BAFF-R Aptamer/STAT3 siRNA Construct Selectively Downregulates STAT3 mRNA in BAFF-R Expressing Lymphomas in Vitro and in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 65-65
Author(s):  
Robert W. Chen ◽  
Pritsana Chomchan ◽  
Deepti Chadalavada ◽  
Jessica Alluin ◽  
jie-Hua Zhou ◽  
...  
Keyword(s):  
B Cell ◽  
Intratumoral Injection ◽  
Negative Control ◽  
Primary Lymphoma ◽  
Delivery Vehicle ◽  
Lymphoma Cells ◽  
Mouse Xenograft ◽  
Xenograft Models

Abstract Abstract 65 Background Specific oncogenes when overexpressed in lymphoma are associated with poor prognosis. Small interfering ribonucleic acids (siRNAs) can be custom designed to inhibit target oncogenes, but delivery of siRNA into lymphoma cells is a difficult task. Aptamers are synthetic in vitro selected nucleic acids that bind with high specificty to their target molecules. B cell activating factor (BAFF) enhances the maturation and survival of B cells via binding to its receptor, BAFF-R, which is expressed on the surface of B cell lymphoid malignancies. We have designed an aptamer against BAFF-R (R1) as a delivery vehicle for siRNA by covalently linking it to an siRNA molecule that inhibits oncogene target STAT3 (si-STAT3). We aim to downregulate STAT3 mRNA both in vitro (primary lymphoma cells) and in vivo (mouse xenograft models) with our aptamer/siRNA construct (R1-STAT3). Methods In vitro experiments used 2 mantle cell lymphoma cell (MCL) lines (Jeko-1, Z138), one diffuse large B cell lymphoma (DLBCL) cell line (Daudi), one negative control cell line (CEM, no BAFF-R expression), and primary lymphoid tumors. Primary lymphoid tumors were obtained from patients with at least 15% peripheral blood lymphoma involvement (chronic lymphoid leukemia-CLL, marginal zone lymphoma-MZL, and MCL). The selection and synthesis of aptamers used SELEX, gel shift, and filter binding assays. The visualization of aptamers utilized Z-axis confocal microscopy and Cy3 labeled aptamers. siRNA against STAT3 was generated using standard algorithms. Conjugation of aptamer to siRNA was done by two separate methods (chimera and stick, Zhou 2009). Downregulation of STAT3 was accessed by qRT-PCR and western blot analysis. We used NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice and MCL cell lines for in vivo mouse xenograft experiments. One million Z138 cells transduced with lentiviral vectors expressing luciferase were injected subcutaneously into the flanks of mice. Tumor engraftment was visualized by palpation and Xenogen imaging. Mice were then treated with R1 alone, R1-STAT3, and a negative control via intratumoral injection. Dosage and schedule of administration were consistent for all experimental groups. Mice were then sacrificed and subcutaneous tumors analyzed for STAT3 expression. We also injected one million Z138 cells transduced with a lentiviral vector expressing luciferase intravenously via tail and visualized engraftment by Xenogen imaging. Results Confocal microscopy showed easy entry of Cy3 labeled R1 aptamers and aptamer/STAT3 siRNA (R1-STAT3) conjugate into lymphoma cell lines (MCL and DLBCL), and primary lymphoma cells (CLL and MCL), but not into CEM cells. Intravenous injection of R1 showed biodistribution of R1 in lung/liver/spleen/BM and tumor site. qRT-PCR for R1 showed the highest concentration of R1 in tumor sites as compared to spleen/liver (4 log increase). These results show R1 aptamer can selectively target BAFF-R expressing lymphomas in vitro and in vivo. Table 1 shows R1-STAT3 conjugates downregulate STAT3 mRNA and STAT3 protein in MCL cells lines, but has no effect on CEM cells. Subcutaneous injection of MCL cells caused subcutaneous tumor formation which was easily palpable. We then performed Intratumoral injection of R1 and R1-STAT3 into subcutaneous tumors and analyzed STAT3 mRNA expression. Table 1 shows downregulation of STAT3 mRNA with R1-STAT3 as compared to R1 injection alone (83% downregulation). These results shows R1-STAT3 can efficiently downregulate STAT3 mRNA in vivo as well as in vitro. Conclusions BAFF-R functions as an efficient delivery vehicle for siRNA therapy in BAFF-R expressing B cell lymphomas. The aptamer/siRNA construct selectively and efficiently downregulates target oncogenes in primary lymphoma cells and in mouse xenograft models. Disclosures: No relevant conflicts of interest to declare.

scholarly journals P03.20 A murine, myc-driven lymphoma model expressing human CD22 enables testing of targeted therapies and their effects on tumor immune microenvironment

