CDC Activities Research Articles

Engineering novel Lec1 glycosylation mutants in CHO–DUKX cells: Molecular insights and effector modulation of N‐acetylglucosaminyltransferase I

Many secreted or cell surface proteins are post-translationally modified by carbohydrate chains which are a primary source of heterogeneity. The Lec1 mutant, which is defective in Golgi N-acetylglucosaminyltransferase I (GnTI) activity, produces relatively homogeneous Man(5) GlcNAc(2) glycan modifications, and is widely used for various applications. To facilitate the investigation of GnTI, its Man5 glycan endproduct, and the impact of Man5 on effector function, the present study has established several novel Lec1 mutants in dhfr(-) CHO-DUKX cells through chemical mutagenesis and lectin selection. A total of nine clonal lines exhibiting the Lec1-phenotype are characterized, six of which harbor non-sense mutations leading to a truncated GnTI, and three (R415K, D291N, and P138L) of which are novel loss-of-function sense mutations. Analysis of the rabbit GnTI structure (Unligil et al., 2000) indicates that D291 is the proposed catalytic base and R415 is a crucial residue in forming the substrate binding pocket, whereas P138 is key to maintaining two β strands in proximity to the substrate binding pocket. Computational modeling reveals that the oligomannose glycan backbone of a glycoprotein (the acceptor substrate) fits nicely into the unoccupied channel of the substrate binding pocket partly through hydrogen bonding with R415 and D291. This finding is consistent with the ordered sequential Bi Bi kinetic mechanism suggested for GnTI, in which binding of UDP-GlcNAc (the donor substrate)/Mn(2+) induces conformational changes that promote acceptor binding. When an anti-human CD20 antibody protein is stably expressed in one CHO-DUKX-Lec1 line, it is confirmed that N-glycans are predominantly Man(5) GlcNAc(2) and they do not contain an α1,6-fucose linked to the innermost GlcNAc. Furthermore, this Man(5) GlcNAc(2) modified antibody exhibits a significantly increased ADCC activity than the wild-type protein, while displaying a lower CDC activity. The data support the hypothesis that modulating GnTI activity can influence antibody effector functions for proteins with an IgG1 immunoglobulin Fc domain.

Induction of Human Anti-Human Antibody Responses (Ab2) after Application of a Humanized Lewis Y Carbohydrate Specific Antibody (Ab1): Connection of Prolonged Disease Stabilization with Ab3 Induction?

Purpose: Detailed analysis of a patient with epithelial Lewis Y (LeY) positive cancer who received twice 50 mg of the humanized Lewis Y carbohydrate specific mAb IGN311 and developed a clinically significant human anti-human antibody (HAHA) response (Ab2). Results: Clinical stabilization of the disease was assigned to in this patient. The HAHA response consisted mainly of IgG1 and was found to be directed against the IGN311 binding site. Consistent with the induction of the HAHA response, CDC activity against Lewis Y positive target cells was completely abolished at day 8 and could not be restored by the second 50 mg infusion indicating complete neutralization of applied IGN311. The ADCC reactivity was also significantly reduced and anti-anti idiotype-specific antibodies (Ab3) were detectable at day 65. Conclusions: Induction of Ab3 antibodies should be considered as an additional factor influencing the efficacy of humanized antibodies. In this context, the potential threat of induced HAHA responses against therapeutic mAbs might have to be reconsidered because they might actually have also beneficial immunological long-term effects leading to an active immunization component induced by therapeutic antibodies.

TG20, a Transgenically-Derived Anti-CD20 Monoclonal Antibody, Exhibits Enhanced Cytotoxicity Against Cells with Low Levels of CD20

Abstract 2730CD20 is a cell-surface glycoprotein that is highly expressed on most B-cells, tightly restricted to the B-cell lineage, and not expressed on either precursor lymphoid cells or the majority of plasma cells. These characteristics make CD20 an appealing target for mAb therapy of B-cell malignancies and B-cell dependent autoimmune conditions, because antibody production is maintained during therapy and B-cell regeneration post-CD20 mAb treatment is facilitated. Among several marketed anti-CD20 mAbs, rituximab is the prototype; a chimeric human–mouse type-1 antibody that has proven efficacy in a wide variety of mature B-cell malignancies, including CD20+ B-cell lymphoma, and has also been approved for the treatment of refractory rheumatoid arthritis. However, patients do not always respond to this therapy, and it has been reported that close to 60% of follicular lymphoma patients previously treated with chemotherapy, while initially responsive, become resistant upon repeat treatment with rituximab monotherapy. Furthermore, the high cost of treatment with rituximab and other anti-CD20 mAbs severely curtails their availability to patients in emerging economies, as well as their use post treatment for maintenance therapy.Transgenic production offers an easily scalable system for the cost-effective manufacturing of large amounts of complex therapeutic proteins. The regulatory approvals of ATryn® (recombinant antithrombin), first by the EMA (August 2006) and by the FDA (February 2009) have provided a strong regulatory validation of this production platform. Produced in the milk of transgenic goats, TG20, a novel chimeric IgG1 strongly binding a specific discontinuous epitope on CD20 (KD = 1–2 × 10−8 M), is being developed as a second-generation anti-CD20 mAb with enhanced cytotoxic activity. Using regulatory sequences from the caprine beta-casein gene to target expression of the TG20 heavy- and light-chains to the lactating mammary gland, 7 independent lines of transgenic goats were generated by somatic cell nuclear transfer. Induced lactation studies indicated that these lines expressed TG20 in their milk at levels varying from 2 g/liter up to ∼ 10 g/liter. Following genetic characterization (Southern blotting, FISH analysis) and expression analysis (protein and peptide mapping, glycosylation profiling, amino-acid sequence analysis), one line was selected for expansion and further development.In-vitro studies comparing TG20 to rituximab showed a significantly increased ability to activate effector cells expressing the FcgRIIIa receptor (CD16a). Not surprisingly, this translates to a significantly increased ability to activate NK cells and to induce target lysis. This increased ADCC has been demonstrated both with CD20+ Raji cells and with primary B-cells isolated from patients with B-CLL (TG20 EC50 = 3.6 ng/ml vs. rituximab EC50 = 59.4 ng/ml). The ability of TG20 to activate the complement system was assessed using human serum as complement and WIL2-S cells as target exhibiting a > 50% increase in CDC activity vs. rituximab (TG20 = 170% CDC activity vs. rituximab = 100%). Finally, studies conducted in Cynomolgus monkeys were performed to assess pharmacokinetics and in-vivo depletion activities of TG20.In conclusion, TG20 is a highly active anti-CD20 mAb showing interesting characteristics in terms of cytotoxicity that makes it a promising agent for indications in which rituximab is poorly active. Furthermore, the use of the cost-efficient transgenic production platform for the large-scale production of this antibody drug candidate may allow expanded access of anti-CD20 therapy to those who currently cannot afford these expensive treatments, especially in emerging market countries. Disclosures:Echelard:GTC Biotherapeutics, Inc.: Employment. Pollock:GTC Biotherapeutics, Inc.: Employment. De Coupade:LFB Biotechnologies: Employment. Groseil Olivier:LFB Biotechnologies: Employment. Brune:LFB Biotechnologies: Employment. Chen:GTC Biotherapeutics, Inc.: Employment. Masiello:GTC Biotherapeutics, Inc.: Employment. Williams:GTC Biotherapeutics, Inc.: Employment. Gavin:GTC Biotherapeutics, Inc.: Employment. Chtourou:LFB Biotechnologies: Employment. Meade:GTC Biotherapeutics, Inc.: Employment.

