors. The dose, frequency and number of infusions appear to be important variables. Post-rituximab therapy immunoglobulin levels return to normal. During this process. IgM levels take a longer time to return to normal levels when compared to IgG or other immunoglobulins. IgM deficiency persists after B cell repopulation to normal levels has occurred. Laboratory animals and humans deficient in IgM can have multiple infections. Specific pharmacologic agents or biologic therapy that address and resolve IgM deficiency are currently unavailable. If the clinical situation so warrants, then prophylactic antibiotics may be indicated and perhaps helpful. Research in this iatrogenic phenomenon will provide a better understanding of not only the biology of IgM, but also the factor(s) that control its production and regulation, besides its influence if any, on rituximab therapy.

Biomarkers Apo10 and TKTL1: Epitope-detection in monocytes (EDIM) as a new diagnostic approach for cholangiocellular, pancreatic and colorectal carcinoma.

OBJECTIVE: The EDIM (Epitope detection in monocytes) blood test is based on two biomarkers Apo10 and TKTL1. Apo10 is responsible for cell proliferation and resistance to apoptosis. TKTL1 plays a major role in anaerobic glycolysis of tumor cells, leading to destruction of the basal membrane and metastasis as well as in controlling cell cycle. For the first time we analyzed Apo10 and TKLT1 in patients with cholangiocellular (CCC), pancreatic (PC), and colorectal carcinoma (CRC).METHODS: Blood samples of 62 patients with CCC, PC, and CRC were measured and compared to 29 control patients. We also investigated 13 patients with inflammatory conditions, because elevated TKTL1 and Apo10 have been previously described in affected individuals. Flow cytometry was used to detect surface antigens CD14/CD16 (activated monocytes/macrophages). Percentages of macrophages harboring TKTL1 and Apo10 were determined. A combined EDIM score (EDIM-CS: TKTL1 plus Apo10) was calculated. Results were correlated with serum tumor markers CEA and CA19-9.RESULTS: Patients with CCC had 100% positive EDIM-CS but CEA and CA19-9 were positive in only 22.2% and 70%, respectively. Patients with PC had 100% positive EDIM-CS but positive tumor markers in only 37.5% (CEA) and 72.7% (CA19-9). Patients with CRC had 100% positive EDIM-CS but only 50% positive CEA. EDIM-CS was positive in 100% (62/62) of all cancer patients and in 0% of healthy individuals. Of the individuals with inflammation, 7.7% had a positive EDIM-CS.CONCLUSION: The sensitivity of the EDIM blood test and the comparison with traditional tumor markers indicate that this new test might improve the detection of carcinomas (CCC, PC and, CRC) and might be relevant for the diagnosis of all tumor entities.