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A30.1-A30
Author(s):  
F Gsottberger ◽  
C Brandl ◽  
S Petkovic ◽  
L Nitschke ◽  
A Mackensen ◽  
...  
Keyword(s):  
Tumor Growth ◽  
B Cell ◽  
Targeted Therapies ◽  
Immune Cell ◽  
Myeloid Cells ◽  
B Cell Lymphoma ◽  
Primary Lymphoma ◽  
Murine Lymphoma

BackgroundThe tumor microenvironment (TME) is composed of various cell types which closely interact via cell cell contacts and cytokines leading to tumor promotion, immune cell inhibition and drug resistance. TME is increasingly recognized for its role in cancer immunotherapies. In B-cell malignancies, myeloid cells play a central role in supporting tumor growth and immune suppression (Roussel et al., 2017, Cancer Immunol Immunother). Despite the importance of a syngeneic TME, preclinical studies with novel drugs have mainly been performed in models lacking a functional immune system. Therefore, we developed an immune competent murine lymphoma model transgenic to human CD22 to study effects of targeted therapies on TME.Materials and MethodsA chimeric CD22 consisting of human extracellular and murine intracellular CD22 (h/mCD22) was introduced in BL6 mice (BL6h/mCD22). Crossbreeding with BL6λ-myc lead to spontaneous development of murine lymphoma that were serially transplanted. Tumor infiltration and TME was characterized by flow cytometry. Mice were treated with Moxetumomab pasudotox, a CD22 targeted immunotoxin and Doxorubicin.ResultsSpontaneously developed tumors in lymphoid organs from BL6h/mCD22 x λ-myc consist of a monomorphic population of h/mCD22+ murine B cells. Three primary lymphoma subclones were isolated from distinct mice and serially transplanted in syngeneic mice. Stable tumor growth was established after subcutaneous (sc) and intravenous (iv) injection. However, TME of sc tumors was infiltrated by less than 1% immune cells, while myc-driven lymphoma in humans usually show substantial immune infiltration. In contrast to sc tumors, systemically growing lymphoma in murine bone marrow (BM) are infiltrated by 30% myeloid cells and 1% T-cells and in murine spleen by 10% and 30%, respectively. Myeloid cells found in these tumors were shown to suppress T cell proliferation in vitro. To test functionality of the h/mCD22 transgene, lymphoma-bearing mice were treated with Moxetumomab, which reduced BM lymphoma infiltration by 20 to 100-fold and infiltration in spleen by 5 to 20-fold in the three lymphoma models. Effects of treatment on TME were analyzed after treatment with Doxorubicin which is known to activate myeloid cells in vivo. Compared to untreated controls, Doxorubicin increased CD11b+ cells in spleen by 1.5-fold. Among these cells, Ly6G+ granulocytic cells increased most substantially.ConclusionsWe established primary, myc-driven h/mCD22+ B-cell lymphoma which stably engraft in syngeneic mice with a TME mimicking myc-driven lymphoma in men. The model responds well to CD22-targeted therapy and Doxorubicin induces expected immunologic changes. Therefore, our unique model provides a platform to test CD22-targeting treatment strategies in an immune competent background.Disclosure InformationF. Gsottberger: None. C. Brandl: None. S. Petkovic: None. L. Nitschke: None. A. Mackensen: None. F. Müller: None.

A Fully Human Anti-CD40 Antagonistic Antibody, CHIR-12.12, Inhibit the Proliferation of Human B Cell Non-Hodgkin’s Lymphoma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3279-3279 ◽  
Author(s):  
Wen-Kai Weng ◽  
Xia Tong ◽  
Mohammad Luqman ◽  
Ronald Levy
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Expression Level ◽  
Primary Lymphoma ◽  
Immunoglobulin Gene ◽  
Dependent Manner ◽  
Lymphoma Cells

