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Risk factors for and postoperative results of the Fontan operation in patients living at high altitude (2500 meters above sea level) in the Andean region remain unknown. This study was conducted to evaluate immediate postoperative outcomes and to assess short- and long-term functional class after the Fontan operation.From June 2003 to February 2019, 104 patients receiving the Fontan procedure at 2640 meters (8661 feet) above sea level were retrospectively studied. Preoperative catheterization, intraoperative variables, and postoperative outcomes were described. Functional class was evaluated in patients living permanently below (group I) and at or higher than 2500 meters (8202 feet) above sea level (group II). Risk factors for mortality were analyzed.Median age at operation was 8.5 ± 4.4 years; pulmonary artery pressure, 16.2 ± 3.6 mm Hg; end-diastolic systemic ventricular pressure, 13.3 ± 3.8 mm Hg; and pulmonary vascular resistance index, 2.1 (interquartile range, 07-3.7) Wood units. Chest tube duration was 8.5 (6-12) days. Mortality was 4.8%, with 0 in the last 5 years. Higher preoperative pulmonary pressure (16.2 ± 3.6 mm Hg vs 21.2 ± 3.40 mm Hg; P = .01), aortic cross-clamp time (P.001), and renal failure (P.01) were associated with mortality. Functional class improved to class I in 86.4%. Overall survival was 90.7% at 10 years of follow-up.Increased pulmonary pressure and pulmonary vascular resistance index are directly related to high altitude. The Fontan-Kreutzer operation performed at high altitude in the Andean region is feasible with good results. We routinely fenestrate all cases to avoid dysfunction in the early postoperative period. Functional status is adequate after the operation.

Supplementary information on cell maintenance, and antibody selection and characterization

FAP-drozitumab antibody-mediated apoptosis does not correlate with DR5.

Characterization and optimization of RG7386 dosing in vivo.

Dysregulated cellular apoptosis and resistance to cell death are hallmarks of neoplastic initiation and disease progression. Therefore, the development of agents that overcome apoptosis dysregulation in tumor cells is an attractive therapeutic approach. Activation of the extrinsic apoptotic pathway is strongly dependent on death receptor (DR) hyperclustering on the cell surface. However, strategies to activate DR5 or DR4 through agonistic antibodies have had only limited clinical success. To pursue an alternative approach for tumor-targeted induction of apoptosis, we engineered a bispecific antibody (BsAb), which simultaneously targets fibroblast-activation protein (FAP) on cancer-associated fibroblasts in tumor stroma and DR5 on tumor cells. We hypothesized that bivalent binding to both FAP and DR5 leads to avidity-driven hyperclustering of DR5 and subsequently strong induction of apoptosis in tumor cells but not in normal cells. Here, we show that RG7386, an optimized FAP-DR5 BsAb, triggers potent tumor cell apoptosis in vitro and in vivo in preclinical tumor models with FAP-positive stroma. RG7386 antitumor efficacy was strictly FAP dependent, was independent of FcR cross-linking, and was superior to conventional DR5 antibodies. In combination with irinotecan or doxorubicin, FAP-DR5 treatment resulted in substantial tumor regression in patient-derived xenograft models. FAP-DR5 also demonstrated single-agent activity against FAP-expressing malignant cells, due to cross-binding of FAP and DR5 across tumor cells. Taken together, these data demonstrate that RG7386, a novel and potent antitumor agent in both mono- and combination therapies, overcomes limitations of previous DR5 antibodies and represents a promising approach to conquer tumor-associated resistance to apoptosis. Mol Cancer Ther; 15(5); 946–57. ©2016 AACR.

Figure S2. Related to Figure 3. In vitro characterization of the FAP-DR5 BsAb RG7386; Table S4. Characterization of RG7386 binding avidities.

Supplementary Figure from A Novel EGFRvIII T-Cell Bispecific Antibody for the Treatment of Glioblastoma

Figure S1. Related to Figure 1. Characterization of FAP-drozitumab BsAbs and antitumor efficacy in the presence of FAP-expressing fibroblasts in in vitro co-culture models; Table S1. BsAb binding to DR5 and FAP antigens; Table S2. Anti-tumor efficacy of FAP-drozitumab bispecific molecules is FAPdependent.

Supplemental Video 3. In vitro visualization of the dynamics of CD20-TCB (white) during interaction between CD8+ T-cells (green) and tumor cells (blue). The video focuses on an example of CD20-TCB-mediated CD8+ T-cell cytotoxicity of tumor cells. T-cells surround the tumor cells and create stable synapses where CD20-TCB is localized (white). Tumor cells are killed upon certain time of T-cell/tumor cell interaction as evidenced by the formation of tumor blebs.

