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Effects of Systemic Administration of Free Payload Compared with Conventional HER2-vc0101 on Indicators of Bone Marrow Toxicity and Bone Marrow Payload Levels in Rats

The approval of ado-trastuzumab emtansine (T-DM1) in HER2+ metastatic breast cancer validated HER2 as a target for HER2-specific antibody–drug conjugates (ADC). Despite its demonstrated clinical efficacy, certain inherent properties within T-DM1 hamper this compound from achieving the full potential of targeting HER2-expressing solid tumors with ADCs. Here, we detail the discovery of PF-06804103, an anti-HER2 ADC designed to have a widened therapeutic window compared with T-DM1. We utilized an empirical conjugation site screening campaign to identify the engineered ĸkK183C and K290C residues as those that maximized in vivo ADC stability, efficacy, and safety for a four drug–antibody ratio (DAR) ADC with this linker–payload combination. PF-06804103 incorporates the following novel design elements: (i) a new auristatin payload with optimized pharmacodynamic properties, (ii) a cleavable linker for optimized payload release and enhanced antitumor efficacy, and (iii) an engineered cysteine site–specific conjugation approach that overcomes the traditional safety liabilities of conventional conjugates and generates a homogenous drug product with a DAR of 4. PF-06804103 shows (i) an enhanced efficacy against low HER2-expressing breast, gastric, and lung tumor models, (ii) overcomes in vitro- and in vivo–acquired T-DM1 resistance, and (iii) an improved safety profile by enhancing ADC stability, pharmacokinetic parameters, and reducing off-target toxicities. Herein, we showcase our platform approach in optimizing ADC design, resulting in the generation of the anti-HER2 ADC, PF-06804103. The design elements of identifying novel sites of conjugation employed in this study serve as a platform for developing optimized ADCs against other tumor-specific targets.

Immunohistochemical evaluation of HER2 expression in vivo cell line and patient-derived xenograft models.

Includes Supplementary Materials and Methods, Data and Figure Legends

PDF file-458KB, Supplementary Figure S1. Representation of A1mcMMAF and its proposed processing., Supplementary Figure S2. Chromatographic characterization of A1mcMMAF., Supplementary Figure S3. LC-MS analysis of A1mcMMAF ADC., Supplementary Figure S4. Anti-5T4 A1 antibody binding to 5T4 orthologs., Supplementary Figure S5. Anti-5T4 A1 antibody binding to cancer cell lines., Supplementary Figure S6. Cytotoxic activity of A1mcMMAF on TIC-enriched TUM622 spheroids., Supplementary Figure S7. In vivo pharmacology in MDAMB435/5T4., Supplementary Figure S8. No activity of A1mcMMAF in a 5T4-negative tumor. Supplementary Table S1. Humanized A1 BIAcore Affinity Constants for Human and Cynomolgus Monkey 5T4 at pH 7.4 Supplementary Table S2. Representative internalization in MDAMB435/5T4. Supplementary Table S3. Comparison of in vitro cytotoxicities of ADC vs. free payload (IC50 values expressed in nM auristatin).

Immigrant founders of venture capital-backed companies have been critical to the entrepreneurial ecosystem. We document the channels through which immigrant founders find their way to the United States and how those channels have changed over time. Immigrants have been an important source of founders for venture capital-backed startups accounting for roughly 20% of all founders over the past 30 years. Immigrants coming to the United States for their education have been the primary source of founders with those coming after being educated abroad and then arriving for work decreasing in importance over time. The importance of undergraduate education as a channel for immigrant founders has increased over time. Immigrant founders coming for education are likely to start their companies in the state in which they were educated, especially states where they received their graduate education, leading to potentially large local economic benefits. The results of this paper have important policy implications for the supply of entrepreneurial talent and efforts to promote entrepreneurial ecosystems.

