Projected effects of climate change on Pseudo-nitzschia bloom dynamics in the Gulf of Maine.

Worldwide, warming ocean temperatures have contributed to extreme harmful algal bloom events and shifts in phytoplankton species composition. In 2016 in the Gulf of Maine (GOM), an unprecedented Pseudo-nitzschia bloom led to the first domoic-acid induced shellfishery closures in the region. Potential links between climate change, warming temperatures, and the GOM Pseudo-nitzschia assemblage, however, remain unexplored. In this study, a global climate change projection previously downscaled to 7-km resolution for the Northwest Atlantic was further refined with a 1-3-km resolution simulation of the GOM to investigate the effects of climate change on HAB dynamics. A 25-year time slice of projected conditions at the end of the 21st century (2073-2097) was compared to a 25-year hindcast of contemporary ocean conditions (1994-2018) and analyzed for changes to GOM inflows, transport, and Pseudo-nitzschia australis growth potential. On average, climate change is predicted to lead to increased temperatures, decreased salinity, and increased stratification in the GOM, with the largest changes occurring in the late summer. Inflows from the Scotian Shelf are projected to increase, and alongshore transport in the Eastern Maine Coastal Current is projected to intensify. Increasing ocean temperatures will likely make P. australis growth conditions less favorable in the southern and western GOM but improve P. australis growth conditions in the eastern GOM, including a later growing season in the fall, and a longer growing season in the spring. Combined, these changes suggest that P. australis blooms in the eastern GOM could intensify in the 21st century, and that the overall Pseudo-nitzschia species assemblage might shift to warmer-adapted species such as P. plurisecta or other Pseudo-nitzschia species that may be introduced.

PCA062, a P-cadherin Targeting Antibody-Drug Conjugate, Displays Potent Antitumor Activity Against P-cadherin-expressing Malignancies.

The cell surface glycoprotein P-cadherin is highly expressed in a number of malignancies, including those arising in the epithelium of the bladder, breast, esophagus, lung, and upper aerodigestive system. PCA062 is a P-cadherin specific antibody-drug conjugate that utilizes the clinically validated SMCC-DM1 linker payload to mediate potent cytotoxicity in cell lines expressing high levels of P-cadherin in vitro, while displaying no specific activity in P-cadherin-negative cell lines. High cell surface P-cadherin is necessary, but not sufficient, to mediate PCA062 cytotoxicity. In vivo, PCA062 demonstrated high serum stability and a potent ability to induce mitotic arrest. In addition, PCA062 was efficacious in clinically relevant models of P-cadherin-expressing cancers, including breast, esophageal, and head and neck. Preclinical non-human primate toxicology studies demonstrated a favorable safety profile that supports clinical development. Genome-wide CRISPR screens reveal that expression of the multidrug-resistant gene ABCC1 and the lysosomal transporter SLC46A3 differentially impact tumor cell sensitivity to PCA062. The preclinical data presented here suggest that PCA062 may have clinical value for treating patients with multiple cancer types including basal-like breast cancer.

Investigating Pseudo-nitzschia australis introduction to the Gulf of Maine with observations and models.

In 2016, an unprecedented Pseudo-nitzschia australis bloom in the Gulf of Maine led to the first shellfishery closures due to domoic acid in the region's history. In this paper, potential introduction routes of P. australis are explored through observations, a hydrodynamic model, and a Lagrangian particle tracking model. Based on particle tracking experiments, the most likely source of P. australis to the Gulf of Maine was the Scotian Shelf. However, in 2016, connectivity between the Scotian Shelf and the bloom region was not significantly different from the other years between 2012 and 2019, nor were temperature conditions more favorable for P. australis growth. Observations indicated changes on the Scotian Shelf in 2016 preceded the introduction of P. australis: increased bottom salinity and decreased surface salinity. The increased bottom salinity on the shelf may be linked to anomalously saline water observed near the coast of Maine in 2016 via transport through Northeast Channel. The changes in upstream water mass properties may be related to the introduction of P. australis, and could be the result of either increased influence of the Labrador Current or increased outflow from the Gulf of St. Lawrence. The ultimate source of P. australis remains unknown, although the species has previously been observed in the eastern North Atlantic, and connectivity across the ocean is possible via a subpolar route. Continued and increased monitoring is warranted to track interannual Pseudo-nitzschia persistence in the Gulf of Maine, and sampling on the Scotian Shelf should be conducted to map upstream P. australis populations.