Abstract Immunotherapy using anti-tumor antibodies has become a feasible alternative for treating patients with lymphoma. These anti-tumor antibodies may target a specific receptor to disrupt proliferative signaling or mediate their anti-tumor effect by antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated killing. The CD40 antigen is a good target for such anti-tumor antibodies for several reasons: CD40 is expressed on the vast majority of the non-Hodgkin’s B cell lymphomas and it has been proposed that the CD40/CD40L interaction provides a critical survival or proliferative signal for B cell lymphoma, especially the low-grade follicular lymphoma. In addition, B lymphoma cell lines become less sensitive to chemotherapy-induced apoptosis after CD40 cross-linking in an in vitro study. Therefore, an anti-CD40 antagonist that disrupts the CD40/CD40L interaction and mediates effector mechanism could have a therapeutic advantage. In this report, we describe a fully human anti-CD40 antagonistic IgG1 monoclonal antibody, CHIR-12.12 that was generated from mice with a human immunoglobulin gene loci (XenoMouse®mice, Abgenix Inc.). We first compared the antigen expression level of CD40 to the level of CD20, the target for rituximab, on primary lymphoma cells. While the expression level of CD40 was similar between different samples of primary follicular lymphoma cells, it was 10 fold less than the level of CD20. The expression of CD40 and CD20 on chronic lymphocytic leukemia/small lymphocytic lymphoma cells (CLL/SLL) was more variable. However, the level of CD20 was still significantly higher than the level of CD40 in all samples tested (2.4 to 13 fold). While CHIR-12.12 binds to primary lymphoma cells similarly to several other anti-CD40 antibodies, CHIR-12.12 did not induce proliferation of these primary tumore cells. By contrast, an agonist anti-CD40 antibody induced proliferation of these lymphoma cells up to 6-fold over baseline. To study the ability of CHIR-12.12 to interrupt the CD40-CD40L interaction, we cultured lymphoma cells with CD40L-transfected feeder cells in the presence of control IgG1, CHIR-12.12 or rituximab. In this system, the lymphoma cells proliferate in response to CD40-CD40L interaction. The addition of rituximab did not influence the proliferation. However, CHIR-12.12 inhibited the proliferation of follicular lymphoma and of CLL/SLL cells in a dose-dependent manner. The inhibition was observed with antibody concentration at 1 μg/ml and reached maximum of 90% inhibition at 10 μg/ml. We then evaluated the ability of CHIR-12.12 to elicit complement-mediated killing or ADCC. In vitro, rituximab induced complement-mediated cytotoxicity, while CHIR-12.12 did not. However, both CHIR-12.12 and rituximab induced effective ADCC of primary follicular lymphoma cells using purified NK cells from a healthy donor. Even though the level of CD40 is 10-fold less than the level of CD20 on the cell surface of these tumor cells, CHIR-12.12 induced the same degree of ADCC killing as did rituximab. Thus, this novel antagonist CHIR-12.12 antibody both blocks tumor-stimulatory CD40/CD40L interaction and mediates ADCC in the presence of a low number of target antigen. Our results support further development of this antibody to treat patients with B cell lymphoma.

Pathologic Co-Expression and Physical Interaction of c-MYC and BCL6 in B-Cell Lymphomas.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2-2 ◽  
Author(s):  
Masumichi Saito ◽  
Ryan T. Phan ◽  
Herbert C. Morse ◽  
Laura Pasqualucci ◽  
Riccardo Dalla-Favera
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
Somatic Hypermutation ◽  
B Cell Lymphoma ◽  
Transgenic Animals ◽  
Primary Lymphoma ◽  
Physical Interaction

Abstract Deregulated expression of the proto-oncogenes BCL6 and c-MYC caused by chromosomal translocation or somatic hypermutation is common in non-Hodgkin B cell lymphoma derived from germinal center (GC) B cells, including diffuse large cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Normal GC B cells express BCL6, whereas, surprisingly, they do not express c-MYC, suggesting that the expression of this oncogene in BL and DLBCL (20% of cases) is ectopic (Klein, U. et al. Proc Natl Acad Sci U S A100, 2639–2644, 2003). Here we report that c-MYC is absent in proliferating GC B cells because it is transcriptionally suppressed by BCL6, as demonstrated by the presence of specific BCL6 binding sites in the c-MYC promoter region and by chromatin immunoprecipitation experiments showing that BCL6 is bound to these sites in vivo. Thus, c-MYC escapes BCL6-mediated suppression in lymphoma leading to the co-expression of the two transcription factors, an event never observed in immunohistochemical and gene expression profile analysis of normal GC B cells. Surprisingly, co-immunoprecipitation experiments and in vitro binding experiments indicate that, when co-expressed, BCL6 and c-MYC are physically bound in a novel complex detectable in DLBCL and BL cell lines as well as in primary lymphoma cases. The formation of the BCL6/c-MYC complex has several significant functional consequences on the function of both c-MYC and BCL6: 1) a two fold, BCL6-binding dependent increase in c-MYC half-life, an event that has been shown to contribute to its oncogenic activation; 2) a synergistic increase in the ability of both BCL6 and c-MYC to suppress MIZ1-activated transcription of the p21CIP cell cycle arrest gene; 3) MYC-dependent inhibition of BCL6 acetylation by p300, an event that physiologically inactivates BCL6 via c-MYC-mediated recruitment of HDAC. Notably, the pathologic co-expression of c-MYC and BCL6 was shown to have pathologic consequences in vivo, since double transgenic BCL6/c-MYC mice display accelerated lymphoma development and the appearance of a novel GC-derived tumor phenotype not recognizable in single transgenic animals and containing the pathologic c-MYC/BCL6 complex. Thus, the pathologic co-expression and illegitimate physical interaction of BCL6 and c-MYC leads to an increase in the constitutive activity of both oncogenes. These results identify a novel mechanism of oncogenic function for BCL6 and c-MYC and a novel tumor-specific protein complex of potential therapeutic interest.

