Treatment with 177 lu-HH1 Increases CD20 Expression in Non-Hodgkin Lymphoma Cells in Vitro and In Vivo

Abstract Immunotherapy (IT) with the anti-CD20 monoclonal antibody rituximab in combination with chemotherapy has resulted in significantly improved response rate and survival in patients with various types of CD20 positive B-cell lymphoproliferative disorders. To be effective, rituximab depends on selective expression of a sufficient number of CD20 antigens per cell. Treatment with rituximab alone or in combination with chemotherapy can, however, result in disappearance of the CD20 expression, which may result in reduced clinical effect of subsequent CD20 targeted treatments. We have discovered that treatment of NHL in vitro and in vivo with the anti-CD37 antibody radionuclide conjugate (ARC) 177Lu-DOTA-HH1 (177Lu-HH1 or Betalutin™) results in an upregulation of the CD20 antigen expression, and therefore represents a rationale for a combination treatment with both agents. The in vitro expression of CD20 in Burkitt's Lymphoma, Daudi, cells 1-7 days after treatment with 177Lu-HH1 increased up to 120 % when compared with cells treated with unlabeled mAb, while Ramos (Burkitt's Lymphoma) and Rec-1 (Mantle Cell Lymphoma) cells showed 10 to 30 % increase, indicating a variation of the antigen upregulation in vitro with different cell lines. An upregulation of CD20 at the same order of magnitude was observed when cells where treated with similar absorbed radiation doses of external beam radiation. Treatment of nude mice with Ramos xenografts with 177Lu-HH1 resulted in a 3 times higher uptake of radiolabeled rituximab in tumor xenografts 5 days after start of treatment than in mice treated with unlabeled HH1 (p < 0.05) while uptake in normal organs was similar in both treatment groups (p > 0.05). SCID mice with intravenously injected Rec-1 cells were treated with NaCl, 100 mg rituximab, 40 MBq/kg 177Lu-HH1 or with the combination of 40 MBq/kg 177Lu-HH1 followed with 100 mg rituximab 5 days later. The combination of 177Lu-HH1 and rituximab resulted in significantly improved survival as compared with NaCl or rituximab alone, and a strong therapeutic gain as compared with 177Lu-HH1 alone (Table 1). In conclusion, 177Lu-HH1 treatment seems to improve uptake of rituximab and increase tumor suppression when used prior to anti-CD20 monoclonal antibody targeting in preclinical models. The reason for the upregulation of CD20 is probably related to the oxidative stress induced by the ARC-treatment, which will be evaluated in further studies. If the upregultation of CD20 is confirmed in clinical studies this effect could affect the way ARC and CD20 immunotherapy would be used in the future. Table 1. Therapy experiment groups and result Group Median ± SD Surviving fraction at the end of the study % Increase in symptom free survival compared to control NaCl + NaCl 64 ± 2 0.1 ---- NaCl + Rituximab 75 ± 10 0.3 15.4 177 Lu-HH1 + NaCl 92 ± 14 * 0.3 43.8 177 Lu-HH1 + Rituximab > 132 * 0.7 > 106.3 *Significantly different from NaCl + NaCl group (p < 0.01) Disclosures Repetto-Llamazares: Nordic Nanovector ASA: Employment, Equity Ownership. Larsen:Nordic Nanovector ASA: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Stokke:Nordic nanovector ASA: Equity Ownership. Generalov:Nordic Nanovector ASA: Employment. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership.

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Combination of the glycoengineered Type II CD20 antibody obinutuzumab (GA101) and The novel Bcl-2 selective Inhibitor GDC-0199 Results in superior In Vitro and In Vivo Anti-tumor activity in models Of B-Cell Malignancies

Blood ◽

10.1182/blood.v122.21.4412.4412 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4412-4412 ◽

Cited By ~ 4

Author(s):

Deepak Sampath ◽

Sylvia Herter ◽

Frank Herting ◽

Ellen Ingalla ◽

Michelle Nannini ◽

...

