De Haard Research Articles

Blocking CD70/CD27 Signaling in Combination with Hypomethylating Agents Eradicates Human CD34+ AML Stem and Progenitor Cells in Vitro and In Vivo

Leukemia stem cells (LSCs) are the origin of acute myeloid leukemia (AML) and are resistant to standard therapeutic regimens resulting in frequent relapse of the disease and poor prognosis especially for AML patients not fit for intensive chemotherapy. Consequently, LSCs represent a major obstacle for AML therapy. We recently identified the interaction of the TNF ligand CD70 and its receptor CD27 on LSCs as a promising therapeutic strategy to target LSCs.In this study, we demonstrate for the first time that treatment with hypomethylating agents (HMA, azacitidine or decitabine) significantly up-regulates CD70 mRNA and protein expression on primary CD34+ stem/progenitor cells from newly diagnosed AML patients independent of the cytogenetic/molecular risk category in vitro (mRNA: 4.3±1.6 fold increase, FACS: 4.0±0.9 fold increase vs. vehicle treatment, n=15). Similarly, CD34+ stem/progenitor cells from AML patients after 5 days decitabine administration (20mg/kg by continuous intravenous infusion over 1 hour repeated daily) exhibited a significant increase in CD70 expression as analyzed by flow-cytometry (6.8±1.1 fold increase vs. diagnostic sample, n=6). In contrast, CD70 expression of CD34+ stem/progenitor cells from healthy donor bone marrow (n=3) was not affected by the treatment.Consequently, co-treatment of CD34+ stem/progenitor cells from AML patients of each risk group (n=3) with the HMA decitabine and a blocking αCD70 monoclonal antibody strongly reduced colony-forming and re-plating capacity in vitro compared to single agent treatment. To confirm and extend our findings towards disease-initiating CD34+ AML LSCs in vivo, we performed patient-derived AML xenografts (PDX) from one patient with intermediate and one patient with adverse cytogenetic risk profile. Combining decitabine treatment with CD70 blockade significantly reduced AML engraftment in blood, spleen and BM compared to controls. Moreover, co-treatment significantly reduced frequency and absolute numbers of CD34+CD38- and CD34+CD38-CD45RA- AML LSCs in the BM as analyzed phenotypically by FACS and eliminated human LSCs as analyzed in limiting dilution PDX experiments compared to single treatments. Importantly, CD34+ hematopoietic stem/progenitor cells from healthy human bone marrow (n=3) were only marginally affected by the co-treatment.In summary, combining HMAs with blocking CD70/CD27 signaling could represent a novel strategy to eradicate human LSCs. DisclosuresVan Rompaey:argenX: Employment. Leupin:argenX: Employment. De Haard:argenX: Employment.

Efficacy and Safety of Intravenous Efgartigimod 10 Mg/Kg in Adult Patients with Primary Immune Thrombocytopenia: Advance, a Phase 3 Clinical Trial in Progress

Efficacy and Safety of Intravenous Efgartigimod 10 Mg/Kg in Adult Patients with Primary Immune Thrombocytopenia: Advance, a Phase 3 Clinical Trial in Progress

Targeting CD70 with Cusatuzumab Eliminates Acute Myeloid Leukemia Stem Cells in Humans

Introduction Acute myeloid leukemia (AML) is a heterogenous hematological malignancy driven by leukemia stem cells (LSCs) (Lapidot et al, 1994). LSCs resistant against conventional chemotherapy represent the major cause of relapse. Elderly or unfit AML patients not eligible for intensive chemotherapy are treated in a palliative setting with hypomethylating agents (HMA) or low dose Ara-C, but responses are modest and not durable. The reason for the low efficacy of HMA treatment is their insufficient action on the disease initiating- and -maintaining LSC population (Craddock et al, 2013). We recently demonstrated that CD34+ AML cells (progenitors and LSC) consistently express the tumor necrosis factor family ligand CD70 as well as its receptor CD27 and that cell-autonomous CD70/CD27-signaling propagates the disease (Riether et al. 2017). The aim of the current study was to evaluate whether resistance to HMA treatment can be overcome by combining HMA with an anti-CD70 monoclonal antibody treatment. Experimental design The effect of HMA treatment on the expression of CD70 on primary human CD34+CD38- AML LSCs was determined in vitro cultures and in patients treated with HMA in vivo. The therapeutic potential of targeting CD70-expressing LSCs in presence and absence of HMA was assessed using the anti-CD70 ADCC-optimized monoclonal antibody (mAb), cusatuzumab, and an effector-dead anti-CD70 mAb in colony formation and re-plating assays as well as patient-derived xenograft models (Silence et al, 2014). The clinical relevance of the findings was determined in a clinical phase 1 trial in previously untreated elderly AML patients with a single dose of cusatuzumab monotherapy followed by a combination therapy with the HMA azacitidine (AZA, NCT03030612). Four different dose levels of cusatuzumab (1, 3, 10 and 20 mg/kg Q2W) were studied; AZA was administered at 75 mg/m² for 7 days every 28 days. Results We found that resistance of AML LSCs to HMA treatment is mediated by the up-regulation of the CD70. The up-regulation of CD70 triggered cell-autonomous CD70/CD27 signaling on AML LSCs. Based on these findings we hypothesized that the upregulation of CD70 by HMA may render LSCs more susceptible to CD70-targeting interventions. Targeting CD70-expressing LSCs by a blocking anti-CD70 mAb and the anti-CD70 mAb cusatuzumab, which blocks CD70/CD27-signaling and additionally mediates ADCC and CDC, eradicated LSCs in colony and re-plating assays in vitro and in xenotransplantation experiments in vivo. HMA in combination with blocking αCD70 mAb synergistically reduced LSC numbers in vivo and this was even more efficient when ADCC-enhanced αCD70 mAb cusatuzumab was added in the presence of NK cells. In order to test the hypothesis that targeting CD70 in combination with HMA eliminates LSCs in AML patients, we initiated a phase 1 dose-escalation trial in previously untreated elderly AML patients with a single dose of cusatuzumab monotherapy followed by a combination therapy with azacitidine. No dose-limiting toxicities (DLT) were observed in the dose-escalation phase 1 trial and responses were observed across the dose levels (1-20 mg/kg). A single dose of cusatuzumab reduced bone marrow blasts in just two weeks in all patients on average by 32%. Cusatuzumab monotherapy significantly reduced LSC numbers and frequencies in all patients analyzed in the bone marrow as assessed in limiting dilution colony assays. Single cell sequencing analysis revealed that cusatuzumab induced gene signatures related to myeloid differentiation and apoptosis in LSCs. In combination with azacitidine, cusatuzumab induced CR/CRi in 10 out of 12 patients. Responses were observed at all dose levels of cusatuzumab and median time to response was 3.3 months. Conclusions Blocking CD70/CD27-signaling and targeting CD70-expressing LSCs by the ADCC-optimized mAb, cusatuzumab, eliminated LSCs in vitro and in xenotransplantation experiments. In a phase 1 study promising activity of cusatuzumab in combination with HMA was observed in AML patients, in which translational data indicate that cusatuzumab selectively eliminates CD70-expressing LSCs. Disclosures Van Rompaey: argenx: Employment, Equity Ownership, Patents & Royalties. Moshir:Argenx: Employment, Equity Ownership. Delahaye:argenx: Employment, Equity Ownership. Gandini:Argenx: Employment, Equity Ownership. Erzeel:argenx: Employment, Equity Ownership. Hultberg:Argenx: Employment. Fung:argenx: Consultancy, Equity Ownership. De Haard:Argenx: Employment, Equity Ownership, Patents & Royalties. Leupin:Argenx: Employment, Equity Ownership, Patents & Royalties.