Subcutaneous Administration of AME-133 Demonstrated Significant Dose Dependent B-Cell Depletion in Cynomolgus Monkeys (Macaca fascicularis)

Abstract 2475 Introduction:Anti-CD20 antibodies like rituximab are traditionally administered by the intravenous routes although clinical trials by subcutaneous routes have been initiated.AME-133 is a monoclonal anti-CD20 antibody, has been engineered to be more potent than rituximab. It has 10–20 fold higher binding affinity for the CD20 epitope and approximately 6-fold greater potency in ADCC assays relative to rituximab. The higher potency may provide benefit to patients with low affinity FcgIIIRa and enable subcutaneous administration (patient convenience). In addition, AME-133 has been engineered to be potentially safer than rituximab; AME-133 is humanized to reduce immunogenicity and the CDC activity has been diminished to potentially reduce side effects associated with tumor lysis syndrome. Phase I/II clinical studies of AME-133 in rituximab pretreated/relapsed, patients with low affinity FcγIIIRa, follicular non-Hodgkin’s lymphoma (NHL) in the US and in Japan have shown excellent safety profile and response rate (RR) greater than 30%. As a prelude to investigating the clinical efficacy and safety of AME-133 in subcutaneous formulation, a safety/PK study was conducted in cynomolgus monkeys. Objective:The objective of this study was to evaluate the systemic toxicity and pharmacokinetics of AME-133 in cynomolgus monkeys at three different dose levels when administered subcutaneously once each week for 14 consecutive weeks. In addition, the effect of AME-133 on B-cell depletion was evaluated. Study Design:A total of 48 naive cynomolgus monkeys (24 male and 24 female) were randomly assigned to four dose groups of AME-133 administered subcutaneously once weekly (0.0 mg/kg (vehicle), 0.6 mg/kg, 1.9 mg/kg and 6.0 mg/kg). Within the dose groups, there were two (6 week and 14 week) dosing schedules. In the 6 week dosing schedule, two-third of animals were sacrificed immediately after 6 weeks, and one-third of animals were sacrificed after 4 to 8 weeks of recovery period. In the 14 week dosing schedule, two-third of animals were sacrificed immediately after 14 weeks, and one-third of animals were sacrificed after 4 to 8 weeks of recovery period. Results:There was no AME-133 related toxicity. All dose levels of AME-133 substantially depleted B-cells in peripheral blood. The extent and duration of B-cell depletion was dose-dependent (Figure 1). B-cell depletion was not affected by gender of the animal. [Display omitted] [Display omitted] All dose levels of AME-133 substantially depleted the B-cells in peripheral blood beginning with the first post-dose time point (4 hours after infusion). The mid and high doses resulted in delayed return to normal during recovery in cynomolgus monkeys in 14 week dosing schedule. Conclusion:This study in cynomolgus monkeys showed AME-133 caused no toxicity, well absorbed and rapidly depleted circulating B-cells when administered subcutaneously. The No Observed Adverse Effect Level (NOAEL) is 6.0 mg/kg, the highest administered dose. These preliminary results justify further investigation of AME-133v administered subcutaneously in follicular lymphoma and other B-cell malignancies. Disclosures:No relevant conflicts of interest to declare.

Potent B-Cell Depletion by IMGN529, a CD37-Targeting Antibody-Maytansinoid Conjugate for the Treatment of B-Cell Malignancies,