Keyword(s):

Cell Death ◽

Single Agent ◽

Employment Equity ◽

Direct Cell ◽

Xenograft Models ◽

Equity Ownership ◽

Anti Cd20 ◽

Agent Treatment

Introduction Obinutuzumab (GA101) is a novel glycoengineered type II, anti-CD20 monoclonal antibody induces a high level of direct cell death. As a result of glycoengineering, GA101 has increased affinity for FcgRIIIa on effector cells resulting in enhanced direct cell death and ADCC induction. GA101 is currently in pivotal clinical trials in CLL, indolent NHL and DLCBL. ABT-199 (GDC-0199) is a novel, orally bioavailable, selective Bcl-2 inhibitor that induces robust apoptosis in preclinical models of hematological malignancies and is currently in clinical trials for CLL, NHL and MM. Based on their complementary mechanisms of action involving increased apoptosis (GDC-0199) or direct cell death (GA101) the combination of anti-CD20 therapy with a Bcl-2 inhibitor has the potential for greater efficacy in treating B lymphoid malignancies. Experimental Methods The combination of GA101 or rituximab with GDC-0199 was studied in vitro utilizing assays that measure direct cell death induction/apoptosis (AxV/Pi positivity) on WSU-DLCL2, SU-DHL4 DLBCL and Z138 MCL cells by FACS and the impact of Bcl-2 inhibition on ADCC induction. In vivo efficacy of the combination of GA101 or rituximab and GDC-0199 was evaluated in SU-DHL4 and Z138 xenograft models. Results GA101 and rituximab enhanced cell death induction when combined with GDC-0199 in SU-DHL4, WSU-DLCL2 and Z138 cell lines. When combined at optimal doses an additive effect of the two drugs was observed. GDC-0199 did not negatively impact the capability of GA101 or rituximab to induce NK-cell mediated ADCC. Combination of GDC-0199 and GA101 induced a greater than additive anti-tumor effects in the SU-DHL4 and Z138 xenograft models resulting in tumor regressions and delay in tumor regrowth when compared to monotherapy. Moreover, continued single-agent treatment with GDC-0199 after combination with GA101 resulted in sustained in vivo efficacy in the SU-DHL4 model. Conclusions Our data demonstrate that the combination of GA101 with GDC-0199 results in enhanced cell death and robust anti-tumor efficacy in xenograft models representing NHL sub-types that is comparable to the combination of rituximab with GDC-0199. In addition, single-agent treatment with GDC-0199 following combination with GA101 sustains efficacy in vivo suggesting a potential benefit in continued maintenance therapy with GDC-0199. Collectively the preclinical data presented here supports clinical investigation of GA101 and GDC-0199 combination therapy, which is currently in a phase Ib clinical trial (clinical trial.gov identifier NCT01685892). Disclosures: Sampath: Genentech: Employment, Equity Ownership. Herter:Roche: Employment. Herting:Roche: Employment. Ingalla:Genentech: Employment. Nannini:Genentech: Employment. Bacac:Roche: Employment. Fairbrother:Genentech: Employment, Equity Ownership. Klein:Roche Glycart AG: Employment.

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A new anti-idiotype antibody capable of binding rituximab on the surface of lymphoma cells

Blood ◽

10.1182/blood-2004-05-1733 ◽

2004 ◽

Vol 104 (8) ◽

pp. 2540-2542 ◽

Cited By ~ 33

Author(s):

Mark S. Cragg ◽

Mike B. Bayne ◽

Alison L. Tutt ◽

Ruth R. French ◽

Stephen Beers ◽

...