Discrepancy of CD20 Protein Expression In IHC and FCM Analyses In Primary B-Cell Lymphoma: Relationship Between FCM-Negative Phenotype and Rituximab Binding with Lymphoma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5087-5087 ◽  
Author(s):  
Takashi Tokunaga ◽  
Akihiro Tomita ◽  
Kazuyuki Shimada ◽  
Junji Hiraga ◽  
Takumi Sugimoto ◽  
...  
Keyword(s):  
B Cell ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Primary Lymphoma ◽  
Newly Diagnosed ◽  
Rt Pcr ◽  
Lymphoma Cells ◽  
Anti Cd20

Abstract Abstract 5087 Background Rituximab is an anti-CD20 chimeric-monoclonal antibody, and its effectiveness for treatment of CD20-positive B-cell lymphomas has been proven over the past 10 years. Although rituximab is now a key molecular targeting drug for CD20-positive lymphomas, some patients with rituximab resistance have emerged. We previously reported that the CD20-protein-negative phenotypic change after using rituximab is one of the critical mechanisms in rituximab resistance (Hiraga J, Tomita A, et al., Blood, 2009., Sugimoto T, Tomita A, et al., Biochem Biophys Res Commun, 2009.). Recently, we have recognized that some newly-diagnosed B-cell lymphomas show CD20-protein-positive in immunohistochemistry (IHC) but -negative in flow cytometry (FCM) analyses. For these patients, so far, neither the molecular mechanisms of CD20 IHC(+)/FCM(−) phenotype, nor the relationship between this phenotype and rituximab resistance are clear. Thus, the clinical significance of introducing rituximab therapy for these patients must be elucidated. Aims Analyses of the molecular backgrounds of CD20 IHC(+)/FCM(−) phenotype in primary B-lymphoma cells, and confirmation of the effectiveness of rituximab therapy for the patients who show CD20 IHC(+)/FCM(−) phenotype. Results Primary B-cell lymphoma (diffuse large B-cell (DLBCL), follicular, MALT, mantle cell, and Burkitt) tissues and cells were analyzed by IHC and FCM. Four newly-diagnosed B-cell lymphoma patients showed IHC CD79(+)/CD20(+) and FCM CD19(+)/CD20(−) phenotype using anti-CD20 antibodies L26 for IHC and B1 for FCM, and all were diagnosed as DLBCL. Chromosomal analysis showed complex karyotypes in 3 out of 3 patients analyzed, and no shared abnormalities were confirmed. Primary lymphoma cells from 3 patients were available for further molecular analyses, and the genomic DNA, the total RNA, and the protein from whole cell lysate were obtained from these lymphoma cells. DNA sequencing analysis indicated no significant genetic mutations on the coding sequences (CDS) of MS4A1 (CD20) gene. Semi-quantitative and quantitative RT-PCR indicated that CD20 mRNA expression was almost normal in 2 patients and ≂~f10 times lower in 1 patient compared to the positive control B-lymphoma/leukemia cells. Almost the same expression tendency with RT-PCR was confirmed in immunoblot analysis using whole cell lysate and the two different anti-CD20 antibodies. The molecular weight of the CD20 protein in immunoblotting corresponded to the wild type in these patients. Rituximab binding assay in vitro was performed using primary lymphoma cells from a patient and the fluorescent-labeled rituximab (Alexa488-rituximab). Interestingly, rituximab binding on the surface of the CD19 positive lymphoma cells was confirmed in vitro. Rituximab containing combination chemotherapy was performed, resulting in complete response in all 4 cases after completing 4 to 8 courses. Conclusions and Discussion CD20 IHC(+)/FCM(−) phenotype was confirmed in newly-diagnosed DLBCL patients. Significant abnormalities in CD20 protein and mRNA expression in immunoblotting and RT-PCR were not confirmed, and genetic mutations on CDS of MS4A1 gene, resulting in the conformation change of CD20 protein, were not detected. The possibility of abnormal post-translational modification or aberrant localization of CD20 protein, leading to interference with antibody binding, can not be excluded. Rituximab binding with CD19-positive primary lymphoma cells was confirmed in a patient, suggesting that CD20 IHC(+)/FCM(-) phenotype does not directly indicate the ineffectiveness of rituximab for these cells. Further investigations, performing in vitro CDC and ADCC assay using primary lymphoma cells, are still warranted to show rituximab effectiveness and sensitivity to those cells. Disclosures: Kinoshita: Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Naoe:Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.

scholarly journals Paics Inhibition Is a Potential Therapeutic Strategy for MYC-Positive DLBCL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 396-396
Author(s):  
Kohta Miyawaki ◽  
Takuji Yamauchi ◽  
Takeshi Sugio ◽  
Kensuke Sasaki ◽  
Hiroaki Miyoshi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Poor Prognosis ◽  
De Novo ◽  
Lymphoma Cells ◽  
Purine Synthesis