The Combination of the BCL-2 Antagonist Venetoclax with the CD70-Targeting Antibody Cusatuzumab Synergistically Eliminates Primary Human Leukemia Stem Cells

Introduction Acute myeloid leukemia (AML) is a very heterogeneous hematological malignancy characterized by the accumulation of myeloid blasts. Treatment options for unfit AML patients greater than 65 year of age are still limited and outcomes are dismal. The current standard of care for older AML patients are hypomethylating agents (HMA) or low dose Ara-C. However, even though inducing hematological remissions in up to 30 percent of the patients, responses are not durable and survival of these patients is only marginally prolonged. The poor durability is due to insufficient action on leukemia stem cells (LSC) which drive and maintain the disease and are resistance to therapy (Craddock et al, 2013 and DiNardo et al, 2019). We described how aberrant CD70/CD27 signaling drives stemness of AML LSCs and identified CD70 as potential new target for the treatment of AML patients (Riether et al, 2017). In a recent Phase 1 clinical trial, treatment of older and unfit AML patients with the ADCC-enhanced humanized monoclonal anti CD70 antibody (mAb) cusatuzumab in combination with HMA demonstrated promising clinical activity and a favorable tolerability profile. The BCL-2 antagonist, venetoclax, targets and eliminates LSCs by suppression of oxidative phosphorylation and demonstrated very promising activity in older AML patients in clinical phase I and II studies in combination with standard of care (Pollyea et al, 2018). However, even with novel agents such as venetoclax, there are still patients that become refractory or relapse. We hypothesized that combining venetoclax and cusatuzumab with distinct but complementary mechanisms of action could successfully eliminate LSCs. Experimental design To test this hypothesis, we performed a drug-combination study according to the Chou-Talalay method (Chou 2010) in CD70-expressing AML cell lines such as MOLM-13, MV4-11, and NOMO-1 cells in vitro. In addition, we tested the effect of the cusatuzumab/venetoclax and the cusatuzumab/venetoclax/HMA combination on colony formation and re-plating capacity of primary CD34+CD38- LSCs from newly diagnosed AML patients. Results We first treated MOLM-13, MV4-11, and NOMO-1 AML cells with vehicle, cusatuzumab alone or in combination with venetoclax or decitabine in a constant ratio in the presence of CFSE-labeled NK cells (ratio 1:1). AML cell numbers were assessed 72 hours later. Cusatuzumab in combination with venetoclax or decitabine and NK cells synergistically eliminated CD70-expressing AML cells in a broad dose range (Figure 1). To assess the effect of the cusatuzumab/venetoclax combination on primary human AML LSCs, we treated CD34+ CD38- LSCs with cusatuzumab or venetoclax monotherapy or in combination in the presence of NK cells and assessed colony formation. Cusatuzumab/venetoclax co-treatment was more efficacious than each monotherapy alone and strongly reduced LSCs and leukemia progenitors (Figure 2). To analyze the effect of the cusatuzumab/venetoclax treatment on LSC function in a more stringent way, we performed serial re-plating experiments in vitro. The impaired colony formation after combination treatment observed after the first plating was maintained during subsequent the re-plating, even though cusatuzumab and venetoclax were not present in the re-plating, indicating an effective reduction of LSCs. Mechanistically, we could show that treatment with venetoclax results in up-regulation of CD70 on LSCs, suggesting that venetoclax renders LSCs more susceptible to cytolytic killing with cusatuzumab. In older AML patients, cusatuzumab and venetoclax each have demonstrated promising clinical activity in combination with HMA. We determined whether addition of HMA to the cusatuzumab/venetoclax co-treatment could more effectively eliminate LSCs. The triplet combination cusatuzumab/venetoclax/decitabine did not further reduce growth of AML cell lines in vitro (Figure 1) nor colony and re-plating capacity of human LSCs compared to the cusatuzumab/venetoclax co-treatment. Conclusions Overall, these results indicate that a combination of cusatuzumab with venetoclax eliminates LSCs synergistically and more efficiently than as a monotherapy. Tolerability and efficacy will be tested in mouse models using both primary AML and cell line xenografts. The results suggest that targeting LSCs by combining venetoclax and cusatuzumab treatments is a promising novel treatment strategy in AML. Disclosures Johnson: Janssen R&D: Employment. Drenberg:Janssen R&D: Employment. Syed:Janssen R&D: Employment. Moshir:Argenx: Employment, Equity Ownership. Hultberg:Argenx: Employment. Leupin:Argenx: Employment, Equity Ownership, Patents & Royalties. De Haard:Argenx: Employment, Equity Ownership, Patents & Royalties.

