bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multivalent Anti-CD20/Anti-CD22 Bispecific Antibody Fusion Proteins Made by the DNL Method Show Potent Lymphoma Cytotoxicity.
Authors: Rossi Edmund A. , Losman Michele J. , Nordstrom Diane L. , Cardillo Thomas , Loo Meiyu , Kopinski John , Qu Zhengxing , Goldenberg David M. , Chang Chien-Hsing 
Published: October 2019

Abstract
Purpose: Multivalent and either monospecific or bispecific antibodies have shown the potential for improved efficacy in a number of preclinical studies. A panel of such agents based on hLL2 (epratuzumab, anti-CD22) and hA20 (humanized anti-CD20) was made and evaluated for cytotoxicity in vitro and anti-tumor activity in vivo.
Methods: The Dock and Lock (DNL) method (Rossi et al., Proc. Natl. Acad. Sci. USA, 2006,103:6841) is a novel platform technology for the site-specific and covalent assembly of modular components. DNL was utilized to generate two hexavalent monospecific (hex-hA20 and hex-hLL2), and two hexavalent bispecific (DNL-1 and DNL-2) stably tethered complexes, each composed of four Fab fragments conjugated to an IgG at the latter’s carboxyl termini of the heavy chain. DNL-1 consists of four hA20 Fab fragments tethered to an hLL2 IgG. DNL-2 consists of four hLL2 Fab fragments tethered to an hA20 IgG.
Results: For each construct, the DNL method produced a single, defined, homogeneous structure that is stable in serum. All of the constituent Fab fragments are functional, with binding affinities similar to the parental MAbs. In vitro analyses using human Burkitt lymphoma cell lines demonstrate that both the monospecific and bispecific constructs induce strong cell adhesion and potently inhibit cell growth without the requirements for further cross-linking. DNL-1, DNL-2 and hex-hA20 each completely inhibited the growth of cultured Daudi cells at concentrations as low as 1 nM and exhibited two to three logs greater potency than that of hA20 IgG or rituximab under the same experimental conditions. Anti-tumor efficacy was demonstrated in SCID mice bearing disseminated Daudi lymphoma. Treatment with 4 μg of hex-hA20 or DNL-2 at 1, 4 and 7 days post intravenous injection of 1 × 107 Daudi cells resulted in significantly increased survival of tumor-bearing mice compared to controls treated with saline.
Conclusion: These findings suggest that the DNL method can be used to make multivalent proteins with potent anti-tumor activity, as shown here with anti-CD20/anti-CD22 bispecific antibody fusion proteins.
The Humanized Anti-CD40 Antibody, SGN-40, Promotes Apoptosis Signaling and Is Effective in Combination with Standard Therapies in Lymphoma Xenograft Models.
Authors: Lewis Timothy S. , Sutherland May S.K. , Jonas Mechthild , Cerveny Charles G. , McCormick Renee , Wahl Alan F. , Drachman Jonathan G. , Grewal Iqbal S. , Law Che-Leung 
Published: October 2019

Abstract
CD40 is a TNF receptor family member that is expressed on B cells and by a wide variety of transformed cells including non-Hodgkin’s lymphoma (NHL), multiple myeloma, and various solid tumors. The humanized anti-CD40 antibody, SGN-40, is a partial agonist that induces apoptosis as well as mediates ADCC against CD40+ NHL B cell lines, contributing to in vivo antitumor activity observed in human lymphoma xenograft models. The current study demonstrates the ability of SGN-40 to initiate multiple signaling cascades upon ligation of CD40 on NHL cell lines. SGN-40 was shown to activate the stress-induced p38 MAP kinase and pro-survival pathways including NF-κB, p42/44 MAP kinase and, to a lesser extent, AKT. Consistent with the apoptosis-inducing activity of SGN-40, cleavage of caspase-3 and its downstream substrate poly (ADP-ribose) polymerase was detected in NHL cell lines. SGN-40 signaling was qualitatively similar to that mediated by trimeric recombinant human CD40 ligand (rhCD40L). However, the overall magnitude of signaling was