Diffuse large B-cell lymphoma (DLBCL) is among the most common hematological malignancies with varying prognosis. As many as forty percent of patients eventually experience relapsed/refractory disease after combinatorial chemo-immunotherapies, R-CHOP, and prognosis after relapse is dismal. MYC is among the most established prognostic factors and associated with clinically-distinct subsets of DLBCL with poor prognosis: double-expressor lymphoma (DEL) and double-hit lymphoma (DHL). MYC is co-expressed with BCL2 in DEL, which consists of 60% of activated B-cell type DLBCL (ABC-DLBCL) cases, while DHL, defined by coexistence of MYC and BCL2/BCL6 rearrangements, were reportedly observed in 15% of germinal center B-cell like DLBCL (GCB-DLBCL). Considering that MYC-positive DLBCLs exhibit dismal outcomes, pharmacological inhibition of MYC activity is highly demanded; however, direct targeting of MYC has been proven challenging. Here we show that PAICS (phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase), which catalyzes a critical step in de novo purine synthesis, functions downstream of MYC in DLBCL cells. We further show MRT252040, a newly-developed PAICS inhibitor, effectively suppresses proliferation of MYC-driven DLBCL cells in vitro and in vivo. Through the nCounter-based transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) tissues from 170 untreated DLBCL patients, we found that MYC and PAICS were co-expressed and their mRNA levels were among the most predictive for poor prognosis after standard R-CHOP therapy. Their expression levels were particularly high in a subset of ABC-DLBCL and extranodal DLBCL, namely in DEL and DHL cases. Importantly, these findings were validated using three independent cohorts (Schmitz et al. NEJM, 2018). MYC and PAICS expression levels were high in most DLBCL lines and low in normal B cells in the lymph nodes, while they were variable in primary DLBCL tissues, revealed by nCounter and immunofluorescence. This trend was more evident in PAICS due presumably to active de novo purine biosynthesis in highly-proliferative cell lines and a subset of DLBCLs, including MYC-positive DLBCLs. These findings were also validated using the DepMap, a publicly-available genome-wide CRISPR/Cas9 dropout screen datasets. PAICS was among the top-ranked essential genes for the survival of DLBCL cell lines. Since co-expression of MYC and PAICS in a subset of DLBCL were indicative of a functional relationship between the two factors, we explored publicly-available ChIP-seq datasets to see if MYC directly regulates PAICS expression. As expected, MYC ChIP-seq signals were highly enriched near the PAICS promoter in a series of cancer cell lines. Furthermore, shRNA-mediated MYC knockdown led to reduced levels of PAICS mRNA in MYC-positive DLBCL cells and significantly slowed their growth. Collectively, these data suggest that PAICS is a direct transcriptional target of MYC, playing a key role in proliferation of MYC-positive DLBCL cells. To assess the feasibility of PAICS-inhibition as a therapeutic option for MYC-positive DLBCLs, we tested MRT252040 for its anti-lymphoma activity in vitro and in vivo. To do so, we first assessed cell cycle status and Annexin positivity upon MRT252040 treatment using a series of DLBCL cell lines. As expected, MRT252040-mediated PAICS inhibition induced cell cycle arrest and apoptosis. Furthermore, MRT252040 treatment significantly delayed proliferation of DLBCL cell lines, namely those harboring MYC rearrangements. Finally, to assess anti-lymphoma activity of MRT252040 in vivo, we tested MRT252040 efficacy using patient-derived xenograft DLBCL. After xenotransplantation, proportions of lymphoma cells per total mononuclear cells in peripheral blood were examined over time by FACS, and MRT252040 (or vehicle) treatment was initiated once lymphoma cells constituted >0.1%. MRT252040-treated mice survived significantly longer than vehicle-treated mice, indicative of therapeutic efficacy of MRT252040 monotherapy against DLBCL in vivo. Our data suggest that MYC regulates the de novo purine synthesis pathway via directly transactivating PAICS expression. We propose that MRT252040, a newly-developed PAICS inhibitor, warrants attention as a novel therapeutic approach for MYC-positive DLBCLs, which otherwise exhibit poor clinical outcomes. Disclosures Ohshima: SRL, Inc.: Consultancy; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Celgene Corp.: Honoraria, Research Funding; NEC Corp.: Research Funding. Akashi:Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

scholarly journals A Novel Fully Human Bispecific CD19 x CD3 Antibody That Kills Lymphoma Cells with Minimal Cytokine Secretion

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1671-1671
Author(s):  
Harbani Malik ◽  
Ben Buelow ◽  
Brian Avanzino ◽  
Aarti Balasubramani ◽  
Andrew Boudreau ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cytokine Secretion ◽  
Lymphoma Cells ◽  
Nog Mice