Abstract 3726CD37 is a B-cell surface antigen which is widely expressed on malignant B cells in non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL). In normal tissues CD37 expression is limited to blood cells and lymphoid tissues. This restricted expression profile makes CD37 an attractive therapeutic target for antibodies and antibody-drug conjugates.We developed a novel anti-CD37 antibody, K7153A, which provides a unique combination of functional properties: it demonstrated strong pro-apoptotic and direct cell killing activity against NHL cell lines and could mediate effector activity such as CDC and ADCC. The antibody-maytansinoid conjugate, IMGN529, was produced by conjugation of K7153A with the potent maytansinoid, DM1, via the non-cleavable linker, SMCC. The direct cytotoxic potency of the K7153A antibody was superior to that of the CD20-directed rituximab and was further enhanced with maytansinoid conjugation in IMGN529. In vivo, IMGN529 demonstrated better anti-tumor activity than the K7153A antibody in established subcutaneous follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and CLL xenograft models in SCID mice. A single administration of IMGN529 showed similar or improved efficacy compared to anti-CD20 antibodies or standard chemotherapy where tested.Immunohistochemical (IHC) staining of formalin fixed paraffin-embedded (FFPE) NHL tissue sections was performed to evaluate CD37 expression. CD37 exhibited a similar prevalence to CD20 in subtypes of NHL such as FL, DLBCL, Burkitt's lymphoma (BL) and mantle cell lymphoma (MCL).B-cell depletion is an important measure of efficacy for targeted therapies, such as CD20-directed antibodies, in B-cell malignancies. CD37 expression in blood cells from healthy human donors was measured by quantitative flow cytometry in comparison to CD20. The greatest CD37 expression was found in B cells at approximately 77,000 antibodies bound per cell (ABC), which was similar to CD20 expression in B cells at 95,000 ABC. In other blood cell types CD37 staining was seen at low levels, about 2,000 – 5,000 ABC, in monocytes, NK cells and T cells. In vitro depletion experiments were performed with purified peripheral blood mononuclear cells (PBMCs) and with whole blood, both derived from several healthy donors. Cells were incubated for 1 hr with 10 μg/mL of either K7153A, IMGN529, CD37-targeting TRU-016, rituximab or the anti-CD52 antibody alemtuzumab, with cell depletion determined relative to counting beads by flow cytometry. The K7153A antibody and the IMGN529 conjugate efficiently and specifically depleted B-cells in a dose-dependent manner in the context of purified PBMCs and whole blood. With purified PBMCs, both K7153A and IMGN529 caused 50–60% depletion of B cells, with little to no depletion of T cells or monocytes. IMGN529 was more potent than rituximab, which led to 30–40% B-cell depletion, or TRU-016, which caused 20–30% B-cell depletion. IMGN529 also was more specific than alemtuzumab, which depleted T-cells and monocytes as well as B cells. With whole blood samples, both K7153A and IMGN529 resulted in 30–40% B-cell depletion with no effect on T cells, NK cells or monocytes. IMGN529 was again more potent than rituximab or TRU-016, which caused approximately 10% B-cell depletion, and was more specific than alemtuzumab, which depleted the majority of T cells in addition to 40% of B cells.IMGN529 embodies a unique B-cell targeted agent as it combines the intrinsic pro-apoptotic, CDC and ADCC activities of its anti-CD37 antibody component with the potent cytotoxic mechanism provided by the targeted delivery of its maytansinoid payload. It is highly active in vitro and in vivo against B-cell lymphoma and CLL cell lines. In addition, it mediates specific B-cell depletion in vitro that is greater than B-cell depletion by CD20-directed rituximab. Together, these findings indicate that IMGN529 is a promising therapeutic candidate for the treatment of B-cell malignancies. Disclosures:Deckert:ImmunoGen, Inc.: Employment. Chicklas:ImmunoGen, Inc.: Employment. Yi:ImmunoGen, Inc.: Employment. Li:ImmunoGen, Inc.: Employment. Pinkas:ImmunoGen, Inc.: Employment. Chittenden:ImmunoGen, Inc.: Employment. Lutz:ImmunoGen, Inc.: Employment. Park:ImmunoGen, Inc.: Employment.

Abstract 4566: AGS-1C4D4: A fully human anti-PSCA antibody inhibits tumor formation and metastasis in orthotopic models of pancreatic cancer

Abstract Prostate stem cell antigen (PSCA) is a cysteine-rich cell surface glycoprotein expressed in about 50% of pancreatic and prostate cancers. AGS-PSCA is a hybridoma-derived fully human IgG1κ monoclonal antibody (MAb) targeting PSCA previously reported to have anti-tumor efficacy in prostate and pancreatic tumor models. AGS-1C4D4 is a CHO-derived antibody generated from the same gene as AGS-PSCA, with similar specificity and binding affinity to PSCA (Kd = 2.0 × 10-10M). Since hybridoma and CHO-derived MAbs displayed disparate glycosylation patterns by mass spectroscopy, we further evaluated their MAb effector functions. AGS-1C4D4 was found to have more potent ADCC activity in vitro on PSCA-expressing pancreatic cell lines, HPAC and Panc0203, using human PBMCs from multiple normal donors. However, both MAbs were similarly effective in mediating CDC on PSCA-expressing recombinant B300.19 cells. Deglycosylation greatly reduced the relative ADCC and CDC activities of both MAbs compared to their intact versions. Additional studies to elucidate the role of the effector functions of AGS-1C4D4 are currently being conducted in vivo using intact and deglycosylated MAbs and will be presented. While neither intact antibody had any direct cytotoxic activity on HPAC cells in vitro, both MAbs significantly inhibited tumor formation, local invasion and metastases to distant sites in orthotopic HPAC xenograft tumor models in vivo. In addition, AGS-1C4D4 significantly inhibited the growth and metastasis of established orthotopic HPAC tumors in combination with Gemcitabine. AGS-1C4D4 is currently being evaluated in a Phase 2 clinical study for pancreatic cancer in combination with Gemcitabine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4566. doi:10.1158/1538-7445.AM2011-4566

Human Monoclonal Antibodies to Sialyl-Lewisa (CA19.9) with Potent CDC, ADCC, and Antitumor Activity

