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Supplementary methods (S1-S4), Supplementary tables (1-4) and Supplementary Figures (1-5)

XLSX file - 30K, IC50 concentrations and CD22 expression levels for cell lines.

Supplementary Material and methods

The development of targeted therapies that affect multiple signaling pathways and stimulate antitumor immunity is greatly needed. About 20% of patients with breast cancer overexpress HER2. Small molecules and antibodies targeting HER2 convey some survival benefits; however, patients with advanced disease succumb to the disease under these treatment regimens, possibly because HER2 is not completely necessary for the survival of the targeted cancer cells. In the present study, we show that a polyinosine/polycytosine (pIC) HER2-homing chemical vector induced the demise of HER2-overexpressing breast cancer cells, including trastuzumab-resistant cells. Targeting pIC to the tumor evoked a number of cell-killing mechanisms, as well as strong bystander effects. These bystander mechanisms included type I IFN induction, immune cell recruitment, and activation. The HER2-targeted pIC strongly inhibited the growth of HER2-overexpressing tumors in immunocompetent mice. The data presented here could open additional avenues in the treatment of HER2-positive breast cancer. Cancer Immunol Res; 4(8); 688–97. ©2016 AACR.

Supplementary Figure illustrating the difference in cytokine mRNA and protein expression following treatment of BT-474 with pIC/PPHAffibody and pIC/PPCys

Antibody–drug conjugates (ADC), potent cytotoxic drugs linked to antibodies via chemical linkers, allow specific targeting of drugs to neoplastic cells. We have used this technology to develop the ADC DCDT2980S that targets CD22, an antigen with expression limited to B cells and the vast majority of non-Hodgkin lymphomas (NHL). DCDT2980S consists of a humanized anti-CD22 monoclonal IgG1 antibody with a potent microtubule-disrupting agent, monomethyl auristatin E (MMAE), linked to the reduced cysteines of the antibody via a protease cleavable linker, maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB). We describe the efficacy, safety, and pharmacokinetics of DCDT2980S in animal models to assess its potential as a therapeutic for the treatment of B-cell malignancies. We did not find a strong correlation between in vitro or in vivo efficacy and CD22 surface expression, nor a correlation of sensitivity to free drug and in vitro potency. We show that DCDT2980S was capable of inducing complete tumor regression in xenograft mouse models of NHL and can be more effective than rituximab plus combination chemotherapy at drug exposures that were well tolerated in cynomolgus monkeys. These results suggest that DCDT2980S has an efficacy, safety, and pharmacokinetics profile that support potential treatment of NHL. Mol Cancer Ther; 12(7); 1255–65. ©2013 AACR.

Supplementary Figures S1-S5, Supplementary Materials and Methods

PDF file - 61K, Supplemental Figure 1. Human, cynomolgus monkey, and rat PBMCs were isolated from whole blood in accordance with BD Vacutainer CPT protocol. Mouse PBMCs were isolated from whole blood by ACK lysis buffer treatment to lyse red blood cells followed by centrifucation to recover PBMCs. Human antibodies were labeled in accordance with the protocol with Zenon Alexa Fluor 647 Human IgG Labeling Kit. Mean fluorescent intensity (MFI) was calculated from the CD20‑positive B cells (human and cynomolgus monkey), CD45RA‑positive B cells (rat), or CD45R‑positive B cells (mouse).

The Avalon Elite catheter (Maquet Cardiopulmonary, Rastatt, Germany) is a bicaval catheter for single-site cannulation that can be used in the initiation of venovenous extracorporeal membrane oxygenation (ECMO) or as a transition from venoarterial ECMO. We report a unique complication of tissue obstructing the outflow aperture during insertion. (Level of Difficulty: Advanced.), Graphical abstract

Objectives The aim of this study was to report the 2-year results of the SURTAVI (Surgical Replacement and Transcatheter Aortic Valve Implantation) trial and confirm the interim Bayesian analysis. Background Transcatheter aortic valve replacement (TAVR) with a self-expanding valve was noninferior to surgery in patients with severe aortic stenosis and intermediate operative risk using Bayesian statistical methods. Novel Bayesian designs have been used to shorten the time to primary endpoint analysis in randomized clinical trials, although the predictive value of Bayesian analysis compared with frequentist approaches remains debated. Methods The SURTAVI trial randomized 1,660 patients. An interim analysis was performed 1 year after the 1,400th patient was treated to estimate the primary 2-year endpoint of all-cause mortality or disabling strokes for all patients. Results The Kaplan-Meier rate for the complete 2-year primary endpoint was 12.7% in the TAVR group and 12.6% in the surgery group (0.0% difference; 95% confidence interval: −3.4% to 3.5%), compared with 12.6% with TAVR and 14.0% with surgery (−1.4% difference; Bayesian credible interval: −5.2% to 2.3%) in the interim Bayesian analysis. A comparison of individual clinical, hemodynamic, and quality-of-life endpoints using Bayesian and frequentist methods found no significant differences. Conclusions The complete analysis of all patients with aortic stenosis at intermediate risk for surgery in the SURTAVI trial confirmed the noninferiority, with respect to the frequency of all-cause mortality or disabling stroke, of TAVR to surgery, as determined in the interim Bayesian analysis. Follow-up will extend out to 10 years.