Phase 2 Study of Efgartigimod, a Novel FcRn Antagonist, in Adult Patients with Primary Immune Thrombocytopenia

Primary immune thrombocytopenia (ITP) is an acquired autoimmune bleeding disorder, characterized by a low platelet count (<100 × 109 /L) in the absence of other causes associated with thrombocytopenia. In most patients, IgG autoantibodies directed against platelet receptors can be detected. They accelerate platelet clearance and destruction, inhibit platelet production, and impair platelet function, resulting in increased risk of bleeding and impaired quality of life. Efgartigimod is a human IgG1 antibody Fc-fragment, a natural ligand of the neonatal Fc receptor (FcRn), engineered for increased affinity to FcRn, while preserving its characteristic pH-dependent binding. Efgartigimod blocks FcRn, preventing IgG recycling, and causing targeted IgG degradation. In this Phase 2 study, 38 patients were randomized 1:1:1 to receive four weekly intravenous infusions of either placebo (N = 12) or efgartigimod at a dose of 5 mg/kg (N = 13) or 10 mg/kg (N = 13). This short treatment cycle of efgartigimod in patients with ITP, predominantly refractory to previous lines of therapy, was shown to be well tolerated, and demonstrated a favorable safety profile consistent with Phase 1 data. Efgartigimod induced a rapid reduction of total IgG levels (up to 63.7% mean change from baseline), which was associated with clinically relevant increases in platelet counts (46% patients on efgartigimod vs 25% on placebo achieved a platelet count of ≥50 × 109 /L on at least two occasions, and 38% vs 0% achieved ≥50 × 109 /L for at least 10 cumulative days), and a reduced proportion of patients with bleeding. Taken together, these data warrant further evaluation of FcRn antagonism as a novel therapeutic approach in ITP.

Argx-110 Targeting CD70, in Combination with Azacitidine, Shows Favorable Safety Profile and Promising Anti-Leukemia Activity in Newly Diagnosed AML Patients in an Ongoing Phase 1/2 Clinical Trial