The carbohydrate antigen sialyl-Lewis(a) (sLe(a)), also known as CA19.9, is widely expressed on epithelial tumors of the gastrointestinal tract and breast and on small-cell lung cancers. Since overexpression of sLe(a) appears to be a key event in invasion and metastasis of many tumors and results in susceptibility to antibody-mediated lysis, sLe(a) is an attractive molecular target for tumor therapy. We generated and characterized fully human monoclonal antibodies (mAb) from blood lymphocytes from individuals immunized with a sLe(a)-KLH vaccine. Several mAbs were selected based on ELISA and FACS including two mAbs with high affinity for sLe(a) (5B1 and 7E3, binding affinities 0.14 and 0.04 nmol/L, respectively) and further characterized. Both antibodies were specific for Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ as determined by glycan array analysis. Complement-dependent cytotoxicity against DMS-79 cells was higher (EC(50) 0.1 μg/mL vs. 1.7 μg/mL) for r7E3 (IgM) than for r5B1 (IgG1). In addition, r5B1 antibodies showed high level of antibody-dependent cell-mediated cytotoxicity activity on DMS-79 cells with human NK cells or peripheral blood mononuclear cells. To evaluate in vivo efficacy, the antibodies were tested in a xenograft model with Colo205 tumor cells engrafted into SCID (severe combined immunodeficient mice) mice. Treatment during the first 21 days with four doses of r5B1 (100 μg per dose) doubled the median survival time to 207 days, and three of five animals survived with six doses. On the basis of the potential of sLe(a) as a target for immune attack and their affinity, specificity, and effector functions, 5B1and 7E3 may have clinical utility.

In vitro and in vivo activities of KRN330, a fully human monoclonal antibody against colon cancer.

432 Background: KRN330 is a novel recombinant human IgG1 monoclonal antibody (mAb) targeting A33 surface differentiation antigen that is uniformly expressed on the surface of 95% of colorectal cancer (CRC) cells. In this study, we characterized the activity of KRN330 for its in vitro properties, as well as for its in vivo antitumor activity. Methods: A kinetic analysis of the interaction between KRN330 and recombinant human A33 was conducted using a Biacore 3000. Western blot analysis was conducted using A33 expressing COLO205 lysates under reducing and non-reducing conditions. Binding of KRN330 to human colorectal cancer tissues were investigated using FITC-labeled KRN330. We also developed more conventional staining methods of A33 and investigated A33 expression using human colon cancer tissue microarray (TMA). ADCC and CDC activities of KRN330 were assessed using a standard 51Cr release assay. A33 expression levels of 14 CRC cell lines were analyzed using flow cytometer. In vivo antitumor activities of KRN330 alone or in combination with chemotherapeutic agents against subcutaneous or intraperiotoneal human CRC (COLO205 and LS174T) models were investigated using mice and rats xenograft model. Results: A kinetic analysis revealed that KRN330 showed a high binding affinity to A33. Western blot analysis also showed that antibody recognized not any protein under reducing condition, but non-reducing condition. A33 staining of TMA with 204 different samples revealed the majority of tumor expressed A33. KRN330 exhibited ADCC activity against A33 expressing human colorectal cancer cell lines which include both K-ras wild and mutated types. KRN330 showed dose-dependent antitumor activities in vivo. KRN330 also significantly prolonged survival of human colon tumor bearing mice. In addition, combination treatment of KRN330 with irinotecan showed increased antitumor activitiy and prolongation of survival, compared to either irinotecan or KRN330 alone. Conclusions: These results suggest that KRN330 is a promising candidate of novel therapy for CRC. The phase I/II study of KRN330 plus irinotecan in patients with second line metastatic CRC is ongoing. [Table: see text]

Abstract 3512: Novel DNA alkylating agent BO-1090 is a potential candidate for oral cavity cancer therapy

Abstract Human death from oral cancer is the 4th most common in male and over all 6th most common cause of cancer death in Taiwan. Surgery, radiotherapy in combination with chemotherapy is the most common line of treatment for oral cavity cancer. Although cisplain, 5FU, Docetexal, Pinyangmycin etc are the common drugs being used in clinic, there is no any specific drug for oral cavity cancer. In that direction BO-1090 a newly synthesized alkylating agent has potential to be more specific drug for oral cancer based on its potent in vitro cytotoxicty to oral cancer cells, such are OECM-1, SAS, OC3 and CAL-27 in comparison to other cancer cell lines, including lung cancer H1299, prostate cancer cell PC3 and neuronal cell line U87. BO-1090 target DNA for its cytotoxic effects that have been confirmed by alkaline gel shift assay, BrdU based DNA synthesis inhibition assay, Comet assay and γH2x detection. BO-1090 has been tested for its therapeutic potential in SAS cell xenograft mouse. At a dose of 60 mg/Kg i.v five times, it significantly delays the tumor growth in comparison to the control group of mice. Its tumor efficacy in SAS cell xenograft is better than the cisplatin at dose of 4 mg/kg i.v three times. Based on these preliminary results it suggests that B0-1090 can be a useful drug either alone or in combination with other knows drugs in oral cavity cancer therapy. As to the modes of actions of BO-1090, here is activation of CHK-2 check point in response to DNA damage by BO-1090 as well as late activation of p53 and p21. The late activation of P53 may be due to DNA replication blocked by BO1090. During propidium iodide staining based cell cycle analysis, BO-1090 induces G1/ S phase arrest in SAS cell that further confirmed by BrdU incorporation based pulse chase experiment. G1/S phase arrest may be due to combined effects of CDC 25A degradation and activation of p53 and p21 in response to BO-1090 induced DNA damage. G1/S phase arrest in cell by BO-1090 is not a common feature of a DNA alkylating agent, most of DNA alkylating agent induce G2/M phase arrest. In Annexin V based apoptosis assay, our results show that BO-1090 induces apoptosis in SAS and OECM-1 cell by time and dose dependent manner through caspase dependent pathway. By using bioinformatic approach and preliminary experiments suggest that PIM-1 may be also one of its molecular targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3512.