Clinical guidelines recommend that the exercise protocol of a stress echocardiogram is selected to induce volitional exhaustion after a target duration of at least 8 minutes. While the Bruce protocol is very commonly used for clinical stress tests, it is known to be "steep", and many patients therefore fail to reach 8 minutes. We studied predictors of failure and developed a method for identifying patients not suitable for Bruce protocol which was accurate and yet simple enough to be used as a point-of-care decision support tool.We studied data out-patients undergoing Bruce protocol stress echocardiograms (n = 11 086) and analyzed predictors of inappropriate early termination (defined as test duration 8 min as per current practice guidelines) using logistic regression. A prediction model was constructed as follows: .5 points were given for each of hypertension, diabetes, smoking, and E/e' 7.9 in the resting echocardiogram; .1 point was added for each 1-unit increment in body mass index; 1 point was added for patient age by decade; 2.0 points were subtracted for male sex (p for all 0.001). In tests on held-out validation data, the model was well calibrated (in plots of predicted vs actual risk) and discriminated failure versus non-failure well (C-statistic .86 for a score of 6.0 points; p 0.001).These data may help to standardize protocol selection in stress echocardiography, by identifying patients pre-hoc where Bruce protocol will be inappropriately steep.

Although early pharmacotherapy is recommended for type 2 diabetes (T2DM), the effectiveness of early treatment in Chinese adults is understudied. Chinese populations have a different pattern of complications to Western populations. We analysed a cohort of Chinese adults with T2DM from 2007 to 2017 in the Hong Kong public health care system. Early and delayed groups was defined as pharmacotherapy within 3 months and 3-12 months after T2DM diagnosis respectively. We fitted Cox proportional hazard models on 3122 pairs of propensity-score matched participants to assess treatment intensification with insulin, all-cause mortality, and complications (myocardial infarction, ischemic heart disease, stroke, heart failure, peripheral vascular disease, neuropathy, amputation, skin ulcer, renal failure, retinopathy, cataracts). We accounted for immortality bias by beginning follow-up at the end of the exposure window (12m post-diagnosis). Early pharmacotherapy was associated with reduced risk of treatment intensification with insulin (HR: 0.758; 95% CI: 0.584-0.984). This finding was consistent using alternative thresholds for early treatment (3m intervals from 0-9 months) and for metformin monotherapy. No statistically significant difference for other clinical outcomes were observed. Longer follow-up and larger sample size are required to evaluate other clinical outcomes. Disclosure Y. Lau: None. Y. Yuen: None. C. Ng: None. J. Quan: None. Funding Research Grants Council of Hong Kong (27112518)

To sustain iron homeostasis, microorganisms have evolved fine-tuned mechanisms for uptake, storage and detoxification of the essential metal iron. In the human pathogen Aspergillus fumigatus, the fungal-specific bZIP-type transcription factor HapX coordinates adaption to both iron starvation and iron excess and is thereby crucial for virulence. Previous studies indicated that a HapX homodimer interacts with the CCAAT-binding complex (CBC) to cooperatively bind bipartite DNA motifs; however, the mode of HapX-DNA recognition had not been resolved. Here, combination of in vivo (genetics and ChIP-seq), in vitro (surface plasmon resonance) and phylogenetic analyses identified an astonishing plasticity of CBC:HapX:DNA interaction. DNA motifs recognized by the CBC:HapX protein complex comprise a bipartite DNA binding site 5′-CSAATN12RWT-3′ and an additional 5′-TKAN-3′ motif positioned 11–23 bp downstream of the CCAAT motif, i.e. occasionally overlapping the 3′-end of the bipartite binding site. Phylogenetic comparison taking advantage of 20 resolved Aspergillus species genomes revealed that DNA recognition by the CBC:HapX complex shows promoter-specific cross-species conservation rather than regulon-specific conservation. Moreover, we show that CBC:HapX interaction is absolutely required for all known functions of HapX. The plasticity of the CBC:HapX:DNA interaction permits fine tuning of CBC:HapX binding specificities that could support adaptation of pathogens to their host niches.

Antibody pretargeting is a promising strategy for improving molecular imaging, wherein the separation in time of antibody targeting and radiolabeling can lead to rapid attainment of high contrast, potentially increased sensitivity, and reduced patient radiation exposure. The inverse electron demand Diels-Alder ‘click’ reaction between trans-cyclooctene (TCO) conjugated antibodies and radiolabeled tetrazines presents an ideal platform for pretargeted imaging due to rapid reaction kinetics, bioorthogonality, and potential for optimization of both slow and fast clearing components. Herein, we evaluated a series of anti-human epidermal growth factor receptor 2 (HER2) pretargeting antibodies containing distinct molar ratios of site-specifically incorporated TCO. The effect of stoichiometry on tissue distribution was assessed for pretargeting TCO-modified antibodies (monitored by 125I) and subsequent accumulation of an 111In-labeled tetrazine in a therapeutically relevant HER2+tumor-bearing mouse model. Single photon emission computed tomography (SPECT) imaging was also employed to assess tumor imaging at various TCO-to-monoclonal antibody (mAb) ratios. Increasing TCO-to-mAb molar ratios correlated with increased in vivo click reaction efficiency evident by increased tumor distribution and systemic exposure of 111In-labeled tetrazines. The pharmacokinetics of TCO-modified antibodies did not vary with stoichiometry. Pretargeted SPECT imaging of HER2-expressing tumors using 111In-labeled tetrazine demonstrated robust click reaction with circulating antibody at ~2 hours and good tumor delineation for both the 2 and 6 TCO-to-mAb ratio variants at 24 hours, consistent with a limited cell-surface pool of pretargeted antibody and benefit from further distribution and internalization. To our knowledge, this represents the first reported systematic analysis of how pretargeted imaging is affected solely by variation in click reaction stoichiometry through site-specific conjugation chemistry.