Synthesis and Evaluation of 64Cu (225Ac)-labeled Rituximab for CD20 Expression

Chimeric monoclonal antibody rituximab, which selectively binds to CD20 surface antigen on B lymphocytes, has become a standard treatment for non-Hodgkin’s lymphoma. This study aims to synthesize 64Cu-DOTA-rituximab and evaluate its potential for targeted alpha therapy by in vitro, in vivo studies, and evaluation of the dosimetry of 225Ac-DOTA-rituximab. DOTA-rituximab immunoconjugate was prepared by incubation of rituximab with DOTA-NHS-ester in 1:5, 1:10, and 1:20 ratios. DOTArituximab conjugate was incubated with dried 64CuCl2 in acetate buffer (pH 5-6), and radiolabeling yield was confirmed by ITLC. For stability test, 64Cu-DOTA-rituximab was incubated in serum or phosphate buffered saline for 48 hours, and % dissociation was analyzed by ITLC. Cell binding assay of 64Cu-DOTA-rituximab was performed using Daudi human lymphoma cell line, and specific binding was determined by comparing non-specific binding with total binding. Binding affinity was presented as %injected dose (%ID). Biodistribution was carried out using BALB/c mouse at 1, 2, 6, 24, and 48 h post-injection, and the data is expressed as %ID/g. Residence time were computed via time activity curves of the acquired biodistribution 5 time series data. In order to calculate 225Ac S-value was simulated by Monte Carlo simulations using mice CT images. The organ absorbed dose for 225Ac-labeled DOTA-rituximab was estimated by Monte Carlo simulated 225Ac S-value. MALDI-TOF indicated that 1.3, 2.6, and 5.5 molecules of DOTA were conjugated to rituximab from 1:5, 1:10, and 1:20 conjugation ratio, respectively. Radiolabeling efficiency was higher than 98%, and 64Cu-DOTA-rituximab was used directly without purification. In vitro analysis of the stability showed that no significant dissociation of radioactivity from the complex was observed until 48 h. 64Cu-DOTA-rituximab showed significant specific binding to Daudi cells upon 3-h incubation having 19.8-24.5 %ID. It was shown that the lower the number of DOTA conjugated to rituximab, the higher the binding affinity to CD20. Biodistribution in BALB/c mouse (n=4) indicated that 64Cu-DOTA-rituximab has prolonged blood circulation up to 48 h, and heart, liver, lungs, spleen, and kidneys had high uptake at early time point. The estimated 64Cu-DOTA-rituximab absorbed dose in liver, lung, spleen, and kidney were 0.099, 0.028, 0.079, and 0.14 mSv/MBq, respectively. The estimated 225Ac-DOTA-rituximab absorbed dose in liver, lungs, spleen, and kidneys were 18.1, 5.54, 1.57, and 29.5 mSv/MBq, respectively. The RBE5 of kidney was 0.33 SvRBE5/MBq. DOTA-rituximab was successfully synthesized and in vitro and showed high binding affinity to CD20-expressing cells. DOTA-rituximab will be successfully applied to targeted alpha therapy by labeling with 225Ac.

Immunomodulating properties of Cinnabsin

Introduction: Complex natural medicines are successfully used in various inflammatory diseases of the upper respiratory tract, but their immunomodulatory activity has not been studied enough, in particular in case of Cinnabsin. Therefore, the aim of the study was to determine the effect of the medicine on the production of cellular and humoral factors of immunity and inflammation by tonsil cells of the children. Materials and methods: The studies were performed in vitro using 24-hour cultures of children`s pharyngeal tonsil cells after adenotomy. Altogether 90 samples of cultures were examined before and after the influence of the medicine. The effect of complex natural medicine Cinnabsin on the cells production of various cytokine groups (γ-interferon, interleukins 1, 4, 10), expression of surface antigens CD14+, CD16+, and cytolytic activity of natural killer cells were investigated. Methods of enzyme immunoassay, light microscopy and cellular cytolysis with xenogenic targets were used. Statistical processing was carried out with the Student`s non-parametric criterion, and the Fisher`s angle transformation method when processing ratios. Results: Cinnabsin, both in dosage 10 μg/ml and 100μg/ml, increased the number of cells with the expression of the antigen CD16+ and partially CD14+, activated the natural cytotoxic cells of the tonsils, reduced the production of IL-1β and increased the production of IL-10 and γ-interferon in cell culture. Conclusion: According to the sum of the effects and the immunomodulation vectors, it is possible to state the modulating effect of the medicine on the activity of various immunity reactions, which allows concluding the pronounced anti-inflammatory and antiviral effect of Cinnabsin.

First-in-Human, Phase 1/2 Trial to Assess the Safety and Clinical Activity of Subcutaneous GEN3013 (DuoBody®-CD3×CD20) in B-Cell Non-Hodgkin Lymphomas

First-in-Human, Phase 1/2 Trial to Assess the Safety and Clinical Activity of Subcutaneous GEN3013 (DuoBody®-CD3×CD20) in B-Cell Non-Hodgkin Lymphomas

Functional Characterization of Gamma Delta T Cells and Allogeneic Activated NK Cells As Effector Cells for Tafasitamab (MOR208)