Obesity and type 2 diabetes (T2D) remain major global healthcare challenges and developing therapeutics necessitate using nonhuman primate models. Here, we present transcriptomic and proteomic analyses of all the major organs of cynomolgus monkeys with spontaneous obesity or T2D in comparison to healthy controls. Molecular changes occur predominantly in the adipose tissues of individuals with obesity, while extensive expression perturbations among T2D individuals are observed in many tissues, such as the liver, kidney, brain, and heart. Immune response-related pathways are upregulated in obesity and T2D, whereas metabolism and mitochondrial pathways are downregulated. Incorporating human single-cell RNA sequencing findings corroborates the role of macrophages and monocytes in obesity. Moreover, we highlight some potential therapeutic targets including SLC2A1 and PCSK1 in obesity as well as SLC30A8 and SLC2A2 in T2D. Our findings provide insights into tissue-specific molecular foundations of obesity and T2D and reveal the mechanistic links between these two metabolic disorders.

Implication Statement: The bone marrow aspirate and biopsy procedure are fundamental to the diagnosis of many hematologic pathologies. We describe a hands-on, anatomy-based workshop that allows learners to practice bone marrow procedures on cadavers. Notably, participants learned how to perform sternal aspirates: a procedure rarely performed in real-life practice. Learners valued the experience and described increased comfort with the procedure after the workshop. This workshop provides a valuable opportunity for trainees to learn a procedural skill in a safe, high fidelity environment. Given its hands-on nature, residency training programs could also adapt it for direct observation and trainee assessment., Énoncé des implications de la recherche : La ponction et la biopsie de la moelle osseuse sont d’une importance capitale pour le diagnostic de nombreuses pathologies hématologiques. Nous décrivons un atelier pratique, axé sur l'anatomie, qui permet aux apprenants de faire des prélèvements de moelle osseuse sur des cadavres. Les participants ont notamment appris à effectuer des ponctions sternales, une intervention qui est plutôt rare dans la pratique réelle. Ils indiquent avoir apprécié l’atelier, grâce auquel ils ont pris confiance pour pratiquer la technique. L’atelier est une occasion précieuse pour les apprenants d'acquérir une habileté technique dans un environnement sûr et haute fidélité. Étant donné son caractère pratique, les programmes de résidence pourraient aussi l’adapter dans un contexte d’observation directe et d’évaluation.

Immigrant founders of venture capital-backed companies have been critical to the entrepreneurial ecosystem. We document the channels through which immigrant founders find their way to the United States and how those channels have changed over time. Immigrants have been an important source of founders for venture capital-backed startups accounting for roughly 20% of all founders over the past 30 years. Immigrants coming to the United States for their education have been the primary source of founders with those coming after being educated abroad and then arriving for work decreasing in importance over time. The importance of undergraduate education as a channel for immigrant founders has increased over time. Immigrant founders coming for education are likely to start their companies in the state in which they were educated, especially states where they received their graduate education, leading to potentially large local economic benefits. The results of this paper have important policy implications for the supply of entrepreneurial talent and efforts to promote entrepreneurial ecosystems. Institutional subscribers to the NBER working paper series, and residents of developing countries may download this paper without additional charge at www.nber.org.

Introduction As medical students, we were always told that medicine is a tough road. The training is gruelling with endless hours of studying, patient care, and considerable sacrifices in our personal lives. Many came into this profession ready for long hours and difficult training. However, until residency, it is tough to envision what the experience really entails. Recently, a debate erupted in the Medical Twitter universe when Dr. Colleen Farrell, an Internal Medicine resident at Bellevue Hospital in New York, wrote a tweet decrying 27-hour resident call shifts as inhumane. Dr. Farrell argued that residents and staff physicians deserve protection against harsh working hours and conditions on par with workers in unionized professions. For instance, the Ontario Nurses Association closely regulates how long nurses can work in a given day with the minimal time nurses must receive for breaks1. The ensuing debate saw numerous residents and staff physicians joining the conversation on either side of the argument. Many physicians argued that extended call shifts are a necessary part of resident training which equips residents to work effectively and independently in future demanding roles. On the other hand, many suggest that the lack of adequate rest and humane working hours leaves residents ill-prepared to make decisions and may hinder patient care. In the end, Dr. Farrell received heavy backlash that led to her taking a break from Twitter. But what does the research say? Does reducing working hours improve resident wellness and patient safety? What is the impact on resident education? Can there be a way to balance resident wellness with competency and quality of care?