Keyword(s):

Monoclonal Antibody ◽

Cell Surface ◽

Immunoglobulin G ◽

Human Immunoglobulin ◽

Lymphoma Cells ◽

Fine Specificity ◽

High Affinity ◽

Anti Cd20

Abstract The chimeric anti-CD20 monoclonal antibody (mAb), rituximab, is an established part of the management of many non-Hodgkin lymphomas. The in vivo action of rituximab remains elusive, and this partially reflects a lack of highly specific reagents to detect rituximab binding at the cell surface. Here we report a new high-affinity mAb (MB2A4) with fine specificity for the idiotype of rituximab. It is able to detect rituximab in vitro, in the presence of high levels of human immunoglobulin G (IgG), in the serum of patients receiving rituximab therapy, and, surprisingly, when rituximab is bound to CD20 on the cell surface. We propose that the anti–idiotype (Id) binds to rituximab molecules bound univalently at the cell surface, facilitated by the relatively high off-rate of rituximab. This reagent provides new insights into the binding of rituximab at the cell surface and demonstrates a mode of binding that could be exploited for the surface detection of other mAbs with clinical and biologic applications.

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Lirilumab Enhances Anti-Tumor Efficacy of Elotuzumab

Blood ◽

10.1182/blood.v124.21.4711.4711 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4711-4711 ◽

Cited By ~ 4

Author(s):

Caroline Sola ◽

Mathieu Blery ◽

Cécile Bonnafous ◽

Elodie Bonnet ◽

Nicolas Fuseri ◽

...

Keyword(s):

Monoclonal Antibody ◽

Nk Cells ◽

Tumor Cells ◽

Therapeutic Efficacy ◽

Nk Cell ◽

Employment Equity ◽

Equity Ownership ◽

Checkpoint Receptors

Abstract Tumor cells that express reduced levels of Major Histocompatibility Complex (MHC) class I molecules may be recognized and killed by Natural Killer cells (NK cells), through a process known as “missing self” recognition. In humans, this is controlled by inhibitory receptors such as Killer Immunoglobulin-like Receptors (KIR) that recognize Human Leukocyte Antigen (HLA)-A, -B or –C. Engagement of KIR by HLA molecules results in inhibitory signaling that reduces NK cell-mediated natural killing and antibody-dependent cellular cytotoxicity (ADCC). Hence, antibodies that block interactions between inhibitory KIR and their HLA ligands are being evaluated as an anti-cancer therapeutic strategy. The anti-KIR2DL1/2/3-specific monoclonal antibody, lirilumab (BMS-986015 / IPH2102), is a fully human IgG4 that blocks binding of KIR to HLA-C, and is being developed for treating hematologic malignancies and solid tumors. Elotuzumab (BMS901608 / HuLuc63) is a humanized IgG1 anti-SLAMF7 (signaling lymphocyte activation molecule family member 7, CS-1) being developed for the treatment of Multiple Myeloma (MM). SLAMF7 is a cell surface glycoprotein highly expressed in myelomatous cells and only at low levels on normal cells. NK cell-mediated ADCC is one of the main mechanisms of action of elotuzumab, but ADCC is negatively regulated by KIR checkpoint receptors. Thus a combination of lirilumab and elotuzumab has strong scientific rationale. The aim of the present study was to assess whether lirilumab would enhance elotuzumab anti-MM activity in vitro with human peripheral blood NK cells and MM cell lines, and in vivo in a newly developed xenogenic mouse model. Two MM cell lines (OPM-2 and U266B1) were identified that express both HLA-C and SLAMF7. These MM cells were capable of activating peripheral blood NK cells from healthy donors in vitro, as assessed by three different endpoints (CD107 mobilization on NK cells surface and intracellular production of the cytokines IFN-g and TNF- a); each of these responses were significantly enhanced, in a dose-dependent manner, by both lirilumab and elotuzumab independently. Moreover, the elotuzumab-mediated functional activation of KIR2D+ NK cells could be further enhanced by the addition of increasing doses of lirilumab. The best combinatorial effect was observed in response to MM cells expressing low densities of SLAMF7. These data suggest that lirilumab treatment may increase the therapeutic efficacy of elotuzumab, particularly in MM patients with low SLAMF7 expression. In these experiments, it was not possible to clearly identify the impact of Fc receptor genotype or HLA-C genotype on the NK cell responses. To assess the therapeutic efficacy of lirilumab and elotuzumab in vivo, we generated a novel strain of double-transgenic mice expressing human KIR2DL3 as well as its ligand, HLA-cw3, on a Rag1-/- background (KIR-cw3-tgRAG mice), to allow engraftment of human MM tumor cells expressing SLAMF7. The OPM-2 MM cell line was subcutaneously engrafted in these mice and when high tumor volumes were reached, mice were treated with lirilumab, elotuzumab or a combination of both. As monotherapy, each of monoclonal antibody had some therapeutic effect while the combination of both resulted in a significantly stronger anti-tumor effect and increased survival of the mice. Median survival of mice treated with huIgG control was 38 days, 41 days with lirilumab, 42 days with elotuzumab and 51 days with both mAbs in combination (10 mice per group). In conclusion, we demonstrate that blockade of KIR checkpoint receptors with lirilumab was able to augment elotuzumab mediated ADCC in vitro and synergized with elotuzumab to mediate potent anti-MM activity in vivo. Taken together, these data provide a rationale for clinical trials to test combination treatment of lirilumab and elotuzumab in MM patients. Disclosures Sola: InnatePharma: Employment, Equity Ownership. Blery:Innate Pharma: Employment, Equity Ownership. Bonnafous:Innate Pharma: Employment, Equity Ownership. Bonnet:Innate Pharma: Employment, Equity Ownership. Fuseri:Innate Pharma: Employment, Equity Ownership. Graziano:Bristol-Myers Squibb: Employment; Bristol-Myers Squibb: Equity Ownership. Morel:Innate Pharma: Employment, Equity Ownership. André:Innate Pharma: Employment, Equity Ownership.