Introduction Tafasitamab (MOR208) is an Fc-enhanced, humanized, monoclonal antibody that binds to the human B cell surface antigen CD19. CD19 is broadly and homogeneously expressed across different B cell malignancies, including diffuse large B cell lymphoma (DLBCL), and amplifies B cell receptor signaling and induces tumor cell proliferation. Tafasitamab is currently in clinical development in patients with relapsed or refractory DLBCL in combination with the immunomodulatory drug lenalidomide (L-MIND study) and the chemotherapeutic agent bendamustine (B-MIND study). The modes of action of tafasitamab comprise antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and direct cytotoxicity (apoptosis). Tafasitamab carries two amino acid exchanges in the Fc region to increase its affinity to Fcγ receptors, including FcγRIIIa. FcγRIIIa plays a key role in mediating ADCC and is expressed on the surface of natural killer (NK) cells, as well as the majority of γδ T cells. MG4101 (a novel therapeutic agent consisting of cryopreserved, ex vivo-expanded, highly activated NK cells) has demonstrated potent anticancer activity in preclinical in vitro and in vivo studies. Currently, MG4101 is in clinical development in patients with malignant lymphoma and advanced solid tumors. Here, we have characterized two FcγRIIIa receptor-expressing cell types, γδ T cells and NK cells (MG4101), as effector cells for tafasitamab in vitro and explored the concept of supplementing MG4101 during tafasitamab therapy using disseminated in vivo models of non-Hodgkin's lymphoma. Methods γδ T cells (CD3+/γδ T cell receptor+) were derived from different donors by stimulation of peripheral blood mononuclear cells with zoledronate/IL-2 for 9-10 days. These were applied as effector cells in in vitro ADCC assays with tafasitamab in Mino and Jeko-1 mantle cell lymphoma (MCL) cell lines, as well as primary patient-derived chronic lymphocytic lymphoma (CLL) and MCL cells. Further, effector cell activity of MG4101 was assessed using tafasitamab-mediated ADCC assays in Raji and Ramos Burkitt's lymphoma cells. The concept of combining tafasitamab with allogeneic effector cell therapy in vivo was studied in two therapeutic survival models of disseminated lymphoma in SCID mice. In the Raji model, tafasitamab (0.05 µg/mouse) was given on Day 1 after intravenous (IV) tumor inoculation, while MG4101 (2x107 cells/mouse) was given on Days 1, 3, 6, 8 and 10. In the Ramos model, tafasitamab (10 mg/kg) and MG4101 (2x107 cells/mouse) were applied twice weekly for 3 weeks starting on Days 3 and 4, respectively, after IV tumor inoculation. Results Tafasitamab in combination with γδ T cells showed distinctly increased ADCC in Mino and Jeko-1 target cells in vitro, compared with a negative control IgG1 antibody. ADCC assays with patient-derived CLL and MCL target cells confirmed tafasitamab-mediated cytotoxic activity and demonstrated a clear enhancement in activity compared with the non-Fc-enhanced version of tafasitamab that was unable to induce substantial cytotoxicity. In vitro ADCC assays with tafasitamab and MG4101 on Raji and Ramos cell lines confirmed potent effector cell activity of the ex vivo-expanded, cryopreserved, allogeneic NK cells. In the disseminated Raji survival model, combination therapy with a single low dose of tafasitamab (0.05 µg) and MG4101 resulted in a distinct increase in survival of the mice with an increased life span (ILS) of 100% compared with monotherapy (ILS of 57% for tafasitamab and 50% for MG4101). Of note, the combination demonstrated a substantial and more than additive enhancement in survival in the more therapeutic Ramos survival model (Figure 1). Combination therapy with tafasitamab (10 mg/kg) and MG4101 NK cells resulted in superior antitumor activity (ILS of 103%) compared with either tafasitamab monotherapy (ILS of 49%) or MG4101 alone (ILS of 25%). Conclusions FcγRIIIa-expressing immune cell types, including NK cells and γδ T cells, are potent effector cells for tafasitamab-mediated ADCC. Combination therapy with tafasitamab and allogeneic MG4101 NK cells in vivo demonstrated a more than additive survival benefit compared with tafasitamab or MG4101 monotherapy in a disseminated therapeutic lymphoma model. Combination of tafasitamab supplemented with immune effector cells could represent a promising new approach for lymphoma therapy. Disclosures Her: GC LabCell: Employment, Patents &amp; Royalties. Cho:GC LabCell: Employment, Patents &amp; Royalties. Hwang:GC LabCell: Employment, Equity Ownership, Patents &amp; Royalties. Boxhammer:MorphoSys AG: Employment, Patents &amp; Royalties. Steidl:MorphoSys AG: Employment. Patra:MorphoSys AG: Employment. Schanzer:MorphoSys AG: Employment. Endell:MorphoSys AG: Employment, Patents &amp; Royalties.