Discovery of Potent and Selective Antibody-Drug Conjugates with Eg5 Inhibitors through Linker and Payload Optimization.

Targeted antimitotic agents are a promising class of anticancer therapies. Herein, we describe the development of a potent and selective antimitotic Eg5 inhibitor based antibody-drug conjugate (ADC). Preliminary studies were performed using proprietary Eg5 inhibitors which were conjugated onto a HER2-targeting antibody using maleimido caproyl valine-citrulline para-amino benzocarbamate, or MC-VC-PABC cleavable linker. However, the resulting ADCs lacked antigen-specificity in vivo, probably from premature release of the payload. Second-generation ADCs were then developed, using noncleavable linkers, and the resulting conjugates (ADC-4 and ADC-10) led to in vivo efficacy in an HER-2 expressing (SK-OV-3ip) mouse xenograft model while ADC-11 led to in vivo efficacy in an anti-c-KIT (NCI-H526) mouse xenograft model in a target-dependent manner.

Pseudo-nitzschia bloom dynamics in the Gulf of Maine: 2012-2016.

The toxic diatom genus Pseudo-nitzschia is a growing presence in the Gulf of Maine (GOM), where regionally unprecedented levels of domoic acid (DA) in 2016 led to the first Amnesic Shellfish Poisoning closures in the region. However, factors driving GOM Pseudo-nitzschia dynamics, DA concentrations, and the 2016 event are unclear. Water samples were collected at the surface and at depth in offshore transects in summer 2012, 2014, and 2015, and fall 2016, and a weekly time series of surface water samples was collected in 2013. Temperature and salinity data were obtained from NERACOOS buoys and measurements during sample collection. Samples were processed for particulate DA (pDA), dissolved nutrients (nitrate, ammonium, silicic acid, and phosphate), and cellular abundance. Species composition was estimated via Automated Ribosomal Intergenic Spacer Analysis (ARISA), a semi-quantitative DNA finger-printing tool. Pseudo-nitzschia biogeography was consistent in the years 2012, 2014, and 2015, with greater Pseudo-nitzschia cell abundance and P. plurisecta dominance in low-salinity inshore samples, and lower Pseudo-nitzschia cell abundance and P. delicatissima and P. seriata dominance in high-salinity offshore samples. During the 2016 event, pDA concentrations were an order of magnitude higher than in previous years, and inshore-offshore contrasts in biogeography were weak, with P. australis present in every sample. Patterns in temporal and spatial variability confirm that pDA increases with the abundance and the cellular DA of Pseudo-nitzschia species, but was not correlated with any one environmental factor. The greater pDA in 2016 was caused by P. australis - the observation of which is unprecedented in the region - and may have been exacerbated by low residual silicic acid. The novel presence of P. australis may be due to local growth conditions, the introduction of a population with an anomalous water mass, or both factors. A definitive cause of the 2016 bloom remains unknown, and continued DA monitoring in the GOM is warranted.

Nicotinamide Phosphoribosyltransferase Inhibitor as a Novel Payload for Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are a novel modality that allows targeted delivery of potent therapeutic agents to the desired site. Herein we report our discovery of NAMPT inhibitors as a novel nonantimitotic payload for ADCs. The resulting anti-c-Kit conjugates (ADC-3 and ADC-4) demonstrated in vivo efficacy in the c-Kit positive gastrointestinal stromal tumor GIST-T1 xenograft model in a target-dependent manner.