The Virion Host Shut-Off (vhs) Protein Blocks a TLR-Independent Pathway of Herpes Simplex Virus Type 1 Recognition in Human and Mouse Dendritic Cells

Molecular pathways underlying the activation of dendritic cells (DCs) in response to Herpes Simplex Virus type 1 (HSV-1) are poorly understood. Removal of the HSV virion host shut-off (vhs) protein relieves a block to DC activation observed during wild-type infection. In this study, we utilized a potent DC stimulatory HSV-1 recombinant virus lacking vhs as a tool to investigate the mechanisms involved in the activation of DCs by HSV-1. We report that the release of pro-inflammatory cytokines by conventional DC (cDC) during HSV-1 infection is triggered by both virus replication-dependent and replication-independent pathways. Interestingly, while vhs is capable of inhibiting the release of cytokines during infection of human and mouse cDCs, the secretion of cytokines by plasmacytoid DC (pDC) is not affected by vhs. These data prompted us to postulate that infection of cDCs by HSV triggers a TLR independent pathway for cDC activation that is susceptible to blockage by the vhs protein. Using cDCs isolated from mice deficient in both the TLR adaptor protein MyD88 and TLR3, we show that HSV-1 and the vhs-deleted virus can activate cDCs independently of TLR signaling. In addition, virion-associated vhs fails to block cDC activation in response to treatment with TLR agonists, but it efficiently blocked cDC activation triggered by the paramyxoviruses Sendai Virus (SeV) and Newcastle Disease Virus (NDV). This block to SeV- and NDV-induced activation of cDC resulted in elevated SeV and NDV viral gene expression indicating that infection with HSV-1 enhances the cell's susceptibility to other pathogens through the action of vhs. Our results demonstrate for the first time that a viral protein contained in the tegument of HSV-1 can block the induction of DC activation by TLR-independent pathways of viral recognition.

GA101-Coated Target Cells Are More Effective Than Rituximab-Coated Target Cells at Activating NK Cells When Complement Is Present.

Abstract 2707Poster Board II-683Rituximab has had a major impact on the treatment of B cell malignancies. The mechanisms responsible for mediating the anti-tumor effects of rituximab are complex. For example, complement can have both positive and negative effects on the ability of rituximab to induce target cell lysis. In particular, we recently reported that rituximab-mediated complement activation results in C3b deposition on the rituximab Fc. C3b then impedes interaction between rituximab and NK cell CD16, thereby limiting NK cell activation and ADCC.GA101 is a type II anti-CD20 monoclonal antibody that mediates enhanced direct cell death induction. It has significantly reduced CDC activity compared to type I anti-CD20 antibodies such as rituximab. In addition, GA101 was engineered to mediate increased ADCC (Umana et al., ASH 2007). The current studies were designed to assess whether the decreased ability of GA101 to activate complement results in an enhanced ability of GA101 to activate NK cells when complement is present. [Display omitted] Peripheral blood mononuclear cells (PBMCs) were obtained from normal donors and added to Raji cells (Burkitt lymphoma cell line) at a 1:1 ratio. Various concentrations of rituximab or GA101 were added along with media, 20% autologous serum or 20% heat-inactivated autologous serum (heated to 57°C for 30 minutes). Samples were cultured for 20 hours. NK cell (CD3−, CD56+) activation, as determined by phenotypic changes, was evaluated by flow cytometry based on prior studies demonstrating that downmodulation of CD16, and upregulation of CD54 and CD69 are reproducible surrogates for mAb-induced NK activation and ADCC. Raji cells coated with either rituximab or GA101 were able to activate NK cells when cultures were performed in media alone or with heat-inactivated serum (left panel). In contrast, serum blocked the ability of rituximab to activate NK cells, but not the ability of GA101 to activate NK cells (right panel). Similar results were found when upregulation of CD69 or downmodulation of CD16 were evaluated as markers of NK activation and using PBMCs from two other donors.We conclude that the presence of complement does not limit the ability of GA101-coated target B cells to activate NK cells. This is in contrast to rituximab-coated target B cells which are unable to activate NK cells in the presence of serum. These results suggest that the decreased ability of GA101 to fix complement could, paradoxically, enhance the efficacy of GA101 by resulting in enhanced activation of NK cells and increased ADCC. Disclosures:No relevant conflicts of interest to declare.

Dysregulation of the cell survival/anti-apoptotic NF-κB pathway by the novel humanized BM-ca anti-CD20 mAb: Implication in chemosensitization

Treatment of patients with relapsed or refractory low grade follicular B-NHL lymphoma with rituximab (chimeric anti-CD20 mAb) has resulted in approximately 50% response rate. The mechanism underlying the failure of rituximab to affect the remaining 50% of the patients is not clear, though their tumors express CD20. The in vivo effector functions of rituximab include ADCC, CDC and seldom apoptosis. In addition, we have reported that rituximab signals the cells and inhibits several intracellular cell survival pathways that are responsible for the immuno and chemo-sensitizing effects of rituximab on resistant B-NHL cell lines. The objective of this study was to develop novel and fully humanized anti-CD20 monoclonal antibodies with enhanced effector functions and molecular signaling that may potentiate their therapeutic efficacy. Novel humanized anti-CD20 monoclonal antibodies were derived from a chimerized form of murine anti-CD20 1K11791, shown to exert a more potent ADCC, CDC and apoptotic activities compared to rituximab. A representative humanized monoclonal antibody, BM-ca was used to examine its biological effect and molecular signaling using Ramos B-NHL cell line as a model. The studies were also performed in parallel with rituximab treatment for comparison. Ramos cells were treated with various concentrations of BM-ca monoclonal antibody. Inhibition of cell proliferation was observed in a concentration-dependent manner, suggesting cell signal perturbations must have occurred. Compared to untreated cells, treatment with BM-ca inhibited both the constitutively activated NF-kappaB and p38 MAPK pathways, as assessed by inhibition of both phospho-p65 and phospho-IkappaBalpha and phospho-p38, respectively, but not the unphosphorylated forms. BM-ca significantly induced the expression of the metastasis suppressor and immune surveillance cancer gene product, Raf-1 kinase inhibitor protein (RKIP). These alterations resulted in inhibition of anti-apoptotic gene products and sensitized Ramos cells to apoptosis by CDDP. In comparison with rituximab, BM-ca showed qualitative and quantitative differences in the above analyses. These findings demonstrate that BM-ca triggers CD20 expressing B-NHL cells resulting in a significant alteration of several gene products that regulate cell growth and chemoresistance.