Incidence and Predictive Risk Factors of Infective Events in Patients With Multiple Sclerosis Treated With Agents Targeting CD20 and CD52 Surface Antigens.

ObjectiveMonoclonal antibodies (MAbs) directed against the CD20 and CD52 antigens are used increasingly in patients with multiple sclerosis (MS). Several life-threatening opportunistic infections have been reported in postmarketing case series. The aim of this study was to investigate the incidence of infections and associated prognostic factors during the first year of treatment in patients receiving anti-CD20 (ocrelizumab or rituximab) or anti-CD52 MAbs (alemtuzumab).MethodsA retrospective study was conducted in patients with MS referring to the Neurodegenerative Diseases Center at the University of Naples Federico II who received MAbs between November 2015 and June 2018.ResultsA total of 163 patients were enrolled. Approximately 40% of patients experienced lymphocytopenia during treatment. Eighty-six infective events were reported in 67 patients (41%). Bacterial infections were significantly more frequent with anti-CD20, whereas viral infections prevailed with alemtuzumab. Cytomegalovirus reactivation rates were significantly higher in the alemtuzumab group than in patients on anti-CD20 (51% vs 6%, P < .001). The overall annualized infection rate was 1.1 per patient-year, higher in patients on anti-CD52 versus those on anti-CD20 regimens (1.5 vs 0.8 per patient-year). Alemtuzumab treatment, prior exposure to ≥2 MS drugs, and iatrogenic immune impairment significantly and independently predicted an infection event (adjusted hazard ratio [aHR], 2.7; P = .013; aHR, 1.7; P = .052; and aHR, 2.9; P = .004; respectively).ConclusionsGiven their considerable infection risk, MS patients receiving MAbs should undergo timely follow up and tailored preventive interventions. Anti-CD52–based treatment, prior exposure to MS drugs, and on-treatment immune impairment are significant predictive factors of infection and their evaluation could help clinicians to stratify a patient’s risk of infection.

Decitabine enhances cytotoxic effect of T cells with an anti-CD19 chimeric antigen receptor in treatment of lymphoma.

Background: CD19-directed chimeric antigen receptor (CAR) T cells have substantial benefit in the treatment of patients with B-cell malignancies. However, despite encouraging therapeutic efficiency, there is limited overall response rate when anti-CD19 CAR-T cells are used to treat patients with relapsed and refractory (R/R) B cell lymphomas. Therefore, it further investigation is urgently needed to improve treatment efficacy.Method: A combined treatment protocol of CAR-T cell with decitabine (DAC) to treat B cell lymphoma was developed and tested on lymphoma cell lines first, and then efficacy and the underlying mechanism were investigated. After ethical approval was granted, the combined treatment protocol was applied to treat two patients with R/R B-cell lymphomas.Results: CAR-T cells were prepared successfully, and they recognized CD19 antigen expressed on lymphoma cell lines specifically. Cell-line studies also showed that CD19 antigen expression was increased by DAC pretreatment, and the function of CAR-T cells was not compromised. The cell-line study further demonstrated that lymphoma cells pretreated by DAC responded more to the treatment of CAR-T cells. Two patients with R/R B cell lymphoma were pretreated with DAC then treated with CAR-T, and both achieved complete remission (CR).Conclusions: The epigenetic modifying drug DAC increases expression of the surface antigen CD19 on lymphoma cells. The DAC pretreatment protocol may lead patients with B cell lymphoma to be more susceptible to adoptive transfer of anti-CD19 CAR-T cells treKeywordsatment.