Outcomes in elderly patients with acute myeloid leukemia (AML) are still adverse, as the majority does not qualify for intensive therapy or allogenic stem cell transplantation (ASCT). DNA hypomethylating agents (HMAs) induce remissions and prolong survival in a fraction of these patients. However, overall prognosis remains dismal and all patients progress due to therapy-resistant leukemia stem cells (LSCs). We recently demonstrated that HMAs upregulate the expression of CD70 on primary human AML LSCs, potentially contributing to HMA resistance and that blocking the cell-autonomous CD70/CD27 signaling inhibits proliferation and myeloid differentiation of LSCs and contributes to HMA resistance. Consequently, combining HMA treatment with a blocking αCD70 monoclonal antibody potently reduced colony formation of AML LSCs in vitro and effectively eliminated human AML LCSs in xenograft experiments. Based on these results, we initiated an open-label, non-controlled, non-randomized Phase 1/2 trial combining the HMA azacitidine (AZA) with ARGX-110, a human monoclonal antibody targeting CD70, in newly diagnosed AML patients unfit for intensive chemotherapy (ARGX-110-1601, NCT03030612).Trial designARGX-110 with optimized antibody-dependent cell-mediated cytotoxicity (ADCC), is administered intravenously at 1, 3, 10 or 20 mg/kg once every two weeks (Q2W), in combination with a standard dose of AZA (75 mg/m² subcutaneously for 7 days every 28 days). A 2-week lead-in of ARGX-110 enables studying the effect of αCD70 antibody monotherapy. Primary objectives in the Phase 1 include determining the maximum tolerated dose of ARGX-110 in combination with AZA and the recommended phase 2 dose (RP2D). Secondary objectives comprise safety, pharmacokinetics and anti-leukemic activity of ARGX-110 alone or in combination with AZA (including overall response rate (ORR), frequencies of LSCs and minimal/measurable residual disease (MRD)).ResultsThe trial is ongoing and as of 16th July 2018, 12 newly diagnosed AML patients were treated in the dose-escalation part of the trial, the results of which will be further updated in December 2018. The median age over the dose cohorts was 75 years (range 64-84) and included intermediate (N=6) and adverse (N=6) ELN risk groups. WHO subtypes were AML with recurrent genetic abnormalities (N=2), MDS-related changes (N=6), AML-NOS (N=3) and therapy-related myeloid neoplasms (N=1).No dose-limiting toxicity was observed and the combination of ARGX-110 with a standard dose of AZA was well tolerated. Hematological toxicities, in line with expected AZA toxicities, were the most frequently reported adverse events (AEs).At the cut-off, the ORR (complete remission [CR] + incomplete recovery [CRi] + partial remission [PR] + morphologic leukemia-free status [MLFS]) was 92% (11/12 patients); among the 11 evaluable subjects, the best response was CR/CRi for 9 patients (82%), 1 patient (9%) reached MLFS, and 1 (9%) PR, with 7 patients still on the trial (median duration on the trial at cut-off was 6.9 months [range: 2-14.4 months]). Significantly, 1 patient reached CR and was discontinued after 5 months to undergo ASCT. Five evaluable patients (45%) achieved MRD negativity by flow cytometry. Molecular MRD negativity was achieved in 3/8 patients with available results, two correlating with flow-MRD negativity. The mean time to response for the first 9 patients was 2.8 months and 3.4 months to reach best response. The 3 patients in the 20 mg/kg cohort did not all reach response at the time of cut-off. Based on pharmacokinetics, safety, and pharmacodynamic data, the RP2D of 10 mg/kg was established. Translational studies assessing LSC frequencies in bone marrow aspirates by limiting dilution colony assays and xenograft experiments revealed that ARGX-110 monotherapy and in combination with AZA significantly reduced LSC frequencies in AML patients.ConclusionsARGX-110 in combination with AZA is well tolerated and leads to a higher ORR compared to historical data for AZA alone. Clinical activity of the combination is observed in different AML subtypes and risk categories. Preliminary data indicate that ARGX-110 alone, and in combination with AZA, efficiently eliminates LSCs. These observations warrant further accelerated investigation of ARGX-110 in combination with AZA in AML patients unfit for intensive therapy. DisclosuresOchsenbein:argenx: Research Funding. Riether:argenx: Research Funding. Bacher:argenx: Research Funding. Höpner:argenx: Research Funding. Hinterbrandner:argenx: Research Funding. Van Rompaey:argenx: Employment. Moshir:argenx: Employment. Delahaye:argenx: Employment. Gandini:argenx: Employment. Erzeel:argenx: Employment. Hultberg:argenx: Employment. Fung:argenx: Consultancy. De Haard:argenx: Employment. Leupin:argenx: Employment.

Argx-110 for Treatment of CD70-Positive Advanced Cutaneous T-Cell Lymphoma in a Phase 1/2 Clinical Trial

Argx-110 for Treatment of CD70-Positive Advanced Cutaneous T-Cell Lymphoma in a Phase 1/2 Clinical Trial

Argx-113, a Novel Fc-Based Approach for Antibody-Induced Pathologies Such As Primary Immune Thrombocytopenia

The neonatal Fc Receptor (FcRn) is a key player in the maintenance of high serum levels of immunoglobulin G (IgG) antibodies. IgGs enter cells by pinocytic uptake and bind FcRn in acidic endosomal compartments. FcRn then recycles IgGs back to the cell surface where they are released into circulation at physiological pH. This half-life prolonging salvage pathway has obvious advantages in IgG-mediated pathogen clearance, but also leads to persistence of disease-causing autoantibodies present in various autoimmune indications like immune thrombocytopenia (ITP). Indeed, the principal characteristic of ITP is autoantibody-mediated peripheral platelet destruction and impaired platelet production in bone marrow which leads to an increased risk of bleeding. Functional inhibition of the FcRn receptor could therefore be a novel therapeutic modality in the treatment of ITP as standard treatment options, like immunomodulatory drugs, typically suffer from a high risk of comorbidities.ARGX-113 is a proprietary antibody Fc-fragment based on argenx’ ABDEG™ technology. ABDEG™ mutations dramatically increase the Fc/FcRn binding both at neutral and acidic pH. This results in constitutive blockage of FcRn function by ARGX-113, and concomitantly leads to fast clearance of pathogenic antibodies in an autoimmune setting.The ABDEG™ concept was validated in various mouse models for IgG-induced autoimmune indications. In all models, a significant reduction of disease severity is observed upon ARGX-113 administration, which parallels with markedly reduced disease-causing antibody levels. Follow-up studies in cynomolgus monkeys demonstrated a highly effective and rapid elimination of immunoglobulins, which outperformed by far the pharmacodynamic effects of a 50-fold higher dose of intravenous immunoglobulin (IVIg), a standard-of-care therapy in many IgG-driven autoimmune indications.A first-in-human Phase I study was initiated to evaluate safety, tolerability, efficacy (reduction in IgG levels), pharmacokinetics and immunogenicity of ARGX-113 following single or multiple intravenous administration(s). Up to 68 healthy volunteers will be enrolled in this double-blind placebo-controlled study (48 active, 20 placebo). At present, the compound showed favorable safety and tolerability across multiple doses and dosing regimens with promising pharmacodynamics effects relating to speed, depth and duration of IgG reduction. Upon multiple dosing, IgG reduction of up to 85% is observed and IgG levels slowly return to baseline when dosing is terminated. Full study results, covering all planned doses and dosing regimens, will be presented focusing on safety, PK/PD relationship, IgG subset response, anti-drug antibody formation and dose/dosing regimen selection for future studies.In addition, the study design and outcome measures of a Phase II study evaluating safety, tolerability and efficacy of ARGX-113 in an ITP setting is being prepared and will be presented. DisclosuresUlrichts:argenx: Employment. Cousin:argenx: Employment. Dreier:argenx: Employment. de Haard:argenx: Employment. Leupin:argenx: Employment.