Three rationally designed pyrrolobenzodiazepine (PBD) drug-linkers have been synthesized via intermediate 19 for use in antibody–drug conjugates (ADCs). They lack a cleavable trigger in the linker and consist of a maleimide for cysteine antibody conjugation, a hydrophilic spacer, and either an alkyne (6), triazole (7), or piperazine (8) link to the PBD. In vitro IC50 values were 11–48 ng/mL in HER2 3+ SK-BR-3 and KPL-4 (7 inactive) for the anti-HER2 ADCs (HER2 0 MCF7, all inactive) and 0.10–1.73 μg/mL (7 inactive) in CD22 3+ BJAB and WSU-DLCL2 for anti-CD22 ADCs (CD22 0 Jurkat, all inactive at low doses). In vivo antitumor efficacy for the anti-HER2 ADCs in Founder 5 was observed with tumor stasis at 0.5–1 mg/kg, 1 mg/kg, and 3–6 mg/kg for 6, 8, and 7, respectively. Tumor stasis at 2 mg/kg was observed for anti-CD22 6 in WSU-DLCL2. In summary, noncleavable PBD-ADCs exhibit potent activity, particularly in HER2 models.

Full-length antibodies lack ideal pharmacokinetic properties for rapid targeted imaging, prompting the pursuit of smaller peptides and fragments. Nevertheless, studying the disposition properties of antibody-based imaging agents can provide critical insight into the pharmacology of their therapeutic counterparts, particularly for those coupled with potent payloads. Here, we evaluate modulation of binding to the neonatal Fc receptor (FcRn) as a protein engineering-based pharmacologic strategy to minimize the overall blood pool background with directly labeled antibodies and undesirable systemic click reaction of radiolabeled tetrazine with circulating pretargeted trans-cyclooctene (TCO)-modified antibodies. Noninvasive SPECT imaging of mice bearing HER2-expressing xenografts was performed both directly (111In-labeled antibody) and indirectly (pretargeted TCO-modified antibody followed by 111In-labeled tetrazine). Pharmacokinetic modulation of antibodies was achieved by two distinct methods: Fc engineering to reduce binding affinity to FcRn, and delayed administration of an antibody that competes with binding to FcRn. Tumor imaging with directly labeled antibodies was feasible in the absence of FcRn binding, rapidly attaining high tumor-to-blood ratios, but accompanied by moderate liver and spleen uptake. Pretargeted imaging of tumors with non-FcRn-binding antibody was also feasible, but systemic click reaction still occurred, albeit at lower levels than with parental antibody. Our findings demonstrate that FcRn binding impairment of full-length IgG antibodies moderately lowers tumor accumulation of radioactivity, and shifts background activity from blood pool to liver and spleen. Furthermore, reduction of FcRn binding did not eliminate systemic click reaction, but yielded greater improvements in tumor-to-blood ratio when imaging with directly labeled antibodies than with pretargeting.

Peat is a type of soft soil with very high organic content, which makes it highly susceptible to extreme differential settlement due to the lack of structure. Although cement stabilisation is highly effective, it is not a common practice due to the high cost of treatment associated with the large amount of binder dosage required. In this paper, the use of a novel material, biochar as a potential partial replacement for cement and as an alternative filler to sand, was investigated. Biochar is more sustainable than traditional construction materials and has carbon sequestration ability. It could potentially be cheaper than cement or sand, depending on the source of feedstock used. Cement treated peat with sand was used as a benchmark to assess the performance of biochar enhanced cement treated peat. Unconfined Compressive Strength (UCS) tests were conducted to evaluate the strength of stabilised peat. The samples with biochar fragments finer than 75 μm performed notably better than samples with sand only, with over 50 % increase in compressive strength. Furthermore, it was found that the samples with 100 kg=m3 cement and 400 kg=m3 biochar had comparable performance to the control sample with 200 kg=m3 cement only, without biochar, highlighting the potential of biochar to partially replace cement. Scanning Electron Microscopy (SEM) and X-ray Diffraction (XRD) tests results showed that the mechanisms behind the performance gain in biochar samples are of both mechanical and chemical nature.

Predictors of left ventricular ejection fraction (LVEF) improvement after transcatheter aortic valve replacement (TAVR) in patients with a preoperative reduced LVEF are limited.This study aimed to investigate the relationship between preprocedural valuvuloarterial impedance (ZVa), which represents the global LV afterload, and LVEF improvement after TAVR.This was a single-center, retrospective study, which included patients with symptomatic aortic valve stenosis (AS) with a reduced baseline LVEF ( 50%) underwent TAVR. Based on the difference in the LVEF before and 1 month after the procedure, they were divided into two groups: improved group (≥ 10% improvement) and non-improved group ( 10% improvement or worsening). Preprocedural ZVa and clinical outcomes were then compared. ZVa was calculated using preprocedural transthoracic echocardiography data.Among 473 cases of TAVR performed from May 2012 to July 2017 at Queen's Medical Center (Honolulu, HI, USA), 99 patients (improved group, n = 42; mean age 82.0 ± 8.6 years vs. non-improved group, n = 57, mean age 81.4 ± 9.5 years) were included. The improved group had a higher baseline ZVa {4.83 (4.15-6.89) mmHg/ml/mIn conclusion, Zva is a simple, noninvasive marker that shows promise as a predictor of LVEF improvement after TAVR in reduced LVEF patients.