lower with SGN-40 compared to rhCD40L, consistent with the partial agonistic properties of SGN-40. In addition, constitutive phospho-AKT levels, a key pro-survival signal, were found to be very low in most high-grade lymphoma cell lines and primary NHL specimens, in contrast to the high levels reported in carcinomas. Low AKT activity may bias lymphoma cells toward apoptosis in response to SGN-40 signaling. To augment SGN-40-induced cell killing, in vitro combination studies with chemotherapeutics have been performed using the Ramos, RL, and HT NHL lines. We now report that SGN-40 has additive activity when combined with cisplatin, melphalan, or mitoxantrone and is synergistic with bleomycin. Furthermore, in vivo activity of combination therapy has been demonstrated in a subcutaneous Ramos lymphoma xenograft model. While tumor growth was delayed by either CHOP (cyclophosphamide, adriamycin, vincristine, prednisone) or SGN-40 alone, the combination of SGN-40 and CHOP was significantly more active. Our results suggest that SGN-40 can be combined with standard lymphoma therapies resulting in improved therapeutic efficacy, and provide a rationale for combination clinical trials involving SGN-40. The molecular mechanisms through which standard chemotherapeutic agents enhance SGN-40-mediated cell killing in target lymphoma B cells are currently being investigated.
HCD122, an Antagonist Human Anti-CD40 Monoclonal Antibody, Inhibits Tumor Growth in Xenograft Models of Human Diffuse Large B-Cell Lymphoma, a Subset of Non-Hodgkins Lymphoma.
Authors: Hsu Ssucheng J. , Esposito Lin A. , Aukerman Sharon L. , Kantak Seema , Mirza Amer M. 
Published: October 2019

Abstract
CD40, a member of the tumor necrosis factor receptor family, is expressed in all human B-cell malignancies and engagement by the CD40 ligand (CD40L) is important for both cell proliferation and cell survival. CD40L has been shown to be co-expressed with CD40 in neoplastic B-cells from Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkins Lymphoma (NHL), suggesting the importance of an autocrine CD40/CD40L loop in these malignancies. HCD122 (formerly known as CHIR-12.12) is a fully human, highly potent, IgG1 antagonist anti-CD40 monoclonal antibody (mAb) that blocks CD40/CD40L interactions in vitro and also mediates ADCC. Previous studies showed that HCD122 can mediate ADCC in vitro and has anti-proliferative and anti-tumor activities as a single agent in CLL, MM, and Burkitts Lymphoma in vitro and in vivo. In this study, the activity of HCD122 on a subtype of NHL, Diffuse Large B-Cell Lymphoma (DLBCL) was examined. The DLBCL derived cell lines, RL and SU-DHL-4, were selected for this study based upon in vivo characterization as well as their sensitivity to Rituximab as reported in the literature. These cell lines were subsequently confirmed for the expression of CD40 and CD20 by flow cytometry. The in vivo anti-tumor effects of HCD122 as single agent was demonstrated in these two xenograft models and was compared to Rituximab, an anti-CD20 antibody therapeutic currently approved for the treatment of relapsed or refractory, low-grade or follicular, NHL. HCD122 when administered intraperitoneally weekly at 1 mg/kg significantly reduced tumor growth with a tumor growth inhibition (TGI) of 85.5% (P<0.01) in the RL model. At the same dose and schedule in the RL model, TGI achieved with Rituximab was 31.7% (P>0.05). In the SU-DHL-4 model, an 85% TGI (P<0.01) was observed at the 1 mg/kg dose of HCD122. In comparison, Rituximab at this dose elicited a 57.6% TGI (P<0.05). Additionally, the downstream CD40/CD40L signal transduction pathways were also examined in order to elucidate the molecular mechanism underlying the HCD122-mediated effects in DLBCL. Taken together, these results support the clinical development of HCD122 for the treatment of DLBCL. Currently HCD122 is in Phase I trials for treatment of CLL and MM.