Purpose:CD40 agonists hold great promise for cancer immunotherapy (CIT) as they enhance dendritic cell (DC) activation and concomitant tumor-specific T-cell priming. However, the broad expression of CD40 accounts for sink and side effects, hampering the efficacy of anti-CD40 antibodies. We hypothesized that these limitations can be overcome by selectively targeting CD40 agonism to the tumor. Therefore, we developed a bispecific FAP-CD40 antibody, which induces CD40 stimulation solely in presence of fibroblast activation protein α (FAP), a protease specifically expressed in the tumor stroma.Experimental Design:FAP-CD40's in vitro activity and FAP specificity were validated by antigen-presenting cell (APC) activation and T-cell priming assays. In addition, FAP-CD40 was tested in subcutaneous MC38-FAP and KPC-4662-huCEA murine tumor models.Results:FAP-CD40 triggered a potent, strictly FAP-dependent CD40 stimulation in vitro. In vivo, FAP-CD40 strongly enhanced T-cell inflammation and growth inhibition of KPC-4662-huCEA tumors. Unlike nontargeted CD40 agonists, FAP-CD40 mediated complete regression of MC38-FAP tumors, entailing long-term protection. A high dose of FAP-CD40 was indispensable for these effects. While nontargeted CD40 agonists induced substantial side effects, highly dosed FAP-CD40 was well tolerated. FAP-CD40 preferentially accumulated in the tumor, inducing predominantly intratumoral immune activation, whereas nontargeted CD40 agonists displayed strong systemic but limited intratumoral effects.Conclusions:FAP-CD40 abrogates the systemic toxicity associated with nontargeted CD40 agonists. This enables administration of high doses, essential for overcoming CD40 sink effects and inducing antitumor immunity. Consequently, FAP-targeted CD40 agonism represents a promising strategy to exploit the full potential of CD40 signaling for CIT.

Pharmacokinetic profiles of CEA TCB in mice, additional information on CD3 signaling, T cell activation, tumor cell lysis and cytokine release upon CEA TCB and untargeted control TCB treatment.

Supplemental Figure 1. Assessment of the binding characteristics and cynomolgus monkey cross-reactivity of CD20-TCB. Supplemental Figure 2. Tumor cell lysis mediated by different CD20-targeting TCB antibodies. Supplemental Figure 3. T-cell and B-cell counts along with E:T ratio upon CD20-TCB treatment of primary bone marrow aspirates from patients with lymphoma and leukemia. Supplemental Figure 4. Quantification of interactions between T-cells and tumor cells from live imaging experiments and kinetic analysis of T-cell activation and exhaustion marker expression. Supplemental Figure 5. Secretion of cytokines, cytotoxic granules and T-cell proliferation upon tumor lysis. Supplemental Figure 6. Assessment of the cytokine-mediated endothelial cell activation. Supplemental Figure 7. Characterization of stem cell humanized mice (HSC-NSG) generated in house.

Purpose: CEA TCB is a novel IgG-based T-cell bispecific (TCB) antibody for the treatment of CEA-expressing solid tumors currently in phase I clinical trials (NCT02324257). Its format incorporates bivalent binding to CEA, a head-to-tail fusion of CEA- and CD3e-binding Fab domains and an engineered Fc region with completely abolished binding to FcγRs and C1q. The study provides novel mechanistic insights into the activity and mode of action of CEA TCB.Experimental Design: CEA TCB activity was characterized on 110 cell lines in vitro and in xenograft tumor models in vivo using NOG mice engrafted with human peripheral blood mononuclear cells.Results: Simultaneous binding of CEA TCB to tumor and T cells leads to formation of immunologic synapses, T-cell activation, secretion of cytotoxic granules, and tumor cell lysis. CEA TCB activity strongly correlates with CEA expression, with higher potency observed in highly CEA-expressing tumor cells and a threshold of approximately 10,000 CEA-binding sites/cell, which allows distinguishing between high- and low-CEA–expressing tumor and primary epithelial cells, respectively. Genetic factors do not affect CEA TCB activity confirming that CEA expression level is the strongest predictor of CEA TCB activity. In vivo, CEA TCB induces regression of CEA-expressing xenograft tumors with variable amounts of immune cell infiltrate, leads to increased frequency of activated T cells, and converts PD-L1 negative into PD-L1–positive tumors.Conclusions: CEA TCB is a novel generation TCB displaying potent antitumor activity; it is efficacious in poorly infiltrated tumors where it increases T-cell infiltration and generates a highly inflamed tumor microenvironment. Clin Cancer Res; 22(13); 3286–97. ©2016 AACR.