Gastric cancer is the second most common cause of cancer-associated death in Asia. The incidence and mortality rates of gastric cancer have markedly increased in the past few decades. Therefore, the identification of novel gastric cancer biomarkers are needed to determine prognosis. The role of serpin peptidase inhibitor clade A member 1 (SERPINA1) has been studied in several types of cancer; however, little is known about its mechanism in gastric cancer. The present study aimed to evaluate SERPINA1 as a potential prognostic biomarker in gastric cancer and to identify the possible mechanisms underlying its action. The expression levels of SERPINA1 in several gastric cancer datasets were assessed, and it was identified that high expression of SERPINA1 was associated to poor clinical outcomes. Furthermore, using histochemical analysis, western blotting, apoptotic analysis, gap closure and invasion assays in cell lines, it was reported that silencing of SERPINA1 inhibited the formation of cellular pseudopodia and did not affect apoptosis, but promoted cell cycle S-phase entry. In addition, overexpression of SERPINA1 increased the migration and invasion of gastric cancer cells, whereas knockdown of SERPINA1 decreased these functions. Moreover, SERPINA1 overexpression increased the protein levels of SMAD4, which is a key regulator of the transforming growth factor (TGF)-β signaling pathway. Taken together, the present data demonstrated that SERPINA1 promotes gastric cancer progression through TGF-β signaling, and suggested that SERPINA1 may be a novel prognostic biomarker from tumor tissue biopsy in gastric cancer.

The approval of ado-trastuzumab emtansine (T-DM1) in HER2+ metastatic breast cancer validated HER2 as a target for HER2-specific antibody–drug conjugates (ADC). Despite its demonstrated clinical efficacy, certain inherent properties within T-DM1 hamper this compound from achieving the full potential of targeting HER2-expressing solid tumors with ADCs. Here, we detail the discovery of PF-06804103, an anti-HER2 ADC designed to have a widened therapeutic window compared with T-DM1. We utilized an empirical conjugation site screening campaign to identify the engineered ĸkK183C and K290C residues as those that maximized in vivo ADC stability, efficacy, and safety for a four drug–antibody ratio (DAR) ADC with this linker–payload combination. PF-06804103 incorporates the following novel design elements: (i) a new auristatin payload with optimized pharmacodynamic properties, (ii) a cleavable linker for optimized payload release and enhanced antitumor efficacy, and (iii) an engineered cysteine site–specific conjugation approach that overcomes the traditional safety liabilities of conventional conjugates and generates a homogenous drug product with a DAR of 4. PF-06804103 shows (i) an enhanced efficacy against low HER2-expressing breast, gastric, and lung tumor models, (ii) overcomes in vitro- and in vivo–acquired T-DM1 resistance, and (iii) an improved safety profile by enhancing ADC stability, pharmacokinetic parameters, and reducing off-target toxicities. Herein, we showcase our platform approach in optimizing ADC design, resulting in the generation of the anti-HER2 ADC, PF-06804103. The design elements of identifying novel sites of conjugation employed in this study serve as a platform for developing optimized ADCs against other tumor-specific targets.

Despite the use of rabbits in biomedical research, including regulatory toxicology and cardiovascular studies, little data exist on heart findings in this species. This study was designed to document myocardial findings in female rabbits and the impact of study-related procedures typical for vaccine toxicology studies. One hundred and forty 6- to 8-month-old female New Zealand White rabbits were divided equally into 2 groups, high and low study procedure groups (group 1 and group 2, respectively). All animals received intramuscular (IM) injections of sterile saline every 2 weeks for 5 times and were necropsied 2 days after the final IM injection. Clinical chemistry, hematology, and urinalysis were evaluated. Blood for stress biomarkers (norepinephrine, epinephrine, cortisol, and corticosterone), C-reactive protein, cardiac troponin I, and creatine kinase were collected at time 0 (just before dose administration) and then at 4, 24, and 48 hr after dose administration in group 1 only. Hearts were assessed histologically. Focal to multifocal minimal inflammatory cell infiltrates were common (∼80%), particularly in the left ventricle and interventricular septum, and were similar to the types of infiltrates identified in other laboratory animal species. Additionally, study-related procedures elevated serum stress biomarkers and exacerbated the frequency and severity of myocardial inflammatory cell infiltrates.