In Vitro Activity of SYK and BCR-ABL Inhibitors in Aggressive Lymphomas.
Authors: Bertoni Francesco , Rinaldi Andrea , Sasso Anna , Gaidano Gianluca , Zucca Emanuele , Uccella Silvia , Pruneri Giancarlo , Ponzoni Maurilio , Tibiletti Maria Grazia , Catapano Carlo , Cavalli Franco 
Published: October 2019

Abstract
The B cell receptor tyrosine kinase SYK is a critical component of the B-cell receptor signalling pathway in normal B cells. We have recently reported that SYK is amplified and over-expressed in mantle cell lymphoma (MCL) and that the growth of MCL and diffuse large B cell lymphoma (DLBCL) cell lines over-expressing SYK is inhibited by piceatannol, a known SYK inhibitor (Bertoni et al, ASH 2005; Rinaldi et al, BJH 2006). Others have reported important SYK expression in splenic marginal zone B cell lymphomas, in DLBCL and peripheral T cell lymphomas (PTCL) (Ruiz-Ballesteros et al, 2005; Mahadevan et al, w Streubel et al, 2006), suggesting SYK targeting agents could be useful for the treatment of various lymphoma subtypes. SYK inhibitors are already in clinical development for treatment of asthma. Here, we report on the activity on lymphoma cell lines and primary cells of the SYK/ZAP-70 inhibitor #1 (Novartis) and of the BCR-ABL inhibitors imatinib (Novartis) and nilotinib (Novartis), which could act as cross-selecting SYK inhibitors (Atwell et al, 2004).
We treated four human MCL and three DLBCL established cell lines with increasing doses of the SYK/ZAP-70 inhibitor #1, imatinib and nilotinib for 72 h. Cell viability was measured with the MTT assay. The two cell lines expressing high levels of SYK, JeKo-1 and SUD-HL-6, were sensitive to the compounds (IC50: Syk/ZAP-70 inhibitor #1, 1–5 μM; imatinib, 15–20 μM; nilotinib, 10 μM). Cells with lower SYK expression were generally less sensitive to all three compounds. To obtain further data on the relevance of SYK inhibition in lymphoma, we treated nine lymphoma primary cells with the piceatannol (Sigma), the SYK/ZAP-70 inhibitor #1 and nilotinib. Responses, defined as <50% decrease in viable cell number, were observed with piceatannol (4/9 samples), the Syk/ZAP-70 inhibitor #1 (3/9 samples) and nilotinib (3/9 samples). Immunoblotting experiments aimed to elucidate the mechanism of action are under-way and data will be presented at the meeting.
In conclusion, our data indicate that pharmacological inhibition of SYK is a therapeutic approach to be further investigated in subsets of aggressive lymphomas.
An Fc-Optimized Monoclonal Antibody Against CD32B, the Human Inhibitory Fc-γ Receptor IIB, in the Immunotherapy of B-Cell Malignanacy.