Potential Impacts of Offshore Wind Farms on North Sea Stratification.

Advances in offshore wind farm (OWF) technology have recently led to their construction in coastal waters that are deep enough to be seasonally stratified. As tidal currents move past the OWF foundation structures they generate a turbulent wake that will contribute to a mixing of the stratified water column. In this study we show that the mixing generated in this way may have a significant impact on the large-scale stratification of the German Bight region of the North Sea. This region is chosen as the focus of this study since the planning of OWFs is particularly widespread. Using a combination of idealised modelling and in situ measurements, we provide order-of-magnitude estimates of two important time scales that are key to understanding the impacts of OWFs: (i) a mixing time scale, describing how long a complete mixing of the stratification takes, and (ii) an advective time scale, quantifying for how long a water parcel is expected to undergo enhanced wind farm mixing. The results are especially sensitive to both the drag coefficient and type of foundation structure, as well as the evolution of the pycnocline under enhanced mixing conditions-both of which are not well known. With these limitations in mind, the results show that OWFs could impact the large-scale stratification, but only when they occupy extensive shelf regions. They are expected to have very little impact on large-scale stratification at the current capacity in the North Sea, but the impact could be significant in future large-scale development scenarios.

MAPK pathway inhibition enhances the efficacy of an anti-endothelin B receptor drug conjugate by inducing target expression in melanoma.

Therapies targeting the mitogen-activated protein (MAP) kinase pathway in melanoma have produced significant clinical responses; however, duration of response is limited by acquisition of drug resistance. Rational drug combinations may improve outcomes in this setting. We assessed the therapeutic combination of an antibody-drug conjugate (ADC) targeting the endothelin B receptor (EDNRB) with small-molecule inhibitors of the MAP kinase signaling pathway in melanoma. Cell lines and tumor models containing either mutant BRAF or NRAS, or wild-type for both, were exposed to small-molecule inhibitors of BRAF and MEK. Expression of EDNRB was analyzed and the therapeutic impact of combining the anti-EDNRB ADC with the BRAF and MEK inhibitors was assessed. Increased expression of EDNRB in response to inhibition of BRAF and/or MEK was observed and augmented the antitumor activity of the ADC. Enhanced target expression and ADC antitumor activity were realized irrespective of the response of the tumor model to the BRAF or MEK inhibitors alone and could be achieved in melanoma with mutant NRAS, BRAF, or neither mutation. Cells that acquired resistance to BRAF inhibition through long-term culture retained drug-induced elevated levels of EDNRB expression. Expression of EDNRB was not enhanced in normal human melanocytes by inhibition of BRAF and the combination of the ADC with MAPK inhibitors was well-tolerated in mice. The anti-EDNRB ADC combines well with BRAF and MEK inhibitors and could have therapeutic use in the majority of human melanoma cases.

Effects of anti-VEGF on pharmacokinetics, biodistribution, and tumor penetration of trastuzumab in a preclinical breast cancer model.

Both human epidermal growth factor receptor 2 (HER-2/neu) and VEGF overexpression correlate with aggressive phenotypes and decreased survival among breast cancer patients. Concordantly, the combination of trastuzumab (anti-HER2) with bevacizumab (anti-VEGF) has shown promising results in preclinical xenograft studies and in clinical trials. However, despite the known antiangiogenic mechanism of anti-VEGF antibodies, relatively little is known about their effects on the pharmacokinetics and tissue distribution of other antibodies. This study aimed to measure the disposition properties, with a particular emphasis on tumor uptake, of trastuzumab in the presence or absence of anti-VEGF. Radiolabeled trastuzumab was administered alone or in combination with an anti-VEGF antibody to mice bearing HER2-expressing KPL-4 breast cancer xenografts. Biodistribution, autoradiography, and single-photon emission computed tomography-X-ray computed tomography imaging all showed that anti-VEGF administration reduced accumulation of trastuzumab in tumors despite comparable blood exposures and similar distributions in most other tissues. A similar trend was also observed for an isotype-matched IgG with no affinity for HER2, showing reduced vascular permeability to macromolecules. Reduced tumor blood flow (P < 0.05) was observed following anti-VEGF treatment, with no significant differences in the other physiologic parameters measured despite immunohistochemical evidence of reduced vascular density. In conclusion, anti-VEGF preadministration decreased tumor uptake of trastuzumab, and this phenomenon was mechanistically attributed to reduced vascular permeability and blood perfusion. These findings may ultimately help inform dosing strategies to achieve improved clinical outcomes.