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Anti-KIR Antibody Enhancement Of Anti-Lymphoma Activity Of Natural Killer Cells As Monotherapy and In Combination With Anti-CD20 Antibodies

Blood ◽

10.1182/blood.v122.21.4417.4417 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4417-4417 ◽

Cited By ~ 1

Author(s):

Holbrook E Kohrt ◽

Ariane Thielens ◽

Aurelien Marabelle ◽

Idit Sagiv Barfi ◽

Caroline Sola ◽

...

Keyword(s):

Board Of Directors ◽

Nk Cell ◽

Dependent Manner ◽

Employment Equity ◽

Advisory Committees ◽

Potential Efficacy ◽

Equity Ownership ◽

Anti Cd20

Natural killer (NK) cells mediate anti-lymphoma activity by spontaneous cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) when triggered by rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B cell lymphomas. The balance of inhibitory and activating signals determines the magnitude of NK cell's efficacy by spontaneous cytoxicity. Using a killer cell immunoglobulin-like receptor (KIR) transgenic murine model, we show that blockade of the interface of inhibitory KIRs with MHC class I antigens on lymphoma by anti-KIR antibodies prevents a tolerogenic interaction and augments NK cell spontaneous cytotoxicity. In combination with anti-CD20 mAbs, anti-KIR treatment induces enhanced NK cell-mediated, rituximab-dependent cytotoxicity against lymphoma in vitro and in vivo in syngeneic and KIR transgenic murine lymphoma models. Specifically targeting murine NK cells in vitro, anti-Ly49C/I F(ab')2 increased anti-CD20 mAb-mediated NK cell degranulation as measured by CD107a mobilization and interferon-γ release, as well as increased cytotoxicity as assessed by chromium release. In the syngeneic EL4-huCD20 lymphoma model, anti-Ly49C/I F(ab')2 enhanced the anti-lymphoma activity of anti-CD20 mAb in vivo (Fig 1A-1B) and was NK cell-dependent with efficacy abrogated by NK cell depletion with anti-Asialo-GM1. To validate these observations and the potential efficacy of a fully human anti-KIR mAb (IPH2101, lirilumab), we demonstrated, in vitro, dose-dependent KIR2DL3 saturation and tumor lysis following blockade of KIR2DL3/HLA-C with lirilumab. In the transgenic KIR murine model, lirilumab therapy improved survival in an NK cell-dependent manner in both a prophylactic and therapeutic HLA+ (221 HLA-Cw3) lymphoma model. In combination, lirilumab therapy synergistically enhanced rituximab's anti-lymphoma efficacy in vivo in an NK cell-dependent manner (Fig 2A-C). These results support a therapeutic strategy of combination, rituximab and KIR blockade through lirilumab, illustrating the potential efficacy of combining a tumor targeting therapy with an NK cell agonist thus stimulating the post-rituximab anti-lymphoma immune response. Disclosures: Thielens: Innate Pharma: Employment, Equity Ownership. Sola:Innate Pharma: Employment, Equity Ownership. Chanuc:Innate Pharma: Employment, Equity Ownership. Fuseri:Innate Pharma: Employment. Bonnafous:Innate Pharma: Employment, Equity Ownership. Vivier:Innate Pharma: Membership on an entity’s Board of Directors or advisory committees. Romagne:Innate Pharma: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Andre:Innate-Pharma: Employment, Equity Ownership. Blery:Innate Pharma: Employment, Equity Ownership.

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IMGN529, a Novel Antibody-Drug Conjugate (ADC) Targeting CD37 Shows Synergistic Activity with Rituximab in Non-Hodgkin Lymphoma (NHL) Models

Blood ◽

10.1182/blood.v126.23.1548.1548 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1548-1548 ◽

Cited By ~ 2

Author(s):

Jutta Deckert ◽

Callum M Sloss ◽

Katie O'Callaghan ◽

Jenny Tsui ◽

Nemisha Dawra ◽

...

Keyword(s):