Representative videos of intravital two-photon (2P) microscopy of tumors treated with CEA TCB. Tumor cells red, T cells green.

List of antibodies used for flow cytometry

Supplemental Video 1. In vitro visualization of the dynamics of untargeted TCB (white) during the interaction between CD8+ T-cells (green) and tumor cells (blue).

Additional details on methods used for SPR assays, NanoString RNAseq and cynomolgus monkey studies

Purpose: Anti-EGF receptor (EGFR) antibodies and small-molecule tyrosine kinase inhibitors have shown activity in epithelial tumors; however, agents that work by blocking the EGFR growth signal are ineffective when the oncogenic stimulus arises downstream, such as in tumors with KRAS mutations. Antibodies of the IgG1 subclass can also kill tumor cells directly through antibody-dependent cell-mediated cytotoxicity (ADCC), and the efficacy of this is determined by the interaction of the Fc portion of the target cell–bound antibody and Fc receptors present on immune effector cells.Experimental Design: We report the development of GA201, a novel anti-EGFR monoclonal antibody with enhanced ADCC properties. GA201 was derived by humanization of the rat ICR62 antibody. The Fc region of GA201 was glycoengineered to contain bisected, afucosylated carbohydrates for enhanced binding to FcγRIIIA.Results:In vitro binding of GA201 to EGFR inhibited EGF ligand binding, EGFR/HER2 heterodimerization, downstream signaling, and cell proliferation to a similar extent as cetuximab. However, GA201 exhibited superior binding to both the low- and high-affinity variants of FcγRIIIA. This resulted in significantly enhanced induction of ADCC compared with cetuximab against both KRAS-wild-type and -mutant tumor cells lines. This enhanced ADCC translated into superior in vivo efficacy in a series of mouse xenograft models. Efficacy of GA201 was further increased when administered in combination with chemotherapy (irinotecan).Conclusions: These data suggest that GA201 may be more effective than cetuximab in patients with EGFR-positive solid tumors and may also represent a first-in-class treatment of patients with KRAS-mutated tumors. Clin Cancer Res; 19(5); 1126–38. ©2012 AACR.

Purpose: Despite promising clinical activity, T-cell–engaging therapies including T-cell bispecific antibodies (TCB) are associated with severe side effects requiring the use of step-up-dosing (SUD) regimens to mitigate safety. Here, we present a next-generation CD20-targeting TCB (CD20-TCB) with significantly higher potency and a novel approach enabling safer administration of such potent drug.Experimental Design: We developed CD20-TCB based on the 2:1 TCB molecular format and characterized its activity preclinically. We also applied a single administration of obinutuzumab (Gazyva pretreatment, Gpt; Genentech/Roche) prior to the first infusion of CD20-TCB as a way to safely administer such a potent drug.Results: CD20-TCB is associated with a long half-life and high potency enabled by high-avidity bivalent binding to CD20 and head-to-tail orientation of B- and T-cell–binding domains in a 2:1 molecular format. CD20-TCB displays considerably higher potency than other CD20-TCB antibodies in clinical development and is efficacious on tumor cells expressing low levels of CD20. CD20-TCB also displays potent activity in primary tumor samples with low effector:target ratios. In vivo, CD20-TCB regresses established tumors of aggressive lymphoma models. Gpt enables profound B-cell depletion in peripheral blood and secondary lymphoid organs and reduces T-cell activation and cytokine release in the peripheral blood, thus increasing the safety of CD20-TCB administration. Gpt is more efficacious and safer than SUD.Conclusions: CD20-TCB and Gpt represent a potent and safer approach for treatment of lymphoma patients and are currently being evaluated in phase I, multicenter study in patients with relapsed/refractory non-Hodgkin lymphoma (NCT03075696). Clin Cancer Res; 24(19); 4785–97. ©2018 AACR.See related commentary by Prakash and Diefenbach, p. 4631

List of all genes analyzed in NanoString gene expression analysis comparing gene expression in tumors of selicrelumab and FAP-huCD40-treated mice

Representative videos of intravital two-photon (2P) microscopy of tumors treated with untargeted TCB. Tumor cells red, T cells green.

List, Source and CEA binding sites of Cell Lines used in the study

Additional data of cynomolgus monkey study showing body weight and plasma cytokine levels over the course of the study

Supplementary material and methods

Assessment of binding of CEA TCB to human and cynomolgus monkey CEA- and CD3-expressing cells, internalization and ADCC, CDC activity.