The development of targeted therapies that affect multiple signaling pathways and stimulate antitumor immunity is greatly needed. About 20% of patients with breast cancer overexpress HER2. Small molecules and antibodies targeting HER2 convey some survival benefits; however, patients with advanced disease succumb to the disease under these treatment regimens, possibly because HER2 is not completely necessary for the survival of the targeted cancer cells. In the present study, we show that a polyinosine/polycytosine (pIC) HER2-homing chemical vector induced the demise of HER2-overexpressing breast cancer cells, including trastuzumab-resistant cells. Targeting pIC to the tumor evoked a number of cell-killing mechanisms, as well as strong bystander effects. These bystander mechanisms included type I IFN induction, immune cell recruitment, and activation. The HER2-targeted pIC strongly inhibited the growth of HER2-overexpressing tumors in immunocompetent mice. The data presented here could open additional avenues in the treatment of HER2-positive breast cancer. Cancer Immunol Res; 4(8); 688–97. ©2016 AACR.

// Simon-Peter Williams 1 , Annie Ogasawara 1 , Jeff N. Tinianow 1 , Judith E. Flores 1 , David Kan 1 , Jeffrey Lau 1 , MaryAnn Go 1 , Alexander N. Vanderbilt 1 , Herman S. Gill 1 , Li Miao 1 , Joshua Goldsmith 1 , Bonnee Rubinfeld 1 , Weiguang Mao 1 , Ron Firestein 1 , Shang-Fan Yu 1 , Jan Marik 1 , Anton G.T. Terwisscha van Scheltinga 1, 2 1 Genentech Research and Early Development, Genentech, Inc., South San Francisco, CA, 94080, USA 2 Department of Clinical Pharmacy and Pharmacology, University Medical Center Groningen, University of Groningen, Groningen, 9700RB, The Netherlands Correspondence to : Simon-Peter Williams, e-mail: williams.simon@gene.com Keywords: antibody-drug conjugates, immunoPET, TENB2, STEAP1, zirconium-89 Received: December 10, 2015 Accepted: March 04, 2016 Published: March 26, 2016 ABSTRACT The efficacy of antibody-drug conjugates (ADCs) targeted to solid tumors depends on biological processes that are hard to monitor in vivo . 89 Zr-immunoPET of the ADC antibodies could help understand the performance of ADCs in the clinic by confirming the necessary penetration, binding, and internalization. This work studied monomethyl auristatin E (MMAE) ADCs against two targets in metastatic castration-resistant prostate cancer, TENB2 and STEAP1, in four patient-derived tumor models (LuCaP35V, LuCaP70, LuCaP77, LuCaP96.1). Three aspects of ADC biology were measured and compared: efficacy was measured in tumor growth inhibition studies; target expression was measured by immunohistochemistry and flow cytometry; and tumor antibody uptake was measured with 111 In-mAbs and gamma counting or with 89 Zr-immunoPET. Within each model, the mAb with the highest tumor uptake showed the greatest potency as an ADC. Sensitivity between models varied, with the LuCaP77 model showing weak efficacy despite high target expression and high antibody uptake. Ex vivo analysis confirmed the in vivo results, showing a correlation between expression, uptake and ADC efficacy. We conclude that 89 Zr-immunoPET data can demonstrate which ADC candidates achieve the penetration, binding, and internalization necessary for efficacy in tumors sensitive to the toxic payload.

The ribosomal maturation factor P (RimP) is a highly conserved protein in bacteria and has been shown to be important in ribosomal assembly in Escherichia coli. Because of its central importance in bacterial metabolism, RimP represents a good potential target for drug design to combat human pathogens such as Mycobacterium tuberculosis. However, to date, the only RimP structure available is the NMR structure of the ortholog in another bacterial pathogen, Streptococcus pneumoniae. Here, we report a 2.2 Å resolution crystal structure of MSMEG_2624, the RimP ortholog in the close M. tuberculosis relative Mycobacterium smegmatis, and using in vitro binding assays, we show that MSMEG_2624 interacts with the small ribosomal protein S12, also known as RpsL. Further analyses revealed that the conserved residues in the linker region between the N- and C-terminal domains of MSMEG_2624 are essential for binding to RpsL. However, neither of the two domains alone was sufficient to form strong interactions with RpsL. More importantly, the linker region was essential for in vivo ribosomal biogenesis. Our study provides critical mechanistic insights into the role of RimP in ribosome biogenesis. We anticipate that the MSMEG_2624 crystal structure has the potential to be used for drug design to manage M. tuberculosis infections.

The tubulysins are promising anticancer cytotoxic agents due to the clinical validation of their mechanism of action (microtubule inhibition) and their particular activity against multidrug-resistant tumor cells. Yet their high potency and subsequent systemic toxicity make them prime candidates for targeted therapy, particularly in the form of antibody–drug conjugates (ADCs). Here we report a strategy to prepare stable and bioreversible conjugates of tubulysins to antibodies without loss of activity. A peptide trigger along with a quaternary ammonium salt linker connection to the tertiary amine of tubulysin provided ADCs that were potent in vitro. However, we observed metabolism of a critical acetate ester of the drug in vivo, resulting in diminished conjugate activity. We were able to circumvent this metabolic liability with the judicious choice of a propyl ether replacement. This modified tubulysin ADC was stable and effective against multidrug-resistant lymphoma cell lines and tumors.