An antibody-drug conjugate targeting the endothelin B receptor for the treatment of melanoma.

PURPOSE: To identify and evaluate targets amenable to antibody therapy in melanoma. EXPERIMENTAL DESIGN: We searched for mRNA transcripts coding for cell-surface proteins with expression patterns similar to that of the melanoma oncogene MITF. One such candidate, the endothelin B receptor (EDNBR), was first analyzed for a functional contribution to tumor growth by conditional induction of shRNA. Second, antibodies were raised to the receptor, conjugated with monomethyl auristatin E, and tested for efficacy against melanoma tumor models generated from cell lines. RESULTS: Conditional knockdown of the receptor in tumor xenograft models resulted in only a modest impact on tumor growth. A monoclonal antibody reactive with the N-terminal tail of EDNBR was found to internalize rapidly into melanoma cells. When conjugated with monomethyl auristatin E, the antibody-drug conjugate (ADC) showed remarkable efficacy against human melanoma cell lines and xenograft tumor models that was commensurate with levels of receptor expression. Comparative immunohistochemistry revealed a range of EDNBR expression across a panel of human melanomas, with the majority expressing levels equivalent to or greater than that in the models responsive to the ADC. CONCLUSION: An ADC targeting the EDNBR is highly efficacious in preclinical models of melanoma.

Effect of immune complex formation on the distribution of a novel antibody to the ovarian tumor antigen CA125.

3A5 is a novel antibody that binds repeated epitopes within CA125, an ovarian tumor antigen that is shed into the circulation. Binding to shed antigen may limit the effectiveness of therapeutic antibodies because of unproductive immune complex (IC) formation and/or altered antibody distribution. To evaluate this possibility, we characterized the impact of shed CA125 on the in vivo distribution of 3A5. In vitro, 3A5 and CA125 were found to form ICs in a concentration-dependent manner. This phenomenon was then evaluated in vivo using quantitative whole-body autoradiography to assess the tissue distribution of (125)I-3A5 in an orthotopic OVCAR-3 tumor mouse model at different stages of tumor burden. Low doses of 3A5 (75 µg/kg) and pathophysiological levels of shed CA125 led to the formation of ICs in vivo that were rapidly distributed to the liver. Under these conditions, increased clearance of 3A5 from normal tissues was observed in mice bearing CA125-expressing tumors. Of importance, despite IC formation, 3A5 uptake by tumors was sustained over time. At a therapeutically relevant dose of 3A5 (3.5 mg/kg), IC formation was undetectable and distribution to normal tissues followed that of blood. In contrast, increased levels of radioactivity were observed in the tumors. These data demonstrate that CA125 and 3A5 do form ICs in vivo and that the liver is involved in their uptake. However, at therapeutic doses of 3A5 and clinically relevant CA125 levels, IC formation consumes only a minor fraction of 3A5, and tumor targeting seems to be unaffected.

Antibody-drug conjugates for the treatment of non-Hodgkin's lymphoma: target and linker-drug selection.