Capacitance of KOH activated carbide-derived carbons

Chemical activation of CDC leads to capacitance values in an organic electrolyte as high as 180 F/g, 30% larger than those of as-produced samples, due to the surface area and microporosity development occurring as a consequence of the activation.

Bispecific Anti-CD20/Anti-CD22 Antibodies with Potent Lymphoma Cytotoxicity.

BACKGROUND: Multivalent bispecific antibodies (bsMAbs) have shown improved efficacy in a number of preclinical studies. We made a pair of hexavalent bsMAbs based on epratuzumab (hLL2, anti-CD22) and veltuzumab (hA20, anti-CD20) by the Dock-and-Lock (DNL) method, and compared their properties to each other and their parental MAbs.METHODS AND RESULTS: DNL is used for the site-specific and covalent assembly of modular components, and was utilized to generate stably tethered hexavalent bispecific complexes, each composed of 4 Fab fragments conjugated to an IgG at the latter's carboxyl termini of the heavy chain. DNL-1 has 4 veltuzumab Fabs tethered to epratuzumab IgG; DNL-2 has 4 epratuzumab Fabs bound to veltuzumab IgG. DNL made each construct as 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 lymphoma cell lines demonstrated that, unlike the parental MAbs, the bsMAbs each induced translocation of both CD22 and CD20 into lipid rafts and also strong cell-cell adhesion. DNL1 or DNL2 treatment resulted in increased apoptosis vs. the parental MAbs alone or combined. DNL1 and DNL2 inhibited the growth of Ramos, Raji and Daudi Burkitt lymphoma cell lines without the requirement of crosslinking, and more potently than the combination of the parental MAbs. For Daudi cells, DNL1 and DNL2 showed similar activity, which was approximately 50-fold more potent than the combination of the parental MAbs. For Raji and Ramos, DNL1 was 8–10-fold more potent than DNL2, which in turn was 8–10-fold more potent than the combined parental MAbs. The results suggest that crosslinking of CD20 and CD22 at the cell surface is required for enhanced cytotoxicity. Veltuzumab, but neither epratuzumab nor either bsMAb, displayed CDC activity. Veltuzumab and DNL2 induced a similarly high degree of ADCC. DNL1 induced ADCC to an intermediate level between veltuzumab/DNL2 and epratuzumab. In an ex-vivo assay using fresh whole blood mixed with either Daudi or Raji, DNL1 and DNL2 each demonstrated selective killing of lymphoma cells over normal B-cells compared to veltuzumab or rituximab; the latter depleted normal B-cells with greater efficiency than the bsMAbs. PK studies in mice demonstrated that despite their large size, the bsMAbs have a significantly shorter serum half-life than the IgGs. Even without CDC activity and with a considerably shorter serum half-life, DNL2 had anti-lymphoma efficacy in the Daudi Burkitt lymphoma model in mice that was equivalent to veltuzumab. DNL1 was less potent than DNL2 in vivo, but more effective than epratuzumab and control bsMAbs comprising either epratuzumab-IgG-AD2 (22-14) or 4 veltuzumab-Fab-DDD2 groups (734-22) combined with 4 non-binding Fab-DDD2 groups or a non-binding IgG-AD2, respectively. The anti-tumor efficacy of both DNL1 and DNL2 was abolished in tumor-bearing mice in which their ADCC potential was diminished by depletion of neutrophils and NK cells. These findings suggest that ADCC is the most critical mechanism of action for lymphoma killing in these murine models. DNL1 is more potent than DNL2 in vitro yet DNL2, having the stronger ADCC activity, is more potent than DNL1 in vivo.CONCLUSIONS: These findings suggest that the DNL method can be used to make a variety of multivalent bsMAbs with potent anti-tumor activity, and having distinct properties dependent on their arrangement and composition. CD20/CD22 bsMAbs appear to have different functions than their parental MAbs, even when these were combined, and appear to be potent anti-B-cell lymphoma therapeutics.

A Humanized Anti-Ganglioside GM2 Antibody, BIW-8962, Exhibits ADCC/CDC Activity against Multiple Myeloma Cells and Potent Anti- Tumor Activity in Mouse Xenograft Models.

BIW-8962 is a humanized anti-ganglioside GM2 (GM2) monoclonal antibody, produced by Poteligent technology to enhance ADCC activity. GM2 is expressed on many cancer cells including multiple myeloma (MM), small cell lung cancer and glioma cells. In this study, we evaluated the anti-myeloma activity of BIW-8962 in preclinical myeloma models both in vitro and in vivo. Expression of GM2 was analyzed in 15 human MM cell lines by FCM. Eleven out of 15 MM cell lines had positive surface expression of GM2. GM2 as a potential target was then verified in primary MM samples obtained from patients. Eleven out of 15 samples were positive for GM2. We then used two GM2 positive MM cell lines (U266B1 and KMS-11) and evaluated ADCC and CDC activity of BIW-8962 in vitro. BIW-8962 exhibited a potent ADCC and less potent CDC activity. In vivo anti-tumor activity of BIW-8962 was then examined using the standard subcutaneous xenograft model; KMS-11 was inoculated in the flank of SCID mice. BIW-8962 (intravenously administered biweekly for 3 weeks) exhibited a potent anti-tumor activity from as low a dose level as 0.1 mg/kg. Furthermore, in a more clinically relevant model, in which OPM-2/GFP (GM2 positive MM cell line) cells were intravenously inoculated into SCID mice with preferentially tumor growth within the bone marrow microenvironment, BIW-8962 (intravenously administered biweekly for 4 weeks, 10 mg/kg) suppressed OPM-2/GFP cell growth and serum M protein elevation, demonstrating in vivo anti-myeloma effect of BIW-8962. Our preclinical investigations rationalize clinical evaluation of BIW-8962 in patients with MM. Currently BIW-8962 is being investigated in a Phase 1 study in patients with multiple myeloma.