A novel disulfide linker was designed to enable a direct connection between cytotoxic pyrrolobenzodiazepine (PBD) drugs and the cysteine on a targeting antibody for use in antibody–drug conjugates (ADCs). ADCs composed of a cysteine-engineered antibody were armed with a PBD using a self-immolative disulfide linker. Both the chemical linker and the antibody site were optimized for this new bioconjugation strategy to provide a highly stable and efficacious ADC. This novel disulfide ADC was compared with a conjugate containing the same PBD drug, but attached to the antibody via a peptide linker. Both ADCs had similar efficacy in mice bearing human tumor xenografts. Safety studies in rats revealed that the disulfide-linked ADC had a higher MTD than the peptide-linked ADC. Overall, these data suggest that the novel self-immolative disulfide linker represents a valuable way to construct ADCs with equivalent efficacy and improved safety. Mol Cancer Ther; 16(5); 871–8. ©2017 AACR.

We present a case of a 56-year-old man with history of coronary artery disease and prior two coronary bypass surgeries, who presented with chronic chest pain and was found to have a fractured sternal wire that migrated through the right ventricle and embolized to the right lower pulmonary artery without evident hemodynamic consequences. The sternal wire migration process, in part due to patient's poor medical compliance, was captured on serial computed tomography scans over a period of several years. .

Antibody-drug conjugates (ADCs) are potent cytotoxic drugs linked to antibodies through chemical linkers, and allow specific targeting of drugs to neoplastic cells. The expression of CD22 is limited to B-cells, and we show that CD22 is expressed on the vast majority of non-Hodgkin's lymphomas (NHLs). An ideal target for an ADC for the treatment of NHL would have limited expression outside the B-cell compartment and be highly effective against NHL. We generated an ADC consisting of a humanized anti-CD22 antibody conjugated to the anti-mitotic agent maytansine with a stable linker (anti-CD22-MCC-DM1). Anti-CD22-MCC-DM1 was broadly effective in in vitro killing assays on NHL B-cell lines. We did not find a strong correlation between in vitro potency and CD22 surface expression, internalization of ADC or sensitivity to free drug. We show that anti-CD22-MCC-DM1 was capable of inducing complete tumor regression in NHL xenograft mouse models. Further, anti-CD22-MCC-DM1 was well tolerated in cynomolgus monkeys and substantially decreased circulating B-cells as well as follicle size and germinal center formation in lymphoid organs. These results suggest that anti-CD22-MCC-DM1 has an efficacy, safety and pharmacodynamic profile that support its use as a treatment for NHL.

Here we describe the generation of an antibody–drug conjugate (ADC) consisting of a humanized anti-CD79b antibody that is conjugated to monomethylauristatin E (MMAE) through engineered cysteines (THIOMABs) by a protease cleavable linker. By using flow cytometry, we detected the surface expression of CD79b in almost all non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia patients, suggesting that anti–CD79b-vcMMAE could be widely used in these malignancies. By using NHL cell lines to simulate a patient population we discovered that a minimal cell-surface expression level of CD79b was required for in vitro activity. Within the subpopulation of cell lines above this minimal threshold, we found that sensitivity to free MMAE, mutation of cancer genes, and cell doubling time were poorly correlated with in vitro activity; however, the expression level of BCL-XL was correlated with reduced sensitivity to anti–CD79b-vcMMAE. This observation was supported by in vivo data showing that a Bcl-2 family inhibitor, ABT-263, strikingly enhanced the activity of anti–CD79b-vcMMAE. Furthermore, anti–CD79b-vcMMAE was significantly more effective than a standard-of-care regimen, R-CHOP (ie, rituximab with a single intravenous injection of 30 mg/kg cyclophosphamide, 2.475 mg/kg doxorubicin, 0.375 mg/kg vincristine, and oral dosing of 0.15 mg/kg prednisone once a day for 5 days), in 3 xenograft models of NHL. Together, these data suggest that anti–CD79b-vcMMAE could be broadly efficacious for the treatment of NHL.

Int. Journal of Political Economy, vol. 32, no. 3, Fall 2002, pp. 5–22.© 2004 M.E. Sharpe, Inc. All rights reserved.ISSN 0891–1016 / 2004 $9.50 + 0.00.The authors are, respectively, Ph.D. candidate in Social and Political Scienceat the University of Cambridge, England, and professor in the Department ofEconomics and at the Schulich School of Business, York University, Toronto,Canada. Without implicating them, the authors thank Geoff Ingham and AlanKerns for their insightful comments.