Abstract P2-05-17: ARGX-111 depletes MET-expressing circulating tumor cells via enhanced ADCC, resulting in inhibition of metastasis

Abstract Several lines of experimental evidence suggest that Hepatocyte Growth Factor (HGF) and its receptor MET play an important role in breast cancer progression and drug resistance. To date, targeted MET inhibitors in clinical development have primarily shown cytostatic rather than cytotoxic effects. Development of a cytotoxic MET inhibitor would serve to complement standard breast cancer therapy, especially when administered in the adjuvant/neo-adjuvant setting. We have developed ARGX-111, a human antibody antagonist of MET function. ARGX-111 blocks both HGF-dependent and -independent signaling, down-regulates tumor cell surface expression of MET and kills MET-overexpressing cells by enhanced antibody-dependent cellular cytotoxicity (ADCC). ARGX-111 was shown to be more efficacious than an ADCC-inactive control antibody in both HGF-dependent and -independent tumor xenograft models. ADCC reporter assays confirmed the cytotoxic effects of ARGX-111 in patient-derived primary tumor specimens, including MET-expressing breast cancer stem-like cells. In an orthotopic mouse model of metastatic mammary carcinoma (MDA-MB-231), adjuvant or neo-adjuvant treatment with ARGX-111 was significantly more effective in depleting circulating tumor cells (CTCs) and suppressing the development of bone and lung metastases than the ADCC-inactive control. Taken together, these results provide a rationale for clinical investigation of ARGX-111 in the early breast cancer setting. An ongoing Phase 1 study (NCT02055066) is examining the effects of ARGX-111 on CTCs, alongside the assessment of its safety and efficacy. Citation Format: Morello V, Hultberg A, De Jonge N, Huyghe L, Hanssens V, Brouckaert P, Saunders M, Dreier T, Thibault A, Rolfo C, Aftimos P, Awada A, Michieli P, de Haard H. ARGX-111 depletes MET-expressing circulating tumor cells via enhanced ADCC, resulting in inhibition of metastasis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-17.

Abstract B036: Monoclonal antibodies against GARP/TGF-β1 complexes inhibit the immunosuppressive activity of human regulatory T cells in vivo

Abstract Regulatory T lymphocytes (Tregs) are essential to prevent auto-immunity, but excessive Treg function contributes to cancer progression by inhibiting anti-tumor immune responses. Tregs exert contact-dependent inhibition of immune cells through the production of active TGF-β1. On the Treg cell surface, TGF-β1 is in an inactive form bound to membrane protein GARP and then activated by an unknown mechanism. Here, we demonstrate that GARP is involved in this activation mechanism. Two anti-GARP monoclonal antibodies were generated that block the production of active TGF-β1 by human Tregs. These antibodies recognize a conformational epitope that requires amino-acids GARP137-139 within GARP/TGF-β1 complexes. A variety of antibodies recognizing other GARP epitopes did not block active TGF-β1 production by Tregs. In a model of xenogeneic graft-versus-host disease in NSG mice, the blocking antibodies inhibited the immunosuppressive activity of human Tregs. These antibodies may serve as therapeutic tools to boost immune responses to infection or cancer, via a mechanism of action distinct from that of currently available immuno-modulatory antibodies. Used alone or in combination with tumor vaccines or antibodies targeting the CTLA4 or PD1/PD-L1 pathways, blocking anti-GARP antibodies may improve the efficiency of cancer immunotherapy. Citation Format: Stéphanie Liénart, Julia Cuende, Michael Saunders, Bas van der Woning, Hans De Haard, Pierre G. Coulie, Sophie Lucas. Monoclonal antibodies against GARP/TGF-β1 complexes inhibit the immunosuppressive activity of human regulatory T cells in vivo. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B036.

Abstract LB-330: Four individually druggable Met hotspots mediate HGF-driven tumor progression

Abstract Activation of Met by HGF plays a key role in tumor progression. Using a newly developed llama platform generating human-like immunoglobulins, we selected 68 different antibodies that competed with HGF for binding to Met. HGF-competing antibodies recognized four hotspots localized in different domains of Met. One hotspot coincides with the previously identified HGF β-chain binding site on blades 2-3 of the SEMA β-propeller. Two hotspots lie within SEMA blade 5 and IPT domains 2-3, respectively, where the HGF α-chain is thought to bind. The last hotspot identifies a new region across the PSI-IPT 1 domains. Individual or combined targeting of these hotspots effectively interrupted HGF-Met signaling in multiple biochemical and biological assays. Selected antibodies directed against SEMA blades 2-3 and the PSI-IPT 1 region inhibited brain invasion and prolonged survival in a glioblastoma multiforme model, prevented metastatic disease following neo-adjuvant therapy in a triple-negative mammary carcinoma model, and suppressed cancer cell dissemination to the liver in a KRAS-mutant metastatic colorectal cancer model. These results identify multiple regions of Met responsible for HGF-mediated tumor progression, unraveling the complexity of HGF-Met interaction and providing new molecular tools for targeting Met activity in cancer. Citation Format: Anna Hultberg, Cristina Basilico, Cristophe Blanchetot, Natalie De Jonge, Valérie Hanssens, Gitte De Boeck, Alessia Mira, Manuela Cazzanti, Virginia Morello, Torsten Dreier, Michael Saunders, Hans De Haard, Paolo Michieli. Four individually druggable Met hotspots mediate HGF-driven tumor progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-330. doi:10.1158/1538-7445.AM2014-LB-330

Abstract 5470: Pre-clinical characterization of ARGX-110: A neutralizing, humanized monoclonal antibody to the human CD70 antigen with enhanced ADCC properties.