Inhibition of NF-Kb Cell Signaling and Induction of the Metastasis Suppressor and Immune Surveillance Cancer Gene RKIP Expression in B-NHL by the Novel Humanized BM-Ca Anti-CD20 Monoclonal Antibody: Implication in Chemo and Immune-Sensitization

Treatment of patients with relapsed or refractory low-grade follicular B-NHL lymphoma with rituximab has resulted in ~50% response rate. The mechanism underlying the failure of rituximab to affect the remaining 50% of the patients is not clear, though their tumors express CD20. The in vivo effector functions of rituximab include ADCC, CDC, and seldom apoptosis. In addition, we have reported that rituximab signals the cells and inhibits several intracellular cell survival pathways that are responsible for the immune and chemo-sensitizing effects of rituximab on resistant B-NHL cell lines (see review Jazirehi and Bonavida, Oncogene 24:2121, 2005). The objective of this study was to develop novel and fully humanized anti-CD20 monoclonal antibodies with enhanced effector functions and molecular signaling that may potentiate their therapeutic efficacy. Novel humanized anti-CD20 monoclonal antibodies were derived from a chimerized form of murine anti-CD20 1K11791, shown to exert more important ADCC, CDC, and apoptotic activities as compared to rituximab (Nishida et al., Int J Oncol. , 31:29, 2007). A representative humanized monoclonal antibody, BM-ca (Biomedics Inc., Tokyo, Japan) was used to examine its molecular cell signaling on a representative Ramos B-NHL cell line. The studies were also performed in parallel with rituximab treatment. Ramos cells were treated with various concentrations of BM-ca monoclonal antibody and it was found to inhibit cell proliferation in a concentration-dependent manner. The optimal concentration of BM-ca mAb (20–40 μg/ml) was used for further molecular analyses. Following treatment of Ramos with BM-ca mAb for 24 hours, cell lysates were assessed for the expression of various gene products by Western. Compared to untreated cells, treatment with BM-ca inhibited constitutively activated NF-kB as assessed by inhibition of phospho-p65, phospho-IkB-α, but not unphosphorylated forms. In addition, the NF-kB upstream kinase phospho-p38 MAPK was also inhibited. Inhibition of NF-kB and phospho-p38 MAPK resulted in downstream inhibition of the anti-apoptotic gene product Mcl-1 and induction of the proapototic gene products Bax and Bak. BM-ca significantly induced the expression of the metastasis suppressor and immune surveillance cancer gene product, Raf-1 kinase inhibitor protein (RKIP). RKIP has been shown to inhibition NF-kB activity and inhibition of NF-kB results downstream in the inhibition of the metastasis-inducer gene product Snail. Snail is a transcription factor that has been shown recently to negatively regulate the expression of RKIP (Beach et al, Oncogene 27:2243, 2008). BM-ca inhibited Snail expression in Ramos cells. In comparison with rituximab, BM-ca showed qualitative and quantitative differences in the regulation of gene expression. These findings demonstrate that BM-ca triggers CD20-expressing B-NHL cells and results in significant alteration of several gene products that regulate chemo and immune-resistance and metastasis.

Anti-EpCAM XmAb antibodies with improved cytotoxicity

12506 Background: The epithelial cell adhesion molecule (EpCAM), also known as epithelial protein 2 (EGP-2) or 17–1A antigen, is a trans-membrane protein expressed on the surfaces of most carcinomas, including those of pancreatic, colorectal, prostate, breast, kidney, lung, and ovarian origins. Moderate affinity antibodies (Abs) such as 17–1A (Kd ∼ 10−7 nM) have been safe in humans albeit with limited clinical efficacy. Attempts to improve clinical efficacy by enhancing antigen affinity (Kd ∼ 10−9 nM) have led to serious clinical toxicity, including pancreatitis. These observations raise the question of whether a moderate affinity Ab with enhanced effector function will be both safe and clinically efficacious. Methods: We applied our proprietary XmAb™ technologies to humanize the 17–1A variable domain and engineer a human IgG1 Fc domain to increase affinity for the activating receptor FcγRIIIa. Ab binding to Ep-CAM or to Fc receptors was tested with Biacore and/or AlphaScreen binding assays. In vitro cytotoxic activity against representative cancer cell lines was measured with Antibody Dependent Cell-mediated Cytotoxicity (ADCC) assays, using human PBMC as effector cells. Results: Humanized anti-EpCAM Abs have affinity for EpCAM similar to the parent 17–1A. Affinity for the activating FcγRIIIa was increased 100-fold relative to a control Ab with an IgG1 Fc domain. As expected, these Abs exhibit dramatically enhanced ADCC against multiple cancer cell lines relative to 17–1A and IgG1 control Abs. Despite their moderate affinity for EpCAM, these novel Abs have in vitro cytotoxicity comparable to the high affinity Ab ING-1. CDC activities of these Abs were similar to chimeric 17–1A. Conclusions: We have demonstrated that antibodies with moderate affinity for EpCAM and increased FcγRIIIa affinity exhibit superior cancer cell killing via an ADCC mechanism. The humanized nature and the increased cytotoxicity of anti-EpCAM XmAb™ antibodies make them promising candidates for clinical development of a novel pan-carcinoma Ab that is superior to 17–1A. [Table: see text]

Fully Human CD38 Antibodies Efficiently Trigger ADCC of Multiple Myeloma Cell Lines and Primary Tumor Cells.