Abstract Introduction Along with CD20 and CD22, the restricted expression of CD19 to the B-cell lineage makes it an attractive target for the therapeutic treatment of B-cell malignancies. Many monoclonal antibodies and antibody drug conjugates specific to CD19 have been described, including bispecific T-cell redirecting antibodies (T-BsAbs). In addition, anti-CD19 chimeric antigen receptor T-cells (CAR-Ts) have been approved to treat leukemia. To date, toxicity from over-activation of T-cells and large-scale production of CAR-Ts still hinder this approach. Bispecific T-cell engaging antibodies redirecting T cells to CD19 circumvent the latter problem but to date have shown similar T-cell over-activation, as well as significant neurotoxicity. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline that uses in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αCD19 HCA and a library of αCD3 Flic antibodies that showed a >2 log range of EC50s for T cell activation in vitro. Of note, the library contained a selectively-activating αCD3 that induced potent T-cell dependent lysis of lymphoma cells (when paired with an αCD19 HCA) with minimal cytokine secretion. To characterize the relative efficacy and potential therapeutic window of this unique molecule, we compared the low-activating (and Fc-containing) CD19 x CD3 to two pan T-cell activating bispecific CD19 x CD3 antibodies (blinatumomab and another developed in-house) in vitro and in vivo for T-cell activation, efficacy in killing lymphoma cells, and toxicity. Methods T-cell activation was measured via flow cytometry (CD69 and CD25 expression) and cytokine ELISA (IL-2, IL-6, IL-10, INF-ɣ, and TNFα) in vitro. Lysis of B-cell tumor cell lines (Raji, Ramos, and Nalm6) was measured via calcein release in vitro. In vivo, NOG mice were engrafted with human peripheral blood mononuclear cells (huPBMC) and human lymphoma cell lines, and the mice treated with weekly injections of T-BsAbs. Tumor burden was evaluated via caliper measurement. Pharmacokinetic (PK) studies were performed in NOG mice using ELISA. Results EC50s for cytotoxicity were in the single-digit nanomolar range for the selective T cell activating T-BsAb and sub-nanomolar for the pan T-cell activating controls. The selective T cell activator showed markedly reduced cytokine release for all cytokines tested compared to the pan T-cell controls even at saturating concentrations. In vivo, established CD19 positive B-cell tumors were cleared in NOG mice in the presence of huPBMC. PK profiles of both molecules generated in-house (selective and pan T-cell activators) were consistent with those of an IgG in mice. No activation of T-cells was observed in vitro or in vivo in the absence of CD19 expressing target cells. Conclusions Both the selectively-activating and the pan T-cell activating control bispecific antibodies killed lymphoma cells in vitro and in vivo in a CD19-dependent manner. While the pan T-cell activating controls showed T-cell activation comparable to other CD3-engaging bispecifics, the selective activator induced significantly reduced cytokine secretion by T-cells and demonstrated a half-life consistent with other IgG antibodies. In summary, our selectively activating CD19 x CD3 T-BsAb shows promise as a lymphoma therapeutic differentiated from current T-cell targeted therapies currently in the clinic and in clinical trials. Disclosures Malik: Teneobio, Inc.: Employment. Buelow:Teneobio Inc.: Employment. Avanzino:Teneobio, Inc.: Employment. Balasubramani:Teneobio, Inc.: Employment. Boudreau:Teneobio, Inc.: Employment. Clarke:Teneobio, Inc.: Employment. Dang:Teneobio, Inc.: Employment. Davison:Teneobio, Inc.: Employment. Force Aldred:Teneobio Inc.: Employment. Harris:Teneobio, Inc.: Employment. Jorgensen:Teneobio, Inc.: Employment. Li:Teneobio, Inc.: Employment. Medlari:Teneobio, Inc.: Employment. Narayan:Teneobio, Inc.: Employment. Ogana:Teneobio, Inc.: Employment. Pham:Teneobio Inc.: Employment. Prabhakar:Teneobio, Inc.: Employment. Rangaswamy:Teneobio, Inc.: Employment. Sankaran:Teneobio, Inc.: Employment. Schellenberger:Teneobio, Inc.: Employment. Ugamraj:Teneobio, Inc.: Employment. Trinklein:Teneobio, Inc.: Employment. Van Schooten:Teneobio, Inc.: Employment.

scholarly journals A novel Bcl-2 inhibitor, BM-1197, Induces apoptosis in Malignant Lymphoma Cells through the Endogenous Apoptotic Pathway

2019 ◽  
Author(s):  
Yue-Li Sun ◽  
Wen-Qi Jiang ◽  
Qiu-Yun Luo ◽  
Da-Jun Yang ◽  
Yu-Chen Cai ◽  
...  
Keyword(s):  
Malignant Lymphoma ◽  
Caspase 3 ◽  
Caspase 9 ◽  
Antitumor Effects ◽  
Lymphoma Cells ◽  
Dual Inhibitor ◽  
Apoptosis Pathway ◽  
Xenograft Models