Cell Lines ◽

Synergistic Activity ◽

Employment Equity ◽

Xenograft Models ◽

Equity Ownership ◽

Induction Of Apoptosis ◽

Anti Cd20 ◽

Cd20 Antibodies

Abstract Introduction: Relapsed/refractory B-cell NHL remains an area of significant medical need. CD37 is highly expressed in many B-cell malignancies, including NHL, making it an ideal target for ADC-based therapy. IMGN529 is a CD37-targeting ADC consisting of a CD37-binding antibody conjugated to the maytansinoid anti-mitotic, DM1. IMGN529 has been shown to have potent, targeted activity against NHL cell lines and xenograft models via antibody-mediated direct cell-killing, effector function and the anti-mitotic activity of the DM1 payload. IMGN529 has shown early signs of clinical activity at tolerable doses in an ongoing phase I trial in adult patients with relapsed/refractory NHL (R/R-NHL) (NCT01534715) (Blood 2014 124:1760). Rituximab, an anti-CD20 monoclonal antibody, is widely used for NHL therapy and remains a component of both front-line (with chemotherapy combinations) and late-line (both as a monotherapy and in combination) regimens. We have previously shown data from an in vitro synergy screen which identified strong anti-NHL synergy for IMGN529 used in combination with anti-CD20 antibodies (Hematol Oncol 2015; 33: 181-243). Methods: The activity and mechanism-of-action of IMGN529 in combination with rituximab was further evaluated in clinically relevant preclinical models of NHL: Cell viability in response to single agents and combinations was tested using the WST-8 assay. In vivo combination studies were carried out using human xenograft models of DLBCL implanted in SCID mice. Induction of apoptosis was measured via Annexin-V flow cytometry and caspase 3/7 cleavage assays. Changes in molecular signaling in response to treatment were measured using western blotting and ELISA. Results: Combination of IMGN529 and the anti-CD20 antibodies rituximab, obinutuzumab and ofatumumab resulted in high synergy scores, identifying a potential class-effect of synergy between IMGN529 and anti-CD20 antibodies. The notable activity of an IMGN529/ rituximab combination was confirmed both in vitro and in vivo using cell line viability and xenograft models of DLBCL (both ABC and GCB subtypes). In these models, the activity of the IMGN529/ rituximab combination was consistently greater than either agent administered as a monotherapy. We examined whether this synergistic reduction in cell viability was due to a reduction in cell growth or an induction of cell death. In multiple NHL cell lines, we found that the combination of IMGN529 and rituximab induced significantly higher levels of Annexin-V positivity and caspase 3/7 activity than either single agent alone, consistent with the pro-apoptotic mechanism of action proposed for IMGN529. To further elucidate the mechanisms underlying the synergy of the combination, we are monitoring the effect on key components of upstream signaling pathways responsible for cell survival and induction of apoptosis, including: the apoptotic inhibitors Bcl-2, Bcl-xL and Mcl-1; MAP-kinase signaling, and the NF-kB and AKT/mTOR axes, all of which have been linked to treatment sensitization by rituximab in NHL cell lines. Conclusions: IMGN529 demonstrates synergistic activity in combination with CD20-targeting antibodies including rituximab. In in vitro and in vivo models of NHL, the combination of IMGN529 and rituximab is more active than either agent alone, and this enhanced activity is associated with an increase in the induction of apoptosis and apoptotic signaling pathways. These results support clinical assessment of IMGN529 in combination with rituximab, and a phase II trial assessing safety and efficacy in R/R-NHL is planned. Disclosures Deckert: ImmunoGen, Inc.: Employment, Equity Ownership. Sloss:ImmunoGen, Inc.: Employment, Equity Ownership. O'Callaghan:ImmunoGen, Inc.: Employment, Equity Ownership. Tsui:ImmunoGen, Inc.: Employment, Equity Ownership. Dawra:ImmunoGen, Inc.: Employment, Equity Ownership. Yi:ImmunoGen, Inc.: Employment, Equity Ownership. Coccia:ImmunoGen, Inc.: Employment, Equity Ownership. Lanieri:ImmunoGen, Inc.: Employment, Equity Ownership. Chicklas:ImmunoGen, Inc.: Employment, Equity Ownership. Romanelli:ImmunoGen, Inc.: Employment, Equity Ownership.

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Docetaxel hydrate induces apoptosis and suppresses tumorigenesis of oral Burkitt’s lymphoma cells (in vitro and in vivo studies)

Padjadjaran Journal of Dentistry ◽

10.24198/pjd.vol31no1.18102 ◽

2019 ◽

Vol 31 (1) ◽

pp. 7

Author(s):

Supriatno Supriatno

Keyword(s):