PDF file - 307K, ADCC activity of GA201 using PMBCs as effector cells at E:T ratios of 1:1, 5:1 (24 hour LDH release measured) and 10:1 (4 hour LDH release measured). Multiple ADCC experiments were performed using a variety of different effector:target cell ratios (E:T). Figure 3 in the manuscript displays the results of ADDC assays using NK-92 cells as effectors at an E:T ratio of 3:1 and using human peripheral blood mononuclear cells (PBMCs) as effectors at an E:T ratio of 25:1. These E:T ratios represent the optimal experimental conditions that generated robust ADCC signal read outs. Supplementary Fig. S1 shows additional ADCC results for the MKN45 cell line using human PBMCs as effector cells at an E:T ratios of 1:1, 5:1 and 10:1. The data shows that under all the tested conditions GA201 exhibited superior cell killing compared with cetuximab. The overall ADCC activity with both antibodies using PMBCs at an E:T ratio of 1:1 was substantially lower than at 5:1 or 10:1.

Supplementary Figure 1 shows more details on the mode of action and design of the used antibodies. Supplementary Figure 2 displays additional APC activation data of the human and murine FAP-CD40 molecules. Supplementary Figure 3 shows additional data for FRC in vitro assays and the OVA vaccination study. Supplementary Figure 4 shows additional data on the in vivo study comparing FAP-huCD40 and SGN40 moIgG1. Supplementary Figure 5 displays additional data on the in vivo comparison of selicrelumab and FAP-huCD40. Supplementary Figure 6 shows the data of a FAP-huCD40 dose escalation study in cynomolgus monkeys.

Supplemental data details

Detection of tumor targeting by in vivo imaging (fluorescent intensity, FLI)of CEA TCB formats having bivalent or monovalent binding to CEA and directly labeled with Alexa647.

Supplemental Material and Methods describing CEA TCB production and purification process, determination of affinity and avidity, genetic characterization of cell lines, determination of CEA binding sites, assessment of T cell proliferation and activation, Jurkat NFAT assay, confocal microscopy, FRET, SDPK, in vivo efficacy and imaging studies. Supplemental Video Legends are also within the file.

7 SUPPLEMENTARY FIGURES INCLUDING THE LEGENDS

Supplemental Video 2. In vitro visualization of the dynamics of CD20-TCB (white) during the interaction between CD8+ T-cells (green) and tumor cells (blue).

Supplemental materials and methods

Purpose:The incidence of human papillomavirus–associated head and neck squamous cell carcinoma (HPV+-HNSCC) is rising worldwide and although current therapeutic modalities are efficient in the majority of patients, there is a high rate of treatment failures. Thus, novel combination approaches are urgently needed to achieve better disease control in patients with HPV+-HNSCC. We investigated the safety and therapeutic efficacy of a novel fibroblast activation protein (FAP)-targeted CD40 agonist (FAP-CD40) in combination with local hypofractionated radiation in a syngeneic HPV+-HNSCC model.Experimental Design:Using an established orthotopic model, we treated tumor-bearing mice with local hypofractionated radiotherapy (2 × 6 Gy) alone or in combination with a systemic administration of the FAP-CD40 antibody. Following up the mice, we evaluated the changes in the tumor microenvironment (TME) by immunofluorescence, FACS, and NanoString RNA analysis.Results:The suboptimal radiotherapy regimen chosen failed to control tumors in the treated mice. The FAP-CD40 administered in monotherapy transiently controlled tumor growth, whereas the combined therapy induced durable complete responses in more than 80% of the tumor-bearing mice. This notable efficacy relied on the radiotherapy-induced remodeling of the TME and activation of the CD8+ T-cell-cDC1 axis and was devoid of the systemic toxicity frequently associated with CD40-targeted therapy. Moreover, the robust immunologic memory developed effectively prevented tumor relapses, a common feature in patients with HNSCC.Conclusions:Our study provides proof of concept, as well as mechanistic insights of the therapeutic efficacy of a bispecific FAP-CD40 combined with local radiotherapy in a FAP+-HNSCC model increasing overall survival and inducing long-term antitumor immunity.

Supplemental Table 1. Overview of CD20-targeting T-cell bispecific molecules evaluated in the current study. Supplemental Table 2. Tumor cell lines and EC50 values of tumor cell lysis. Supplemental Table 3. Assessment of the activity in ex vivo primary tumor samples derived from aggressive lymphoma and leukemia patients. Supplemental Table 4. Absolute values of cytokines measured by multiplex analysis of blood samples reported as heatmap in Figure 3e, 4b, 5d, and 6b. Supplemental Table 5. Average score of perivascular CD3 positive T-cells in the lung (assessed by blinded pathologist analysis) referring to T-cell staining of lungs from tumor-bearing, HSC NSG mice 7 days after the first treatment and 24 h after the second treatment (treatment groups are indicated on Figure 6D panels).