Abstract Background: Bcl-2 family members play an important role in the development of malignant lymphoma and can induce drug resistance in anticancer treatment. The development of small molecules targeting Bcl-2 family protein can be new strategy for malignant lymphoma treatment. In this study, we investigate the antitumor effect and the cellular mechanism of a novel Bcl-2/Bcl-xL dual inhibitor BM-1197 in DCBCL and Burkitt lymphoma cells. Methods: CCK-8 assay was used to detect cell viability. Apoptosis was determined by Hoechst 33258 staining and flow cytometry. The activity of caspase-3/caspase-9 was determined using the caspase-3/ caspase-9 activity kit. Western blotting analysis was performed to evaluate the change of protein expression. The functional analysis was evalueated via immunoprecipitation and siRNA interference. Human malignant lymphoma xenograft models in nude mice were established for in vivo efficacy detection. Results: We find that BM-1197 exerts potent growth-inhibitory activity against lymphoma cells which harbor Bcl-2 and Bcl-xL high expression in vitro and has synergistic effect with chemotherapeutic drugs. Mechanistically, we see that the intrinsic apoptosis pathway is activated upon BM-1197 treatment. BM-1197 affects the protein interaction of Bak/Bcl-xl, Bim/Bcl-2, Bim/Bcl-xl, PUMA/Bcl-2 and induced conformational change in the Bax protein.which results in activation of Bax and release cytochrome c, and activated caspase -9, -3, -7 and finally induce cell apoptosis. Furthermore, our data demonstrates that BM-1197 exhibits strong anti-tumor effects against established human malignant lymphoma xenograft models. Conclusions: Our study demonstrated BM-1197 exerts potent antitumor effects both in vitro and in vivo, and provides promising preclinical data for further development of BM-1197 in malignant lymphoma.

Novel Fc-Engineered Antibodies to CD37 with Pro-Apoptotic and Enhanced ADCC Activity

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3916-3916 ◽  
Author(s):  
Karl-Heinz Heider ◽  
Elinborg Ostermann ◽  
Herbert Lamche ◽  
Zaruhi Kuepcue ◽  
Alexander Jacobi ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Burkitt Lymphoma ◽  
Apoptosis Induction ◽  
Adcc Activity ◽  
Lymphoma Cells ◽  
Parental Antibody ◽  
Apoptotic Activity

Abstract Abstract 3916 Introduction: Treatment of B-cell malignancies with rituximab, an antibody specific for the B-cell antigen CD20, in combination with chemotherapy has been established as standard clinical practice during recent years. Despite the impressive clinical benefit of rituximab-chemotherapy combination treatment, there remains a substantial need for improved treatment modalities. Here we describe two novel Fc-engineered antibodies to CD37, mAb 37.1 and mAb 37.2, which mediate potent pro-apoptotic and ADCC activity against malignant B-cells. The tetraspanin CD37 is predominantly expressed on the cell surface of normal and malignant B-cells and therefore qualifies as target for antibody mediated tumor therapy. Methods: MAb 37.1 is an Fc-engineered, mouse-human chimeric IgG1-type of antibody which binds CD37 with low nanomolar affinity as determined by FACS scatchard analysis. MAb 37.2 is a humanized version derived from mAb 37.1. Affinity to Fc-receptors was determined by BIAcore analysis. ADCC and pro-apoptotic activity was determined using Ramos Burkitt lymphoma cells and primary CLL cells. ADCC assays were performed using PBMCs from healthy donors as effector cells. Apoptosis induction was determined by AnnexinV/PI staining. The effect in blood from healthy individuals was assessed in whole blood assays ex vivo. In vivo anti-tumor efficacy was assessed in a Ramos xenograft model in immuno-compromised mice. Results: Both CD37 antibodies are able to induce apoptosis of Ramos Burkitt lymphoma cells and primary CLL cells in vitro. Direct comparison of mAb 37.1 to Rituximab demonstrated superior apoptosis induction both on Ramos and primary CLL cells. The pro-apoptotic activity of mAb 37.1 is not dependent on cross-linking of the antibody, representing a novel mode of CD37-specific apoptosis induction. Phenotypic analysis of mAb 37.1 treated Ramos cells revealed early aggregation of tumor cells resembling homotypic aggregation. In order to further improve the cytotoxic potential of the antibodies an Fc-engineering approach was chosen to enhance binding to FcgRIIIa. By applying a two amino acid substitution in the Fc-region of the antibody the affinities of mAb 37.1 and mAb 37.2 to human FcgRIIIa was increased by 40 to 80-fold, depending on the FcgRIIIa allotype, as compared to the parental antibody. This increase directly translates into a strongly enhanced ADCC activity compared to the parental antibody as detected in an in vitro ADCC assay with Ramos and primary CLL cells, resulting in superior ADCC activity compared to Rituximab. The potent B-cell specific depleting activity of mAb 37.1 and mAb 37.2 was confirmed with naïve B-cells and spiked Ramos Burkitt lymphoma cells in vitro in whole blood conditions, whereas no significant effects on T-cells and monocytes were observed. Rituximab, which was used as comparator antibody in parallel, showed no significant reduction of viable B-cells in this assay format. Finally, the in vivo anti-tumor effect of CD37 mAbs was assessed using a Ramos xenograft. Treatment of established subcutaneous tumors with doses ranging from 2.5 to 25 mg/kg in a twice weekly treatment schedule resulted in tumor growth inhibition up to 100%. Conclusion: MAb 37.1 and mAb 37.2 represent novel, Fc-engineered antibodies specific for CD37 with potent pro-apoptotic and enhanced ADCC activity against lymphoma cell lines and primary CLL cells. Based on these data mAb 37.1 and mAb 37.2 are considered as promising candidates for treatment of patients with CD37-positive B-cell malignancies. Disclosures: Heider: Boehringer Ingelheim: Employment. Ostermann:Boehringer Ingelheim: Employment. Lamche:Boehringer Ingelheim: Employment. Kuepcue:Boehringer Ingelheim: Employment. Jacobi:Boehringer Ingelheim: Employment. Konopitzky:Boehringer Ingelheim: Employment. Hirt:Boehringer Ingelheim: Employment. Reichardt:Boehringer Ingelheim: Employment. Borges:Boehringer Ingelheim: Employment.