B Cell Lymphoma ◽

Burkitt’S Lymphoma ◽

Burkitt's Lymphoma ◽

Control Group ◽

Double Staining ◽

Lymphoma Cells ◽

Raji Cells ◽

Burkitt’S Lymphoma Cells

Introduction: Burkitt’s lymphoma (BL) is one of the tumours with high malignancy and rapid cell growth, derived from B-cell lymphoma. BL typically found in children at dengue-endemic and HIV-AIDS areas with low socioeconomic levels. This study was aimed to analyse the induction of apoptosis and the suppression of tumorigenesis of oral Burkitt’s lymphoma (Raji) cells using docetaxel hydrate in vitro and in vivo. Methods: In the present study, the pure experimental laboratory with post-test only control group design was carried out. Raji cell cultures were incubated with docetaxel hydrate by doses of 0, 1.25 x 10-2, 2.5 x 10-2, and 5.0 x 10-2 M; and IC50 carboplatin (3.1 x 10-6 M) as a positive control. Induction of apoptotic was analysed by double staining of acridine orange-ethidium bromide. Tumorigenesis assay was performed by inoculating Raji cells in nude mice flanks at 1 x 106 cells/mice. Tumour treatment was delivered by various doses of docetaxel hydrate peroral. Results: Apoptosis cells were significantly increased in Raji cells treated with docetaxel hydrate by doses of 2.5 x 10-2 and 5.0 x 10-2 M. The tumour volume in mice given doses of 2.5 x 10-2 and 5.0 x 10-2 M was markedly decreasing compared to control (dose of 0). Conclusion: Docetaxel hydrate has a high antitumour potency by inhibiting tumorigenesis and increasing apoptosis of Burkitt’s lymphoma cells. Keywords: Docetaxel hydrate, double staining, Burkitt’s lymphoma cell, apoptosis, tumorigenesis

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Phenotype Does Not Always Equal Function: HDAC Inhibitors and UM171, but Not SR1, Lead to Rapid Upregulation of CD90 on Non-Engrafting CD34+CD90-Negative Human Cells

Blood ◽

10.1182/blood.v130.suppl_1.659.659 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 659-659

Author(s):

Kevin A. Goncalves ◽

Megan D. Hoban ◽

Jennifer L. Proctor ◽

Hillary L. Adams ◽

Sharon L. Hyzy ◽

...

Keyword(s):

In Vitro Culture ◽

Small Molecule ◽

Hdac Inhibitors ◽

Employment Equity ◽

Nsg Mice ◽

Cd34 Cells ◽

Equity Ownership ◽

Human Hscs

Abstract Background. The ability to expand human hematopoietic stem cells (HSCs) has the potential to improve outcomes in HSC transplantation and increase the dose of gene-modified HSCs. While many approaches have been reported to expand HSCs, a direct comparison of the various methods to expand transplantable HSCs has not been published and clinical outcome data for the various methods is incomplete. In the present study, we compared several small molecule approaches reported to expand human HSCs including HDAC inhibitors, the aryl hydrocarbon antagonist, SR1, and UM171, a small molecule with unknown mechanism, for the ability to expand phenotypic HSC during in vitro culture and to expand cells that engraft NSG mice. Although all strategies increased the number of phenotypic HSC (CD34+CD90+CD45RA-) in vitro, SR1 was the most effective method to increase the number of NOD-SCID engrafting cells. Importantly, we found that HDAC inhibitors and UM171 upregulated phenotypic stem cell markers on downstream progenitors, suggesting that these compounds do not expand true HSCs. Methods. Small-molecules, SR1, HDAC inhibitors (BG45, CAY10398, CAY10433, CAY10603, Entinostat, HC