Although new treatment modalities have recently been added to the standard regimens for multiple myeloma, the clinical outcome for patients with advanced disease is often limited. Monoclonal antibodies are increasingly used for tumor therapy, and may also represent interesting options for multiple myeloma patients. CD38 is one of the most promising target antigens on malignant plasma cells, which are evaluated in preclinical and early clinical studies as targets for antibody therapy. CD38 is a type II transmembrane protein with ectoenzymatic activity, which is involved in calcium mobilization. Human CD38 is predominantly expressed by bone marrow precursor cells and by terminally differentiated plasma cells. Multiple myeloma cells show moderate to high expression levels - making CD38 a potential candidate as target for immunotherapy.A panel of 42 fully human CD38 antibodies was generated by immunizing human Ig transgenic mice. Immunofluorescence studies with CD38 transfected cells demonstrated antigen-specific, high affinity binding, and cross-blocking experiments revealed four distinct epitope groups. Seven antibodies, representing each of the four groups, were selected for further analyses. ADCC and CDC activity against CD38-positive myeloma cell lines (AMO-1 and JK6), and against freshly-isolated primary multiple myeloma cells was investigated. Human whole blood served as effector source, which was then fractionated into plasma (containing human complement), mononuclear (MNC) or granulocytic (PMN) effector cells. All antibodies mediated concentration-dependent killing of both multiple myeloma cell lines - using human mononuclear cells as effector source. Also complement-dependent killing of freshly isolated myeloma cells was observed. However, none of the antibodies recruited PMN for tumor cell lysis. Importantly, CD38 antibodies also killed freshly isolated tumor cells from a rare patient with a CD38/138- positive plasma cell leukemia, which was chemotherapy- refractory at the time of analysis. Furthermore, CD38 antibodies effectively prevented outgrowth of CD38-positive tumor cells in SCID mouse xenograft models. Antibody 005 was significantly more effective in these assays compared to the remaining panel of CD38 antibodies.In conclusion, CD38 antibodies efficiently mediated killing of multiple myeloma cell lines as well as freshly isolated tumor cells and prevented tumor outgrowth in xenografted SCID mice. Antibody 005 was superior in mediating CDC and ADCC via MNC - particularly at low antibody concentrations.

Distinct roles for the NF-κB1 and c-Rel transcription factors in the differentiation and survival of plasmacytoid and conventional dendritic cells activated by TLR-9 signals

AbstractReticuloendotheliosis viral oncogene homolog/nuclear factor of kappa light polypeptide gene enhancer in B cells 1 (Rel/NF-κB) activation is a ubiquitous outcome of engaging Toll-like receptors (TLRs), yet the cell-type–specific functions of this pathway in response to particular microbial signals remain poorly defined. Here we show that NF-κB1 and C-Rel, Rel/NF-κB proteins induced in conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) by cytosine-phosphate-guanosine (CpG) DNA, a TLR-9 ligand, serve markedly different functions in these DC subsets. With the exception of impaired interleukin-12 (IL-12) production, cultured Nfkb1–/–C-Rel–/– cDCs responded relatively normally to CpG DNA. In contrast, CpG-treated Nfkb1–/–C-Rel–/– pDCs, which were still able to produce type I interferon and regulated on activation normal T-cell expressed and secreted (RANTES), but not IL-6 or IL-12, failed to acquire an activated dendritic phenotype and underwent apoptosis. Although the TLR-9–mediated death of Nfkb1–/–C-Rel–/– pDCs, which coincided with a failure to up-regulate the prosurvival proteins B-cell lymphoma apoptosis regulator xL (Bcl-xL) and A1, was blocked by Bcl-2 transgene expression, this inhibition of apoptosis still failed to rescue the differentiation defects. This indicated that these NF-κB transcription factors independently regulate TLR-9–mediated pDC morphogenesis and survival. Collectively, these findings establish that NF-κB1 and c-Rel, while largely dispensable for TLR-9–induced cDC activation, are critical for regulating differentiation and survival programs during pDC activation.

TRU-015, a novel CD20-directed biologic therapy, demonstrates significant anti-tumor activity in human tumor xenograft models

2549 Background: Small Modular Immuno-Pharmaceuticals (SMIPs) represent a novel proprietary biologic compound class that retain Fc mediated effector functions and are smaller than monoclonal antibodies. TRU-015, a SMIP that binds to CD20, depletes B cells in vitro and in animal models. TRU-015 has potent ADCC and apoptotic activity in vitro. By design, TRU-015 has attenuated CDC activity. We have evaluated the activity of TRU-015 in established human tumor xenograft models in nude mice. Methods: Nude mice were injected sub cutaneously with either Ramos or Daudi tumor cells. When large palpable tumors (> 200 mm3) were evident, mice (N ≥ 8/group) were treated with TRU-015 or comparator molecules. Groups were evaluated for median survival time (MST), tumor volume and percentage of tumor-free animals. Results: In nude mice bearing Ramos tumors >200mm3, TRU-015 treated mice demonstrated a significant enhancement in their median survival time (MST) in comparison to control mice (MST 44.5 d vs 11 d, p < 0.0001). In a more demanding model (tumor size > 370 mm3), TRU-015 treated mice also demonstrated a significant improvement in their MST in comparison to control mice (64.5 d vs 11 d, p < 0.0001). In nude mice bearing Daudi tumors, TRU-015 treated mice also showed a significant improvement in MST (40.5 d) in comparison to both rituximab (18 d, p<0.02) and control mice (10d, p<0.02). In addition, TRU-015 treated mice had decreased mean tumor volumes and a greater percentage of complete tumor regressions as compared to the other treatment groups in these tumor models. Conclusions: TRU-015 is effective in treating large, established tumors in these human tumor xenograft models. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Trubion Trubion