Antibody–drug conjugates (ADC), potent cytotoxic drugs linked to antibodies via chemical linkers, allow specific targeting of drugs to neoplastic cells. We have used this technology to develop the ADC DCDT2980S that targets CD22, an antigen with expression limited to B cells and the vast majority of non-Hodgkin lymphomas (NHL). DCDT2980S consists of a humanized anti-CD22 monoclonal IgG1 antibody with a potent microtubule-disrupting agent, monomethyl auristatin E (MMAE), linked to the reduced cysteines of the antibody via a protease cleavable linker, maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB). We describe the efficacy, safety, and pharmacokinetics of DCDT2980S in animal models to assess its potential as a therapeutic for the treatment of B-cell malignancies. We did not find a strong correlation between in vitro or in vivo efficacy and CD22 surface expression, nor a correlation of sensitivity to free drug and in vitro potency. We show that DCDT2980S was capable of inducing complete tumor regression in xenograft mouse models of NHL and can be more effective than rituximab plus combination chemotherapy at drug exposures that were well tolerated in cynomolgus monkeys. These results suggest that DCDT2980S has an efficacy, safety, and pharmacokinetics profile that support potential treatment of NHL. Mol Cancer Ther; 12(7); 1255–65. ©2013 AACR.

The HER2 receptor tyrosine kinase is amplified in approximately 20% of human breast cancer and is associated with poor clinical outcome. The humanized antibodies trastuzumab and pertuzumab are approved for use in both early and metastatic HER2-positive breast cancer, and are most often given with chemotherapy. Antibody-drug conjugates (ADCs) are anti-tumor agents designed to deliver potent cytotoxic drugs selectively to target-expressing tumor cells. Trastuzumab emtansine is a HER2-directed ADC comprised of trastuzumab covalently linked to the microtubule inhibitor DM1, through the stable MCC linker. Trastuzumab emtansine is approved for use in HER2-positive metastatic breast cancer as a single agent in patients who have received prior trastuzumab and a taxane. We are now exploring new HER2-directed ADCs (‘2nd generation ADCs’) with different mechanisms of action (MOA) than trastuzumab emtansine by investigating ADCs utilizing DNA-damaging agents, such as pyrrolobenzodiazepine (PBD) dimers and cyclopropylbenzindole (CBI) dimers, as the cytotoxic drug components. These agents have been conjugated to either trastuzumab or the humanized anti-HER2 antibody 7C2 (hu7C2) using both uncleavable and cleavable linkers. As free drugs and ADCs, the PBDs and CBIs show similar or greater potency in cell proliferation assays in vitro compared to DM1 and trastuzumab emtansine. However, unlike DM1, these agents are not strong substrates of Pgp/MDR1. Moreover, the PBDs and CBIs are active on non-dividing cells, whereas microtubule inhibitors such as DM1 do not affect non-dividing cells. Robust anti-tumor activity was observed in vivo in the fo5 HER2 transgenic tumor transplant model with the 2nd generation ADCs. Efficacious doses resulting in tumor stasis or regression ranged from 0.25-3 mg/kg administered as a single injection. In contrast, doses of trastuzumab emtansine required for stasis/regression in this model are 10 and 15 mg/kg, respectively. Efficacious doses were well-tolerated in the mouse xenograft models. Further tolerability studies of the 2nd generation ADCs were performed in rats. As rats are a non-binding species for trastuzumab and hu7C2, these studies assessed antigen-independent toxicities. Maximum tolerated doses for the different ADCs ranged from 2.5-15 mg/kg administered as a single injection, compared to 46 mg/kg for trastuzumab emtansine (Poon et al., 2013), likely reflecting both the different MOA and greater potency of the cytotoxic agents utilized in the 2nd generation HER2-directed ADCs. Overall, our findings demonstrate robust in vitro and in vivo activity of HER2 ADCs comprised of DNA-active agents, allowing for further development of a HER2-directed ADC distinct from trastuzumab emtansine. Citation Format: Gail D. Lewis Phillips, Guangmin Li, Jun Guo, Jeffrey Lau, Shang-Fan Yu, Thomas Pillow, Byoung-Chul Lee, Jack Sadowsky, Melissa Schutten, Carter Fields, Mark X. Sliwkowski. Preclinical development of 2nd generation HER2-directed antibody-drug conjugates. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1207.

To determine the value of the intent to include internal mammary nodes (IMNs) in the radiation therapy (RT) volume for patients receiving adjuvant locoregional (breast or chest wall plus axillary and supraclavicular fossa) RT for breast cancer.2413 women with node-positive or T3/4N0 invasive breast cancer, treated with locoregional RT from 2001 to 2006, were identified in a prospectively maintained, population-based database. Intent to include IMNs in RT volume was determined through review of patient charts and RT plans. Distant relapse free survival (D-RFS), breast cancer-specific survival (BCSS), and overall survival (OS) were compared between the two groups. Prespecified pN1 subgroup analyses were performed.The median follow-up time was 6.2 years. Forty-one percent of study participants received IMN RT. The 5-year D-RFS for IMN inclusion and exclusion groups were 82% vs. 82% (p = 0.82), BCSS was 87% vs. 87% (p = 0.81), and OS was 85% vs. 83% (p = 0.06). In the pN1 subgroup, D-RFS was 90% vs. 88% (p = 0.31), BCSS was 94% vs. 92% (p = 0.18), and OS was 91% vs. 88% (p = 0.01). After potential confounding variables were controlled for, women who received IMN RT did not have significantly different D-RFS (hazard ratio [HR] = 1.02 (95% confidence interval [CI], 0.84-1.24; p = 0.85), BCSS (HR = 0.98 (95% CI, 0.79-1.22; p = 0.88), or OS (HR = 0.95; 95% CI, 0.78-1.15; p = 0.57). In the pN1 subgroup, IMN RT was associated with trends for improved survival that were not statistically significant: D-RFS (HR = 0.87; 95% CI, 0.63-1.22; p = 0.42), BCSS (HR = 0.85; 95% CI, 0.57-1.25; p = 0.39), and OS (HR = 0.78; 95% CI, 0.56-1.09; p = 0.14).After a median follow-up time of 6.2 years, although intentional IMN RT was not associated with a significant improvement in survival, this population-based study suggests that IMN RT may contribute to improved outcomes in selected patients with N1 disease.