Toxin, LMK235, PCI-34051, Pyroxamide, Romidepsin, SAHA, Scriptaid, TMP269, Trichostatin A, or Valproic Acid) and UM171 were titrated and then evaluated at their optimal concentrations in the presence of cytokines (TPO, SCF, FLT3L, and IL6) for the ability to expand human mobilized peripheral blood (mPB)-derived CD34+ cells ex vivo . Immunophenotype and cell numbers were assessed by flow cytometry following a 7-day expansion assay in 10-point dose-response (10 µM to 0.5 nM). HSC function was evaluated by enumeration of colony forming units in methylcellulose and a subset of the compounds were evaluated by transplanting expanded cells into sub-lethally irradiated NSG mice to assess engraftment potential in vivo . All cells expanded with compounds were compared to uncultured or vehicle-cultured cells. Results. Following 7 days of expansion, SR1 (5-fold), UM171 (4-fold), or HDAC inhibitors (>3-35-fold) resulted in an increase in CD34+CD90+CD45RA- number relative to cells cultured with cytokines alone; however, only SR1 (18-fold) and UM171 (8-fold) demonstrated enhanced engraftment in NSG mice. Interestingly, while HDAC inhibitors and UM171 gave the most robust increase in the number and frequency of CD34+CD90+CD45RA- cells during in vitro culture, these methods were inferior to SR1 at increasing NSG engrafting cells. The increase in CD34+CD90+CD45RA- cells observed during in vitro culture suggested that these compounds may be generating a false phenotype by upregulating CD90 and down-regulating CD45RA on progenitors that were originally CD34+CD90-CD45RA+. We tested this hypothesis by sorting CD34+CD90-CD45RA+ cells and culturing these with the various compounds. These experiments confirmed that both HDAC inhibitors (33-100 fold) and UM171 (28-fold) led to upregulation of CD90 on CD34+CD90-CD45RA+ cells after 4 days in culture. Since approximately 90% of the starting CD34+ cells were CD90-, these data suggest that most of the CD34+CD90+CD45RA- cells in cultures with HDAC inhibitors and UM171 arise from upregulation of CD90 rather than expansion of true CD34+CD90+CD45RA- cells and may explain the disconnect between in vitro HSC phenotype and NSG engraftment in vivo . This was further confirmed by evaluation of colony forming unit frequency of CD34+CD90-CD45RA+ cells after culture with compounds. Conclusions. We have showed that AHR antagonism is optimal for expanding functional human HSCs using the NSG engraftment model. We also demonstrated that UM171 and HDAC inhibitors upregulate phenotypic HSC markers on downstream progenitors. This could explain the discrepancy between impressive in vitro phenotypic expansion and insufficient functional activity in the NSG mouse model. Therefore, these data suggest caution when interpreting in vitro expansion phenotypes without confirmatory functional transplantation data, especially as these approaches move into clinical trials in patients. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership. Hoban: Magenta Therapeutics: Employment, Equity Ownership. Proctor: Magenta Therapeutics: Employment, Equity Ownership. Adams: Magenta Therapeutics: Employment, Equity Ownership. Hyzy: Magenta Therapeutics: Employment, Equity Ownership. Boitano: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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The CT-Element of The C-Myc Gene Does Not Predispose to Chromosomal Breakpoints in Burkitt'S Lymphoma