Autophagy is a type of cellular catabolic degradation process that occurs in response to nutrient starvation or metabolic stress, and is a valuable resource for highly proliferating cancer cells. Autophagy also facilitates the resistance of cancer cells to antitumor therapies. However, the involvement of autophagy in regulating CXCL10 expression in gastric cancer (GC) cells and T lymphocyte migration remains unclear. In this study, we aimed to investigate the effect of autophagy inhibition on CXCL10 expression and T lymphocyte infiltration in GC and elucidate the underlying mechanism. Analysis of public databases revealed a positive correlation between CXCL10 expression and both prognosis of patients with GC and the expression profile of T lymphocyte markers in the GCs. Chemotaxis and spheroid infiltration assays revealed that CXCL10 induced T lymphocyte migration and infiltration into GC spheroids, an in vitro three-dimensional cell culture model. In addition, in vitro autophagy inhibition in GC cells increased CXCL10 expression under both normal and hypoxic culture conditions. Further investigation on the underlying mechanism showed that in vitro autophagy inhibition suppressed the JNK signaling pathway and further enhanced CXCL10 expression in GC cells. Collectively, our results provide novel insights for understanding the role of autophagy in regulation of intra-tumor immunity.

Background Fondaparinux is commonly used for treatment of heparin‐induced thrombocytopenia (HIT) despite lack of approval for this indication. High quality randomized controlled trials of this agent are unlikely to be forthcoming. Objectives The objective of this systematic review is to update the literature on the efficacy and safety of fondaparinux for treatment of confirmed and probable HIT based on the available evidence. Methods Primary articles were identified using Web of Science and PubMed database searches for English‐language studies from January 2006 to November 2017. Selected studies enrolled consecutive adult patients who received fondaparinux as the primary anticoagulant to treat acute HIT; confirmed the diagnosis by serological testing with a serotonin‐release assay; heparin‐induced platelet activation assay or enzyme‐linked immunosorbent assay; provided clinical criteria used to define HIT and reported clinically important outcomes. Results A total of 9 studies were identified with 154 HIT positive patients. Ten experienced a new thrombotic event while receiving fondaparinux (6.5%, 95% CI, 3.4 to 11.7%) and 26 experienced major bleeding (16.9%, 95% CI, 11.7 to 23.6%). Mortality due to thrombosis or bleeding was reported in 5 patients (3.2%, 95% CI, 1.2 to 7.6%). Conclusions Fondaparinux appears to be an effective and safe anticoagulant for treatment of acute HIT despite the absence of randomized trials. Caution should exercised when using fondaparinux in patients with renal insufficiency.

The Staphylococcus aureus virulence factor clumping factor A (ClfA) is a component of an investigational S. aureus prophylactic vaccine. ClfA enables S. aureus to bind to fibrinogen and platelets during the initial stages of invasive disease. Here we demonstrate that ectopic expression of ClfA is sufficient to render nonpathogenic Lactococcus lactis lethal in a murine model of systemic infection. In contrast, L. lactis expressing ClfAY338A, which cannot bind fibrinogen, did not cause death in the mice. Pathogenicity was also prevented by immunization with ClfA. This model was then used to define a preclinical correlate of protection by measuring functional antibody in a S. aureus fibrinogen binding inhibition assay (FBI) and correlating that titer with protective outcomes. Although many humans have pre-existing antibodies that bind to ClfA, only sera with a threshold functional titer in the FBI were protective in this preclinical model. This confirms that fibrinogen binding is critical for ClfA-mediated pathogenesis and demonstrates that functional antibodies against ClfA are sufficient to protect against ClfA-mediated pathogenesis in vivo, enabling the definition of a preclinical correlate of protection for ClfA-containing vaccines based on FBI titer.