Authors: Stavenhagen Jeffrey B. , Gorlatov Sergey , Tuaillon Nadine , Rankin Christopher T. , Veri Maria-Concetta , Inzunza David , Burke Stephen , Huang Ling , Li Hua , Thomas Shannon , Johnson Syd , Ravetch Jeffrey , Koenig Scott , Bonvini Ezio 
Published: October 2019

Abstract
Monoclonal antibodies (mAbs) are widely used in the treatment of lymphoma and autoimmune diseases. The activity of several mAbs depends on the binding of the Fcγ regions to low-affinity Fcγ receptors (FcγR) expressed on effector cells, chiefly mononuclear phagocytes, cells that express both activating and inhibitory FcγRs. FcγRIIB (CD32B), the inhibitory FcγR, is expressed by normal and malignant B lymphocytes and is itself an immunotherapeutic target. A recently characterized anti-human CD32B-specific mAb (2B6) was Fc engineered to customize the FcγR binding profile for increased binding to the activating receptor FcγRIIIA (CD16A), resulting in an improved activating-to-inhibitory binding ratio. Fc-optimized versions of 2B6 showed enhanced cytotoxicity in vitro and increased potency in animal tumor models, including transgenic mice harboring the human low-affinity allele of the activating receptor, CD16A-158phe. The enhancement in tumor depletion correlated with an increased binding to FcγRIV, a mouse functional homolog to human CD16A. The expression of FcγRIV is limited to monocyte and macrophages, consistent with the role of these cells in tumor cell killing in vivo. These data support CD32B as a target for immunotherapy of B cell lymphoproliferative disorders and Fc optimization to increase the activating-to-inhibitory FcγR-binding ratio as a means to designing improved antibodies.
MLL-AF9 Initiates Leukemia from a Single Committed Hematopoietic Progenitor.
Authors: Krivtsov Andrei V. , Stubbs Matthew C. , Wright Renee , Feng Zhaohui , Kung Andrew L. , Armstrong Scott A. 
Published: October 2019

Abstract
Recent experiments have demonstrated that MLL-translocation associated fusion proteins can transform either hematopoietic stem cells (HSC) or granulocyte macrophage progenitors (GMP) into leukemia stem cells. However, it may be that leukemogenic process differs when HSC are the cell of origin as compared to myeloid progenitors. We transduced either HSC or GMP with a retrovirus expressing MLL-AF9 and GFP followed by single cell sorting of transduced cells. Approximately 80% of singly sorted MLL-AF9 transduced GMP (MLL-AF9-GMP) and about 25% of MLL-AF9 transduced HSC (MLL-AF9-HSC) could be serially re-plated over 9 passages. Upon transplantation into syngeneic mice, 83% (n=12) of MLL-AF9-HSC single cell derived clones induced AML with a median latency 70 days. Approximately 30% (n=20) of MLL-AF9-GMP single cell derived clones induced AML, with median latency 112 days. When MLL-AF9-GMP single cell derived clones were co-infected with an empty retrovirus (to provide additional oncogenic events as a result of retroviral integration) before transplantation into recipient mice, 93% of the transplanted mice (n=15) developed AML with mean latency 65 days, similar to leukemia initiated from HSC. This suggests that either single GMP or HSC can be transformed into leukemia initiating cells. However, extra mutations appear to be required to induce leukemia from committed progenitors. Consistent with this hypothesis, southern blot analysis performed on leukemias initiated from 5,000 and 15,000 MLL-AF9 transduced HSC or GMP demonstrated polyclonal AML arising from HSC compared to oligoclonal AML arising from GMP. Next, we used bioluminescent imaging to follow disease kinetics. When 15,000 MLL-AF9 transduced HSC were injected into recipient mice, the disease accumulated in a linear fashion over 42 days. However, when 15,000 MLL-AF9 transduced GMP were injected the disease developed more slowly over 75 days. Immunophenotypic analysis of the resultant leukemias demonstrated that the HSC-derived and GMP-derived leukemias were quite similar, with a GMP-like population containing LSC in both cases. Globally, the two cell types were also very similar with their gene expression profile being more similar to GMP than any other progenitor or stem cell population. However, we found that in addition to the previously reported 363-gene “self-renewal associated signature” LSC derived from HSC also possessed high-level expression of genes such as Flt3, Mcl1, and Notch-1. Preliminary analysis also suggests that gene expression differences between HSC and GMP-derived leukemia stem cells may have prognostic significance in human AML. These data suggest that AML derived from different cells of origin, while globally quite similar, require a different number of genetic events, and have gene expression differences that may influence drug response.
CD34+CD19−, CD34+CD19+ and CD34−CD19+ Cells May All Have Leukemia-Initiating Potential in NOD/Scid Mice.