Analytical Cellular Pathology ◽

10.1155/2006/695262 ◽

2006 ◽

Vol 28 (1-2) ◽

pp. 31-35

Author(s):

Achim Weber ◽

Marina I. Gutierrez ◽

David Levens

Keyword(s):

Fragile Site ◽

Burkitt’S Lymphoma ◽

Burkitt's Lymphoma ◽

Chromosomal Translocations ◽

Wild Type ◽

Chromosomal Breakpoints ◽

Myc Gene

Background: Chromosomal translocations are causally related to the development of many tumors. In Burkitt's lymphoma, abnormalities involving the c-myc gene are essential. The CT-element of the c-myc promoter adopts non-B-conformation in vivo and in vitro, and therefore provides a potential fragile site. Methods: We have developed a LM-PCR-based approach to test if chromosomal breakpoints indeed cluster in this region. Results: Amplifying both, wild-type as well as the translocated c-myc gene by LM-PCR, it was shown that chromosomal breakpoints did not cluster within the CT-element. Conclusions: Therefore, the CT-element is not especially susceptible to the formation of breakpoints leading to chromosomal translocations in Burkitt's lymphoma.

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Highly Differentiated Anti-CD47 Antibody, AO-176, Potently Inhibits Hematologic Malignancies Alone and in Combination

Blood ◽

10.1182/blood-2019-126298 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1844-1844

Author(s):

John Richards ◽

Myriam N Bouchlaka ◽

Robyn J Puro ◽

Ben J Capoccia ◽

Ronald R Hiebsch ◽

...

Keyword(s):

Multiple Myeloma ◽

Combination Therapy ◽

Tumor Growth ◽

Growth Inhibition ◽

Tumor Cells ◽

Tumor Growth Inhibition ◽

Employment Equity ◽

Equity Ownership

AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.

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