Abstract The expression of CD70 is normally restricted to activated B- and T- cells, as well as mature dendritic cells. Overexpression has been documented in a variety of solid and hematological tumors, where it is thought to play a role in evasion of immune surveillance. ARGX-110 is a defucosylated, humanized IgG1 monoclonal antibody that selectively targets and neutralizes CD70, a ligand of CD27. A detailed biological and functional characterization of ARGX-110 was carried out to support its clinical development for the treatment of cancers dependent on signaling through the CD70/CD27 axis. In competitive equilibrium binding experiments, ARGX-110 bound to human and cynomolgus monkey CD70 with affinities of approximately 0.4 nmol/L and 0.6 nmol/L, respectively. ARGX-110 inhibited in a dose-dependent fashion CD70/CD27-induced IL-8 release in Raji cells with IC50 values of 0.1 nmol/L. CD70 is minimally internalized (&amp;lt;40%) by most hematological cell lines, thus providing a plausible rationale to develop a therapeutic mAb designed to enhance ADCC. In cell depletion studies, ARGX-110 was associated with comparable to better cell lysis compared to Rituximab. Cell lysis by ARGX-110 was not dependent on the internalization rate of the ARGX-110/CD70 complex in hematological cell lines. Independence from CD70 copy numbers at the cell surface was also demonstrated. In a Burkitt lymphoma (CD70+ Raji cells) SCID mouse model, ARGX-110 administered intraperitoneally twice weekly at doses ranging from 0.001 to 10 mg/kg starting one day after inoculation resulted in a dose-response effect on animal survival. In cynomolgus monkeys, ARGX-110 did not demonstrate any toxicity upon repeated administration of high doses of 30 mg/kg. B, T and NK cell numbers remained unaltered. ARGX-110 is currently in phase I clinical trial for patients with CD70+ malignancies. Citation Format: Karen Silence, Hans De Haard, Torston Dreier, Peter Ulrichts, Mahan Moshir, Sofie Gabriels, Alain Thibault. Pre-clinical characterization of ARGX-110: A neutralizing, humanized monoclonal antibody to the human CD70 antigen with enhanced ADCC properties. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5470. doi:10.1158/1538-7445.AM2013-5470 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Popular discourse on nutrition, health and indulgence in Flanders, 1945–1960

Ever since Belgian scientists started to investigate the working classes’ diet at the beginning of the twentieth century, popular media has shown traces of researchers’ scientific findings in one way or another. This article investigates whether or not nutritional knowledge was translated into comprehensible food practices on a household level in postwar Flanders. The culinary pages of De Boerin/ Bij de Haard, a widespread monthly magazine published by the Belgian Farmer Women's Association, was subjected to a quantitative and qualitative content analysis. The analysis showed that even though De Boerin/ Bij de Haard revealed several traces of a certain nutritional knowledge, scientific discourse was not translated into ready-to-use recipes or cooking techniques during the immediate postwar years. It was only in the 1950s that women were educated on the function and importance of various nutrients and that nutritional knowledge was actually converted into rather specific daily menus and new ways of preparation. Interestingly, the references to health were countered by references to indulgence as well, especially during festive occasions. The article finishes by exploring whether or not both types of references could be combined in one way or another.

Generation of a Family-specific Phage Library of Llama Single Chain Antibody Fragments That Neutralize HIV-1

Recently, we described llama antibody fragments (VHH) that can neutralize human immunodeficiency virus, type 1 (HIV-1). These VHH were obtained after selective elution of phages carrying an immune library raised against gp120 of HIV-1 subtype B/C CN54 with soluble CD4. We describe here a new, family-specific approach to obtain the largest possible diversity of related VHH that compete with soluble CD4 for binding to the HIV-1 envelope glycoprotein. The creation of this family-specific library of homologous VHH has enabled us to isolate phages carrying similar nucleotide sequences as the parental VHH. These VHH displayed varying binding affinities and neutralization phenotypes to a panel of different strains and subtypes of HIV-1. Sequence analysis of the homologs showed that the C-terminal three amino acids of the CDR3 loop were crucial in determining the specificity of these VHH for different subtype C HIV-1 strains. There was a positive correlation between affinity of VHH binding to gp120 of HIV-1 IIIB and the breadth of neutralization of diverse HIV-1 envelopes. The family-specific approach has therefore allowed us to better understand the interaction of the CD4-binding site antibodies with virus strain specificity and has potential use for the bioengineering of antibodies and HIV-1 vaccine development.