Purpose: Individually targeting B-cell antigens with monoclonal antibody therapeutics has improved the treatment of non-Hodgkin lymphoma (NHL). We examined if the antitumor activity of rituximab, CD20-specific antibody, could be improved by simultaneously targeting CD40 with the humanized monoclonal antibody dacetuzumab (SGN-40). Experimental Design: Dacetuzumab was dosed with rituximab to determine the in vivo activity of this combination in a subcutaneous Ramos xenograft model of non-Hodgkin lymphoma (NHL). The effect of dacetuzumab on rituximab antibody-dependent cell mediated–cytotoxicity (ADCC), antiproliferative, and apoptotic activities were evaluated in vitro using NHL cell lines. Western blotting and flow cytometry were used to contrast the signaling pathways activated by dacetuzumab and rituximab in NHL cells. Results: The dacetuzumab-rituximab combination had significantly improved antitumor activity over the equivalent dose of rituximab in the Ramos xenograft model (P = 0.0021). Dacetuzumab did not augment rituximab-mediated ADCC activity; however, these antibodies were additive to synergistic in cell-proliferation assays and produced increased apoptosis in combination. Rituximab signaling downregulated BCL-6 oncoprotein in a cell line–specific manner, whereas dacetuzumab strongly downregulated BCL-6 in each cell line. Dacetuzumab induced expression of the proapoptotic proteins TAp63 and Fas, whereas rituximab did not affect basal expression of either protein. Finally, rituximab partially blocked dacetuzumab-mediated upregulation of the prosurvival protein BCL-xL. Conclusions: Targeting CD40 with dacetuzumab enhanced the antitumor activity of rituximab in cell line and xenograft NHL models. The distinct but complementary apoptotic signal transduction profiles of dacetuzumab and rituximab are an important mechanism behind the improved activity of this combination. Clin Cancer Res; 17(14); 4672–81. ©2011 AACR.

The primary function of B cells, critical components of the adaptive immune response, is to produce antibodies against foreign antigens, as well as to perform isotype class switching, which changes the heavy chain of an antibody so that it can interact with different repertoires of effector cells. CD40 is a member of the tumor necrosis factor superfamily of cell surface receptors that transmits survival signals to B cells. In contrast, in B cell cancers, stimulation of CD40 signaling results in a heterogeneous response in which cells can sometimes undergo cell death in response to treatment, depending on the system studied. We found an association between sensitivity to CD40 stimulation and mutation of the tumor suppressor p53 in a panel of non-Hodgkin's lymphoma cell lines. Consistent with p53's tumor suppressor role, we found that higher levels of intrinsic DNA damage and increased proliferation rates, as well as higher levels of BCL6, a transcriptional repressor proto-oncogene, were associated with sensitivity to CD40 stimulation. In addition, CD40 treatment-resistant cell lines were sensitized to CD40 stimulation after the introduction of DNA-damaging agents. Using gene expression analysis, we also showed that resistant cell lines exhibited a preexisting activated CD40 pathway and that an mRNA expression signature comprising CD40 target genes predicted sensitivity and resistance to CD40-activating agents in cell lines and mouse xenograft models. Finally, the gene signature predicted tumor shrinkage and progression-free survival in patients with diffuse large B cell lymphoma treated with dacetuzumab, a monoclonal antibody with partial CD40 agonist activity. These data show that CD40 pathway activation status may be useful in predicting the antitumor activity of CD40-stimulating therapeutic drugs.

Summary Kawasaki disease (KD) is a leading cause of non-atherosclerotic coronary artery aneurysms and, less commonly, peripheral artery aneurysms. We report an 81-year-old Japanese man from Hawaii with a history of an abdominal aortic aneurysm, bilateral iliac aneurysms, and an ambiguous right atrial cystic mass. The patient developed new-onset atrial fibrillation during lithotripsy. Angiography and magnetic resonance imaging revealed giant coronary artery aneurysms of the right coronary artery (RCA) and left anterior descending artery, and a thoracic aortic aneurysm. The RCA aneurysm was greater than 2 in. in diameter at the time of operation. Although we cannot confirm whether the patient had KD during childhood, this is the most likely diagnosis in the absence of a connective tissue disorder, systemic vasculitis, or atherosclerotic risk factors. This patient may represent the oldest case of KD, predating the earliest known case by more than 20 years. This case sheds light on the historical epidemiology of KD and its clinical course, especially regarding late vascular sequelae.