Antibody-drug conjugates (ADC), potent cytotoxic drugs covalently linked to antibodies via chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Here, we systematically examine the potential targets and linker-drug combinations that could provide an optimal ADC for the treatment for non-Hodgkin's lymphoma. We identified seven antigens (CD19, CD20, CD21, CD22, CD72, CD79b, and CD180) for potential treatment of non-Hodgkin's lymphoma with ADCs. ADCs with cleavable linkers mediated in vivo efficacy via all these targets; ADCs with uncleavable linkers were only effective when targeted to CD22 and CD79b. In target-independent safety studies in rats, the uncleavable linker ADCs showed reduced toxicity, presumably due to the reduced release of free drug or other toxic metabolites into the circulation. Thus, our data suggest that ADCs with cleavable linkers work on a broad range of targets, and for specific targets, ADCs with uncleavable linkers provide a promising opportunity to improve the therapeutic window for ADCs in humans.

Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index.

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.

Armed antibodies targeting the mucin repeats of the ovarian cancer antigen, MUC16, are highly efficacious in animal tumor models.

MUC16 is a well-validated cell surface marker for serous adenocarcinomas of the ovary and other gynecologic malignancies that is distinguished by highly repetitive sequences ("mucin repeats") in the extracellular domain (ECD). We produced and compared two monoclonal antibodies: one (11D10) recognizing a unique, nonrepeating epitope in the ECD and another (3A5) that recognizes the repeats and binds multiple sites on each MUC16 protein. 3A5 conjugated to cytotoxic drugs exhibited superior toxicity against tumor cells in vitro and in tumor xenograft models compared with antibody-drug conjugates of 11D10. Importantly, drug conjugates of 3A5 were well tolerated in primates at levels in excess of therapeutic doses. Additionally, the presence of circulating CA125 in a rat model did not exacerbate the toxicity of 3A5 drug conjugates. We conclude that targeting the repeat MUC16 domains, thereby increasing cell-associated levels of drug-conjugated antibody, provides superior efficacy in vitro and in vivo without compromising safety.

Antibody-drug conjugates targeted to CD79 for the treatment of non-Hodgkin lymphoma.

Targeting cytotoxic drugs to cancer cells using antibody-drug conjugates (ADCs), particularly those with stable linkers between the drug and the antibody, could be an effective cancer treatment with low toxicity. However, for stable-linker ADCs to be effective, they must be internalized and degraded, limiting potential targets to surface antigens that are trafficked to lysosomes. CD79a and CD79b comprise the hetrodimeric signaling component of the B-cell receptor, and are attractive targets for the use of ADCs because they are B-cell-specific, expressed in non-Hodgkin lymphomas (NHL), and are trafficked to a lysosomal-like compartment as part of antigen presentation. We show here that the stable-linker ADCs anti-CD79b-MCC-DM1 and anti-CD79b-MC-MMAF are capable of target-dependent killing of nonHodgkin lymphoma cell lines in vitro. Further, these 2 ADCs are equally effective as low doses in xenograft models of follicular, mantle cell, and Burkitt lymphomas, even though several of these cell lines express relatively low levels of CD79b in vivo. In addition, we demonstrate that anti-CD79b ADCs were more effective than anti-CD79a ADCs and that, as hypothesized, anti-CD79b antibodies downregulated surface B-cell receptor and were trafficked to the lysosomal-like major histocompatibility complex class II-positive compartment MIIC. These results suggest that anti-CD79b-MCC-DM1 and anti-CD79b-MC-MMAF are promising therapeutics for the treatment of NHL.

Identification and immunotherapeutic targeting of antigens induced by chemotherapy.

Cancer cells differ from normal cells in their response to chemotherapy. We exploited this dissimilarity by identifying and targeting tumor-specific, cell-surface proteins whose expression is induced by the chemotherapeutic irinotecan (CPT-11; Camptosar). A cytotoxin-armed antibody reactive with one of these drug-induced surface proteins, the LY6D/E48 antigen, originally identified as the target of a monoclonal antibody reactive with squamous cell carcinomas, caused complete regression of colorectal tumor xenografts in mice treated with CPT-11, whereas either agent alone was less effective. These results suggest that a positive therapeutic index may be generated for other drug combinations by immunotherapeutic targeting of chemotherapy-induced antigens.