Down-regulation of MyoD by Calpain 3 Promotes Generation of Reserve Cells in C2C12 Myoblasts

Calpain 3 is a calcium-dependent cysteine protease that is primarily expressed in skeletal muscle and is implicated in limb girdle muscular dystrophy type 2A. To date, its best characterized function is located within the sarcomere, but this protease is found in other cellular compartments, which suggests that it exerts multiple roles. Here, we present evidence that calpain 3 is involved in the myogenic differentiation process. In the course of in vitro culture of myoblasts to fully differentiated myotubes, a population of quiescent undifferentiated "reserve cells" are maintained. These reserve cells are closely related to satellite cells responsible for adult muscle regeneration. In the present work, we observe that reserve cells express higher levels of endogenous Capn3 mRNA than proliferating myoblasts. We show that calpain 3 participates in the establishment of the pool of reserve cells by decreasing the transcriptional activity of the key myogenic regulator MyoD via proteolysis independently of the ubiquitin-proteasome degradation pathway. Our results identify calpain 3 as a potential new player in the muscular regeneration process by promoting renewal of the satellite cell compartment.

General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold

Nanobodies, single-domain antigen-binding fragments of camelid-specific heavy-chain only antibodies offer special advantages in therapy over classic antibody fragments because of their smaller size, robustness, and preference to target unique epitopes. A Nanobody differs from a human heavy chain variable domain in about ten amino acids spread all over its surface, four hallmark Nanobody-specific amino acids in the framework-2 region (positions 42, 49, 50, and 52), and a longer third antigen-binding loop (H3) folding over this area. For therapeutic applications the camelid-specific amino acid sequences in the framework have to be mutated to their human heavy chain variable domain equivalent, i.e. humanized. We performed this humanization exercise with Nanobodies of the subfamily that represents close to 80% of all dromedary-derived Nanobodies and investigated the effects on antigen affinity, solubility, expression yield, and stability. It is demonstrated that the humanization of Nanobody-specific residues outside framework-2 are neutral to the Nanobody properties. Surprisingly, the Glu-49 --> Gly and Arg-50 --> Leu humanization of hallmark amino acids generates a single domain that is more stable though probably less soluble. The other framework-2 substitutions, Phe-42 --> Val and Gly/Ala-52 --> Trp, are detrimental for antigen affinity, due to a repositioning of the H3 loop as shown by their crystal structures. These insights were used to identify a soluble, stable, well expressed universal humanized Nanobody scaffold that allows grafts of antigen-binding loops from other Nanobodies with transfer of the antigen specificity and affinity.

A Century's Perspective: Essays on Russian Literature in Honour of Olga Raevsky Hughes and Robert P. Hughes by Lasar Fleishman , Hugh McLean (review)

REVIEWS 713 This second volume also includes sensible items on Chekhov's use of punctuation (Rosamund Bardett), Chekhov and Katherine Mansfield (Sally Dalton-Brown), Chekhov's use of numbers and numerals (Eric de Haard), peasant women's sexualities in thewritings ofGleb Uspenskii and Chekhov (HenriettaMondry), and Brian Friel's Irish version ofTri sestry (KevinWindle). Michael Pursglove considers whether Grigorovich's novel Pereselentsy may have influenced Chekhov's 'Step", Harai Golomb offers a detailed analysis of an extract from Chaika, and David Gillespie observes that Iurii Trifonov and Nikita Mikhalkov 'both remain profoundly compromised because they could not ? would not ? rise above the demands of the day towards conformity' (P- x45) There are, alas, more than 100 misprints in the two volumes, including (in vol. 1) Sharova (forShavrova, pp. 62, 71),Aleksei (forAleksandr, p. 63), Feodotik (forFedotik, pp. 109, 112), Martin (for Marian, p. 129), dush (p. 176), and dotopiat' (for dotiapat', p. 179).There is also the occasional unfortunate ambiguity, such as 'his beloved inside' (vol. 1,p. 7) and 'he helps Ania leave past her behind' (vol. 1,p. 32). A macabre reader might interpret 'the stiff statistician' (vol. 1, p. 15) as a counter of corpses. Above all, however, Joe Andrew and Robert Reid are to be congratulated most warmly for their enthusiasm, their enterprise and their achievement. Bristol Gordon McVay Fleishman, Lasar and McLean, Hugh (eds). A Century'sPerspective: Essays on Russian Literature inHonour of Olga RaevskyHughes and Robert P. Hughes. Stanford Slavic Studies, 32. Stanford, CA, 2006. 564 pp. Tables. Notes. Bibliographies. Price unknown. This festschrift volume of twenty-nine contributions inEnglish and inRussian commemorates Olga Raevsky-Hughes and Robert Hughes, two distinguished Slavic specialists working for many years for the Department of Slavic Languages and Literatures at the University of California at Berkeley. Olga Raevsky-Hughes's scholarship on Boris Pasternak and Russian twentieth century emigre literature together with her two-volume collection of essays on Christianity and the Eastern Slavs (co-edited with Robert Hughes et al.) received wide acclaim. Robert Hughes's work on Russian modernist poets, on Pushkin and his three volumes of Khodasevich's essays on Pushkin are models of outstanding scholarship and editorial work. The collection pays tribute to the two scholars' diversity of interests, including essays and letters penned by Russian modernist authors, biographical notes on various authors and publishers, and insightfuldiscussions of important theoretical and creative works. The articles are not grouped thematically, nor will all secure wide readership, yet several thought-provokingmaterials should not be overlooked by anyone interested in Silver Age culture, especially Joan Delaney Grossman's article on Briusov's 1921 play Dictator (pp. 67-80), Irina Masing Delic's essay on Nabokov's 1957novel Pnin (pp. 351-68), and Elena Obatnina's article on Remizov (pp. 377-98). 714 SEER, 86, 4, OCTOBER 2008 Grossman defines Dictator as a hybrid of the science fiction and classical tragedy genres and sees Briusov's prophetic vision inDictator of Soviet totali tarian future as debunking the myth of Briusov's opportunism and active cooperation with the new regime. Grossman presents Briusov as a tragic figuredisplaced in Soviet Russia and self-fashioned in the clothes of Pushkin's Evgenii as the madman-poet confronting the Bronze Horseman. The trope that views Pushkin's image of the Bronze Horseman as symbol of Soviet modernity is prevalent inmany works of writers of the 1920s to 1940s, but Grossman's article implies that Briusov was a creator of this mythologeme. Masing-Delic's superb article views Pushkin's 1832 work Fairy Tale about Tsar Saltan as an important subtext to Pnin. Masing-Delic concludes, 'InPnin, Nabokov shows how to preserve foreverGvidon's island of Buian with all its magic creatures, not least squirrels, how to protect the sense of life's magic [...] ? by giving us two facets of his self: the remembering one who "commu nicates" with squirrels and lives inwonderland and the one who organises thesememories. [...] Nabokov obviously transcends both' (p. 366). In a simi lar vein, Greta Slobin informatively interpretsRemizov's autobiographical story cycleMusic Teacher (1924-39) as 'a writer's quest for connection, for overcoming the rupture of history' (p.413) and explores its linkswith works penned by...