The purpose of this study was to assess the influence of pupil size and optical blur on measurements obtained with isolated-check visual evoked potential (icVEP). Two stimulus conditions of icVEP, +15 and −15% contrasts, were studied in normal subjects with normal (N), miotic (M), and dilated (D) pupils. The effects of optical blur were studied in subjects with normal pupil. Response to visual stimuli was quantified by a signal-to-noise (SNR) ratio. In 30 normal subjects, the mean age was 26.0 ± 3.4 years. Mean pupil diameters were N = 4.2 ± 0.6 mm, M = 2.7 ± 0.6 mm, and D = 7.3 ± 0.9 mm. For both +15 and −15% contrast levels, mean SNR values were reduced for dilated and constricted pupils when compared with normal pupils. Mean SNR values for optical blur with a +2 or +3 diopter lens placed over the distance correction were reduced when compared with SNR measurements obtained with best-corrected visual acuity under both +15 and −15% contrast levels. Statistical significance was found in comparisons of N versus M (P

In present study, the potential drug delivery of nanoformulations was validated via the comparison of cellular uptake of nanoparticles in various cell lines and in vivo pulmonary cellular uptake in intratracheally (IT) dosed rat model. Nanoparticles were prepared by a bench scale wet milling device and incubated with a series of cell lines, including Caco-2, RAW, MDCK and MDCK transfected MDR1 cells. IT dosed rats were examined for the pulmonary cellular uptake of nanoparticles. The processes of nanoparticle preparation did not alter the crystalline state of the material. The uptake of nanoparticles was observed most extensively in RAW cells and the least in Caco-2 cells. Efflux transporter P-gp did not prevent cell from nanoparticles uptake. The cellular uptake of nanoparticles was also confirmed in bronchoalveolar lavage (BAL) fluid cells and in bronchiolar epithelial cells, type II alveolar epithelial cells in the intratracheally administrated rats. The nanoparticles uptake in MDCK, RAW cells and in vivo lung epithelial cells indicated the potential applications of nanoformulation for poorly soluble compounds. The observed limited direct uptake of nanoparticles in Caco-2 cells suggests that the improvement in oral bioavailability by particle size reduction is via increased dissolution rate rather than direct uptake.

Purpose To introduce a rapid and objective electrophysiological technique that can assess visual function in the magnocellular pathway, which is thought to be affected in early-stage glaucoma. Methods Low-contrast bright or dark isolated-checks were luminance-modulated against a static background at 10 Hz in order to drive preferentially the magnocellular ON or OFF pathway. Visual evoked potentials were recorded during 1-s epochs of stimulation and responses at the stimulus frequency were measured. Artifact rejection features ensured that eight valid runs were obtained per eye. Signal-to-noise ratios (SNR) were derived based on a multivariate statistic. In order to demonstrate its functionality, a small group of patients with glaucoma (N = 18, Snellen acuity of 20/30 or better) and control observers (N = 16) were tested. A participant failed the test if either eye yielded an SNR ≤ 1. Receiver-operating-characteristic curve analysis was used to estimate the accuracy of group classification. Results The instrument was found to elicit reliable responses from control observers. For the 15% bright condition, all control observers yielded significant isolated-check VEPs (icVEPs), whereas the majority of patients failed to do so, indicating significant losses in central visual function. This condition produced the highest classification accuracy (94%), followed by the 10% dark condition (91%). Conclusions Both ON and OFF divisions of the magnocellular pathway can be assessed rapidly through the application of the icVEP technique. This measure of central visual function may be of value in the detection of glaucomatous deficits and may complement tests of peripheral function.

The use of predictive preclinical models in drug discovery is critical for compound selection, optimization, preclinical to clinical translation, and strategic decision-making. Trophoblast glycoprotein (TPBG), also known as 5T4, is the therapeutic target of several anticancer agents currently in clinical development, largely due to its high expression in tumors and low expression in normal adult tissues. In this study, mice were engineered to express human TPBG under endogenous regulatory sequences by replacement of the murine Tpbg coding sequence. The gene replacement was considered functional since the hTPBG knockin (hTPBG-KI) mice did not exhibit clinical observations or histopathological phenotypes that are associated with Tpbg gene deletion, except in rare instances. The expression of hTPBG in certain epithelial cell types and in different microregions of the brain and spinal cord was consistent with previously reported phenotypes and expression patterns. In pharmacokinetic studies, the exposure of a clinical-stage anti-TPBG antibody-drug conjugate (ADC), A1mcMMAF, was lower in hTPBG-KI versus wild-type animals, which was evidence of target-related increased clearance in hTPBG-KI mice. Thus, the hTPBG-KI mice constitute an improved system for pharmacology studies with current and future TPBG-targeted therapies and can generate more precise pharmacokinetic and pharmacodynamic data. In general the strategy of employing gene replacement to improve pharmacokinetic assessments should be broadly applicable to the discovery and development of ADCs and other biotherapeutics.