Abstract 2721 Poster Board II-697 The role of the CD40 pathway in B-cell cancers is rather enigmatic due to having polar opposite roles in tumor biology. Stimulation of the CD40 receptor can reduce cell viability of NHL cell lines and xenograft tumor models, whereas, in sharp contrast, constitutive CD40 stimulation can actually promote proliferation of NHL cell lines, as well as drive lymphomagenesis and transformation of primary B-cells. Therefore, targeting the CD40 pathway has been challenging due to a knowledge gap in understanding the factors that may dictate a pro- or anti-tumor role. To address this directly, we employed a large panel of NHL cell lines and xenografts models to simulate a patient population and to define the molecular parameters to test in the context of clinical trials. Using baseline gene expression profiling, we found that the in vitro activity of an antibody that stimulates the CD40 pathway, Dacetuzumab (SGN-40), correlated strikingly with a transcriptional profile that was representative of an inactive CD40 pathway. Resistant cells, on the other hand, displayed a transcriptional profile indicative of an activated CD40 pathway. Consistent with these observations, constitutive phosphorylation of ERK, a node downstream of CD40 signaling, was present predominantly in cell lines that were resistant to Dacetuzumab. Fifteen genes, based on their association with in vitro activity and their biology as components of the CD40 network, were selected as a classifier from these pre-clinical data and independently tested in a panel of cell lines and xenografts models. Testing the selected gene classifier by qRT-PCR from baseline fixed tumor tissue of 39 patients treated with Dacetuzumab in phase I and II clinical trials revealed the ability to predict sensitivity in patients, as determined by tumor shrinkage >10%, with an overall accuracy of 80%. Consistent with the observed tumor shrinkage, the classifier positive, predicted to respond to Dacetuzumab, patients also exhibited a prolonged progression free survival (PFS) compared to classifier negative patients, predicted not to respond to Dacetuzumab (HR = 0.23, p = 0.001). Finally, using a novel microarray approach to genome-wide expression profiling on fixed tumor tissue revealed that CD40 pathway activation was indeed an important predictor of response. Overall, these data suggest that sensitivity to Dacetuzumab can be predicted by pre-determining the CD40 pathway activation status of tumors. Disclosures: Dornan: Genentech, Inc.: Employment, Equity Ownership. Burington:Genentech, Inc.: Employment, Equity Ownership. Yue:Genentech, Inc.: Employment, Equity Ownership. Shi:Genentech, Inc.: Employment, Equity Ownership. Advani:Seattle Genetics, Inc.: Research Funding. Yu:Genentech, Inc.: Employment, Equity Ownership. Lau:Genentech, Inc.: Employment, Equity Ownership. Yu:Genentech, Inc.: Employment, Equity Ownership. De Vos:Seattle Genetics, Inc.: Research Funding. Forero-Torres:Seattle Genetics, Inc.: Research Funding. Modrusan:Genentech, Inc.: Employment, Equity Ownership. Koeppen:Genentech, Inc.: Employment, Equity Ownership. Yang:Genentech, Inc.: Employment, Equity Ownership. Du:Genentech, Inc.: Employment, Equity Ownership. Elkins:Genentech, Inc.: Employment, Equity Ownership. Polson:Genentech, Inc.: Employment, Equity Ownership. Offringa:Genentech, Inc.: Employment, Equity Ownership. Whiting:Seattle Genetics, Inc.: Employment, Equity Ownership. Ebens:Genentech, Inc.: Employment, Equity Ownership.

SGN-40, an anti-CD40 monoclonal antibody, is a humanized IgG1 antibody that binds to CD40, mediates effector cell functions (ADCC/ADCP) and activates downstream signaling pathways. SGN-40 has shown activity in a phase I single agent multi-dose trial in non Hodgkin’s lymphoma, with greatest activity in diffuse large B-cell lymphoma (Advani et al. 2008, ICML). Previous in vitro studies implicated down-regulation of the germinal center expressed protein Bcl-6, upregulation of p53 family member TAp63a, and FAS death receptor induction as potential mechanisms leading to lymphoma cell death (Lewis et al. 2007, AACR, ASH). In order to further define the apoptosis signaling mechanism, we assessed the ability of SGN-40 to inhibit proliferation and promote apoptosis across a large panel of non-Hodgkin’s lymphoma cell lines. SGN-40 reduced cell viability in 58% (18/31) of cell lines tested. To identify the genes that may be promoting apoptosis and/or inhibiting proliferation, we compared gene expression levels before and after SGN-40 exposure in both sensitive and resistant cell lines, as well as in normal B-cells. SGN-40 strikingly and specifically upregulated FAS on the cell surface of sensitive cell lines. Furthermore, the addition of soluble FAS-Fc dampened SGN-40-induced apoptosis in a subset of sensitive cell lines, suggesting a dependence on a FAS-FASL interaction. These data imply that FAS-dependent apoptosis may directly contribute to the anti-tumor effect of SGN-40. Our data also demonstrate that SGN-40 sensitivity is dependent on the point in B-cell development at which the NHL cell lines were transformed. Sensitive cell lines had a gene signature characteristic of minimal activation of CD40 signaling prior to SGN-40 exposure, whereas resistant cell lines had a signature consistent with prior constitutive signaling downstream of CD40. Thus, SGN-40 appears to elicit its apoptotic properties through activation of CD40 signaling in NHL cell lines not previously exposed to CD40L signaling in the germinal center environment at the time of lymphocyte transformation (GCB lymphomas). In order to develop a clinically feasible assay from FFPE tissue, we developed a 14-gene signature by Stepwise Linear Modeling, utilizing genes from the CD40 pathway activation and GCB gene sets; the classifier gave >96% accuracy (30/31) on the ‘training’ set of cell lines and 75% (3/4) accuracy on a ‘test’ set of xenografts. Overall, our data provides unique insights into SGN-40 mechanisms of action, provides a testable hypothesis of the clinical mechanism of action, and a potential diagnostic test to identify patients more likely to benefit from SGN-40. Efforts are currently underway to test the clinical relevance of these findings.