Cross-Species Investigations Reveal Role, Mechanism and Predictive Power of Paracrine, Secondary Senescence in B-Cell Lymphoma Therapy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3715-3715
Author(s):  
Jan R. Dörr ◽  
Maja Milanovic ◽  
Yong Yu ◽  
Julia Kase ◽  
Dido Lenze ◽  
...  
Keyword(s):  
B Cell ◽  
Predictive Power ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Brdu Incorporation ◽  
Lymphoma Cells ◽  
Long Term Outcome

Abstract Abstract 3715 Apoptosis and cellular senescence operate as anti-tumor safeguard mechanisms. Unlike apoptotic cells, senescent cells remain viable, and, hence, may crosstalk to other cells in their vicinity over extended periods of time. In fact, cells that entered oncogene-induced senescence or anticancer therapy-induced senescence (TIS) present with a senescence-associated secretory phenotype (SASP), a massive production of secretable factors, which reportedly reinforces senescence through an intracellular mechanism. Utilizing the Eμ-myc transgenic mouse lymphoma model, we provide evidence for an outcome-relevant paracrine, DNA damage-independent secondary senescence program (SecS) in vitro and in vivo. Apoptosis-blocked (bcl2-infected) lymphoma cells from different genetic backgrounds were treated with the DNA-damaging anticancer agent adriamycin in vitro or the alkylating agent cyclophosphamide upon lymphoma formation in mice in vivo. TIS and SecS was detected based on senescence-associated b-galactosidase activity (SA-b-gal), Ki67 staining and BrdU incorporation. The secretome of senescent cells was analyzed by proteomics, gene expression and protein arrays. Overall and progression free survival in mice and patients was assessed by Kaplan-Meier analysis. Transcriptome and secretome analyses followed by functional studies found extracellular matrix proteins, especially small leucine-rich proteoglycans (SLRP), but not NF-kB-dependent cytokines and chemokines, to induce SecS in proliferating lymphoma cells in a paracrine fashion, and linked a “high secretor” status to stronger SecS induction. Dissecting senescence-mediating pathways in recipient cells by biochemical, genetic and pharmacological means unveiled an essential role for the LDL receptor-related protein 1 (LRP1), a receptor for SLRP and other SASP components, through the cell-cycle inhibitor p21CIP1 in SecS. Accordingly, mice harboring TIS-capable but genetically SecS-defective lymphomas (e.g. lacking LRP1 or p21CIP1 expression) experienced inferior long-term outcome to therapy. Not only the recipient cell-based LRP1 status but also the genetically and biologically distinct donor cell-based secretor gene signature stratified outcome in mice. Strikingly, humanized versions of both classifiers were predictive in a large cohort of diffuse large B-cell lymphoma (DLBCL) patients, where they identified – although composed of different gene sets – largely overlapping patient subgroups with superior prognosis, again suggesting SecS as the critical underlying treatment effector principle. Our study highlights the predictive power of senescence for treatment outcome in DLBCL, and provides functional examples (which will be discussed at the meeting) for SASP-related non-genotoxic pro-senescent therapies. Disclosures: No relevant conflicts of interest to declare.

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