To date the most common forms of biologics in development have been mAb, recombinant proteins, and vaccines. However, numerous new modalities are now being developed. One of these new modalities is bispecific antibodies, which are able to recognize two distinct targets at the same time and might bridge two targets together, which presents an opportunity for therapeutic gains (such as bringing cancer cells within closer proximity to cytotoxic T lymphocytes).When one considers species selection, if both complementarity-determining regions (CDRs) do not have cross reactivity to the same species, one needs to understand how to appropriately assess the safety if the full pharmacological effect cannot be generated in one species. Therefore, these new biologic modalities offer great promise for the treatment of diseases but also challenges in developing the nonclinical toxicology testing paradigm to ensure the safety of the patients in the clinical trials. Keywords: antibody drug conjugates (ADCs); DNA vaccines; mAb; nonclinical safety testing programs; pharmacokinetics; stem cells; gene therapy

Antibody-drug conjugates (ADCs) have emerged as an important class of cancer therapeutics. The FDA approval of Kadcyla (T-DM1), a single agent for treatment of HER2-positive advanced metastatic breast cancer, was a significant milestone in the field of targeted therapy, as the first and only ADC for treatment of solid tumors. Despite the 3-month improvement over standard of care in the median survival, almost all the patients eventually became refractory to T-DM1. We have identified several possible areas for improvement: 1) The potency of T-DM1 as confirmed by the Phase III clinical data is restricted to high HER2 tumors which leaves moderate or low HER2 expressing patients without access to T-DM1 treatment; 2) The 48% overall response rate is indicative of intrinsic resistance to T-DM1 and all T-DM1 treated patients eventually relapse. 3) The randomized lysine conjugation in T-DM1 generates heterogeneity of the product. We have developed a novel, site-specific anti-HER2 ADC (NG-HER2 ADC) and evaluated it in comparative preclinical studies with T-DM1. The results show that the NG-HER2 ADC is ∼ 10 fold more potent than T-DM1 in HER2 3+ xenograft models of breast and gastric cancers. Our proprietary cleavable and permeable linker-payload can mediate bystander effect and this enables potent anti-tumor activity in non-HER2 amplified breast cancer and heterogeneous low HER2 NSCLC PDX models, where T-DM1 is ineffective. Our ADC can overcome T-DM1 resistance in in vitro and in vivo models.. Our site-specific ADC at HNSTD of 9 mg/kg in cynomolgus monkeys showed high AUC, long half-life and had normal clinical observations with no marked neutropenia. On the contrary, conventional conjugates with cleavable linker payloads typically have severe bone marrow toxicity as DLT above 5 mg/kg. The therapeutic index for NG-HER2 ADC is significantly greater than T-DM1 in all models tested. NG-HER2 ADC has a projected clinical efficacious dose of ∼1 mg/kg, compared to 3-5 mg/kg for T-DM1, based on PK/PD modeling. In addition, the activity of the NG-HER2 ADC shows increased infiltration of CD8 positive effector cells, an essential component for immuno-oncology (IO) efficacy, in a syngeneic HER2 overexpressing model. This property potentially allows the combination of the ADC with IO drugs to improve the long-term, overall survival. Our data provides preclinical proof of concept for NG-HER2 ADC with best-in-class potential and is currently being tested in preparation for clinical trials for treatment of HER2 solid tumors. Citation Format: Dangshe Ma, Bitha Narayanan, Kim Marquette, Edmund Graziani, Frank Loganzo, Manoj Charati, Nadira Prashad, Nathan Tumey, Jon Golas, Christine Hosselet, George Hu, Frank Barletta, Alison Betts, Judy Lucas, Chris O’Donnell, Lioudmila Tchistiakova, Hans-Peter Gerber, Puja Sapra. Creating a superior, site-specific anti-HER2 antibody-drug conjugate (NG-HER2 ADC) for treatment of solid tumors. [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 868.