Authors: Le Viseur Christoph , Hotfilder Marc , Rosemann Annegret , Stam Ronald , Schrauder Andre , Shultz Leonard , Schrappe Martin , Pieters Rob , Vormoor Josef 
Published: October 2019

Abstract
Current data on the leukemic stem cell (LSC) compartment in childhood acute lymphoblastic leukemia (ALL) are conflicting. The traditional hypothesis supposed that childhood ALL originates in a lymphoid progenitor cell and this is assumed to be consistent with the overall good treatment responses in pediatric patients. In accordance with this hypothesis, our previous studies failed to detect involvement of immature CD34+CD19− progenitor cells in ALL/t(12;21) (Hotfilder et al., Blood 2002) while high-risk ALL/t(9;22) and t(4;11) appears to originate in a more primitive CD34+CD19− cell (Hotfilder et al., Cancer Res 2005). In order to characterize the leukemia-initiating cell in vivo, we established a mouse xenograft model by serial intrafemoral transplantation of NOD/scid mice with flow sorted subpopulations from childhood ALL. Samples were taken from the bone marrow of children with ALL/t(12;21) (n=1), t(4;11) (n=3) and t(11;19) (n=1) and B-cell precursor ALL without a marker translocation (n=2). Primary transplantations were performed with freshly thawed unsorted cells, followed by secondary, tertiary and quaternary transplantations with flow sorted populations. Human leukemic engraftment was defined by a proportion of >5% human CD45+ cells in the murine bone marrow that simultaneously express CD34 and/or CD19. From the bone marrow of leukemic mice, we isolated different leukemic populations and successfully re-transplanted 2×103 − 1×105 CD34+CD19− cells, 2×104 − 6×106 CD34+CD19+ lymphoid progenitors and 3×104 − 2×106 more differentiated CD34−CD19+ blasts onto secondary, tertiary and quaternary mice (average purity after flow sorting: >96%). So far, we detected leukemic engraftment in 60 of 161 (37%) transplanted mice (with many mice - having only recently been transplanted - still alive). These include 7 of 36 (19%) mice engrafted with CD34+CD19− cells, 33 of 72 (46%) mice engrafted with CD34+CD19+ cells and 20 of 53 (38%) mice engrafted with CD34−CD19+ cells. With as few as 2 × 103 CD34+CD19− cells being sufficient to re-initiate the leukemia, this intrafemoral ALL-NOD/scid mouse model represents a very sensitive functional assay for candidate LSC in childhood ALL. We have initiated limiting dilution experiments with the different subpopulations to quantify LSC frequency in the different compartments and to exclude that low levels of contaminating blasts with an immunophenotype different from the main transplanted cell population blurred the results. We are also currently investigating whether there is heterogeneity in the CD34+CD19− compartment in respect to standard and high-risk ALL. Altogether, our data indicate that all three subpopulations, CD34+CD19−, CD34+CD19+ and CD34−CD19+ cells, may have the capacity to transfer the leukemia onto NOD/scid mice and that lymphatic LSC may not loose their self-renewal potential with differentiation.
Immortalization of Human HSPC by a Self-Renewal Signal Coupled with Telomere Maintenance.
Authors: Wei Junping , Wunderlich Mark , Krejci Ondrej , Alvarez Sara , Cigudosa Juan , Mulloy James C. 