Promiscuous Modification of the Nuclear Poly(A)-binding Protein by Multiple Protein-arginine Methyltransferases Does Not Affect the Aggregation Behavior

The mammalian nuclear poly(A)-binding protein, PABPN1, carries 13 asymmetrically dimethylated arginine residues in its C-terminal domain. By fractionation of cell extracts, we found that protein-arginine methyltransferases (PRMTs)-1, -3, and -6 are responsible for the modification of PABPN1. Recombinant PRMT1, -3, and -6 also methylated PABPN1. Our data suggest that these enzymes act on their own, and additional polypeptides are not involved in recognizing PABPN1 as a substrate. PRMT1 is the predominant methyltransferase acting on PABPN1. Nevertheless, PABPN1 was almost fully methylated in a Prmt1(-/-) cell line; thus, PRMT3 and -6 suffice for methylation. In contrast to PABPN1, the heterogeneous nuclear ribonucleoprotein (hnRNP) K is selectively methylated only by PRMT1. Efficient methylation of synthetic peptides derived from PABPN1 or hnRNP K suggested that PRMT1, -3, and -6 recognize their substrates by interacting with local amino acid sequences and not with additional domains of the substrates. However, the use of fusion proteins suggested that the inability of PRMT3 and -6 to modify hnRNP K is because of structural masking of the methyl-accepting amino acid sequences by neighboring domains. Mutations leading to intracellular aggregation of PABPN1 cause the disease oculopharyngeal muscular dystrophy. The C-terminal domain containing the methylated arginine residues is known to promote PAPBN1 self-association, and arginine methylation has been reported to inhibit self-association of an orthologous protein. Thus, arginine methylation might be relevant for oculopharyngeal muscular dystrophy. However, in two different types of assays we have been unable to detect any effect of arginine methylation on the aggregation of bovine PABPN1.

Modular Structure of the Receptor Binding Proteins of Lactococcus lactis Phages

Lactococcus lactis is a gram-positive bacterium widely used by the dairy industry. Several industrial L. lactis strains are sensitive to various distinct bacteriophages. Most of them belong to the Siphoviridae family and comprise several species, among which the 936 and P335 are prominent. Members of these two phage species recognize their hosts through the interaction of their receptor-binding protein (RBP) with external cell wall saccharidices of the host, the "receptors." We report here the 1.65 A resolution crystal structure of the RBP from phage TP901-1, a member of the P335 species. This RBP of 163 amino acids is a homotrimer comprising three domains: a helical N terminus, an interlaced beta-prism, and a beta-barrel, the head domain (residues 64-163), which binds a glycerol molecule. Fluorescence quenching experiments indicated that the RBP exhibits high affinity for glycerol, muramyl-dipeptide, and other saccharides in solution. The structural comparison of this RBP with that of lactococcal phage p2 RBP, a member of the 936 species (Spinelli, S., Desmyter, A., Verrips, C. T., de Haard, J. W., Moineau, S., and Cambillau, C. (2006) Nat. Struct. Mol. Biol. 13, 85-89) suggests a large extent of modularity in RBPs of lactococcal phages.

Receptor-Binding Protein of Lactococcus lactis Phages: Identification and Characterization of the Saccharide Receptor-Binding Site

Phage p2, a member of the lactococcal 936 phage species, infects Lactococcus lactis strains by binding initially to specific carbohydrate receptors using its receptor-binding protein (RBP). The structures of p2 RBP, a homotrimeric protein composed of three domains, and of its complex with a neutralizing llama VH domain (VHH5) have been determined (S. Spinelli, A. Desmyter, C. T. Verrips, H. J. de Haard, S. Moineau, and C. Cambillau, Nat. Struct. Mol. Biol. 13:85-89, 2006). Here, we show that VHH5 was able to neutralize 12 of 50 lactococcal phages belonging to the 936 species. Moreover, escape phage mutants no longer neutralized by VHH5 were isolated from 11 of these phages. All of the mutations (but one) cluster in the RBP/VHH5 interaction surface that delineates the receptor-binding area. A glycerol molecule, observed in the 1.7-A resolution structure of RBP, was found to bind tightly (Kd= 0.26 microM) in a crevice located in this area. Other saccharides bind RBP with comparable high affinity. These data prove the saccharidic nature of the bacterial receptor recognized by phage p2 and identify the position of its binding site in the RBP head domain.