Published: October 2019

Abstract
Expression of the leukemia-associated fusion protein AML1-ETO (AE) in human hematopoietic stem and progenitor cells (HSPC) results in long-term cytokine-dependent cells that maintain CD34 expression, multipotential differentiation and the ability to engraft NOD/SCID mice. These cells gradually lose telomere length and are not immortal. To determine whether expression of telomerase would cooperate with AE in immortalization of human HSPC, we transduced AE-expressing CD34+ cells with the telomerase gene, hTERT. In four independent AE cell cultures, hTERT expression allowed the cells to proliferate beyond the crisis point. Cells retained B lymphoid, myeloid and erythroid differentiation potential, displayed continuous replating ability in methylcellulose assays and engrafted in NOD/SCID mice. The hTERT-expressing cells had a significant increase in progenitor cell numbers, more cells in S phase, and increased resistance to gamma radiation-induced apoptosis compared to control cells. No genetic changes were identified by G-banding or comparative genomic hybridization during the passage through crisis, implying that these signals are sufficient for the immortalization. Similar results were obtained using a long-term culture established with the related core binding factor (CBF) fusion gene CBFβ-MYH11 (CM). By microarray analysis we identified a number of genes implicated in stem cell self-renewal, including HOXB4, GATA3 and STAT3, that are upregulated in CBF-expressing cells when compared to control CD34+ cells. Ectopic hTERT expression did not affect the level of expression of these self-renewal genes. We hypothesize that the continual rescue of progenitor cells during culture, as a result of telomerase maintenance of telomeres, is responsible for the proliferation and survival phenotype observed in hTERT expressing AE and CM cells. This is supported by our findings that control cultures contained significantly more DNA damage foci as detected by immunofluorescence staining for phosphorylated H2AX. In addition, signal-free chromosome ends were significantly more abundant in control cultures as demonstrated by a telomeric FISH probe when compared to the hTERT cultures.
Senescence-associated β-gal staining was prominent in control cells as they slowed proliferation after 7 months of growth, but hTERT-expressing cells did not display SA-β-gal staining and continued proliferating indefinitely, for over two years at this point in time. We conclude that the self-renewal signal supplied by a CBF oncogene cooperates with telomere maintenance initiated by hTERT for the immortalization of human HSPC. This system permits a molecular dissection of the signals involved in HSPC self-renewal and leukemogenesis and in the contribution of hTERT expression to this process.
Mesenchymal Stem Cells Produce Nitric Oxide, a Key Molecule for T Cell Suppression, upon Interaction with Activated T Cells.
Authors: Sato Kazuya , Ozaki Katsutoshi , Oh Iekuni , Meguro Akiko , Tatara Reine , Hatanaka Keiko , Nagai Tadashi , Muroi Kazuo , Ozawa Keiya 
Published: October 2019

Abstract
The molecular mechanisms by which mesenchymal stem cells (MSCs) suppress T cell proliferation are poorly understood, and whether a soluble factor plays a major role remains controversial. We demonstrated that nitric oxide (NO) is important for T cell suppression by MSCs (ASH 2005, 403a). Here we further demonstrate that the production of NO in the presence of MSCs is dependent on CD4 or CD8 T cells but not on CD19 B cells. MSCs inhibits B cell proliferation induced by LPS, suggesting that mechanisms of supppression by MSCs are different between T cells and B cells. Inducible NO synthase was exclusively detected in MSCs co-cultured with activated T cells, indicating that the producer of NO is MSC. Experiments with transwell system revealed that separation by transwell membrane reduces the induction of NO and T cell suppression. RAW246.7 macrophage cell line showed a similar transwell-mediated inhibition, suggesting that the inhibition by transwell is a common feature of NO and that direct contact is critical for efficient NO production and T cell suppression. Furthermore, inhibitors of prostaglandin synthase or NO synthase restored the proliferation of T cells, whereas an inhibitor of indoleamine 2,3-dioxygenase and a transforming growth factor-β-neutralizing antibody had no effect. In the view of that NO is upstream of PGE2, NO may be a key regulator of T cell suppression induced by MSCs. Finally, we used inducible NO synthase knockout mice to reconfirm all results here. MSCs from knockout mice did not produce NO even in the presence of activated wild type T cells and had a reduced ability to suppress T cell proliferation. Meanwhile, proliferation of splenocytes from knockout mice was suppressed in the presence of wild type MSCs and NO production was readily detected, confirming that NO produced by MSCs plays a critical role in T cell suppression.