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Capitalizing on their extraordinary specificity, monoclonal antibodies (mAbs) have become one of the most reengineered classes of biological molecules. A major goal in many of these engineering efforts is to add new functionality to the parental mAb, including the addition of cytotoxins and imaging agents for medical applications. Herein, we present a unique peptide-binding site within the central cavity of the fragment antigen binding framework region of the chimeric, anti-epidermal growth factor receptor mAb cetuximab. We demonstrate through diffraction methods, biophysical studies, and sequence analysis that this peptide, a meditope, has moderate affinity for the Fab, is specific to cetuximab (i.e., does not bind to human IgGs), and has no significant effect on antigen binding. We further demonstrate by diffraction studies and biophysical methods that the meditope binding site can be grafted onto the anti-human epidermal growth factor receptor 2 mAb trastuzumab, and that the antigen binding affinity of the grafted trastuzumab is indistinguishable from the parental mAb. Finally, we demonstrate a bivalent meditope variant binds specifically and stably to antigen-bearing cells only in the presence of the meditope-enabled mAbs. Collectively, this finding and the subsequent characterization and engineering efforts indicate that this unique interface could serve as a noncovalent “linker” for any meditope-enabled mAb with applications in multiple mAb-based technologies including diagnostics, imaging, and therapeutic delivery.

Cancer-associated mutations in the spliceosome gene SF3B1 create a neomorphic protein that produces aberrant mRNA splicing in hundreds of genes, but the ensuing biologic and therapeutic consequences of this missplicing are not well understood. Here we have provided evidence that aberrant splicing by mutant SF3B1 altered the transcriptome, proteome, and metabolome of human cells, leading to missplicing-associated downregulation of metabolic genes, decreased mitochondrial respiration, and suppression of the serine synthesis pathway. We also found that mutant SF3B1 induces vulnerability to deprivation of the nonessential amino acid serine, which was mediated by missplicing-associated downregulation of the serine synthesis pathway enzyme PHGDH. This vulnerability was manifest both in vitro and in vivo, as dietary restriction of serine and glycine in mice was able to inhibit the growth of SF3B1MUT xenografts. These findings describe a role for SF3B1 mutations in altered energy metabolism, and they offer a new therapeutic strategy against SF3B1MUT cancers.

In the spring semester of 2020, I chose to create a program which featured works directly related to social injustices. In addition, I incorporated regular discussions about social injustice into the rehearsals. I also created questionnaires to assess how the students responded to this process. This document examines possible approaches to the planning and programming of a social justice themed choral concert. The process includes selecting a social injustice to focus on, choosing appropriate repertoire, and structuring the program. The paper also examines how to incorporate discussions about these critical issues into the classroom. Finally, there is a detailed description of the works I chose for one particular concert program. The second part of the paper describes the process I used for assessing the impact of this work through questionnaires both for students in the class and choral educators. This section outlines the procedure that I used and contains a brief analysis of the results. The questionnaires and the results are published as three separate appendices. Lastly, the document contains several appendices which will provide educators with resources that may be useful for creating a similar experience. This section contains numerous examples of programs, a survey of social justice themed choral music, a similar survey of poetry, and a template for creating a concert program.

Intratumor heterogeneity is an important mediator of poor outcomes in many cancers, including breast cancer. Genetic subclones frequently contribute to this heterogeneity; however, their growth dynamics and interactions remain poorly understood. PIK3CA and HER2 alterations are known to coexist in breast and other cancers. Herein, we present data that describe the ability of oncogenic PIK3CA mutant cells to induce the proliferation of quiescent HER2 mutant cells through a cell contact-mediated mechanism. Interestingly, the HER2 cells proliferated to become the major subclone over PIK3CA counterparts both in vitro and in vivo. Furthermore, this phenotype was observed in both hormone receptor-positive and -negative cell lines, and was dependent on the expression of fibronectin from mutant PIK3CA cells. Analysis of human tumors demonstrated similar HER2:PIK3CA clonal dynamics and fibronectin expression. Our study provides insight into nonrandom subclonal architecture of heterogenous tumors, which may aid the understanding of tumor evolution and inform future strategies for personalized medicine.

Therapeutic antibodies exhibit side effects from binding non-target tissues that limit their effectiveness or cause harm, including acneiform folliculitis and gastrointestinal upset (cetuximab and matuzumab) and cardiotoxicity (trastuzumab). These antibodies are approved to treat tumors wherein proteases are aberrantly expressed, including matrix metalloprotease-9 (MMP-9). To limit the side effects of these otherwise potent therapeutics, \"masks\" that block antibody binding systemically and that are removed by protease exposure are designed, developed and explored to enhance tumor specificity. Herein, a design is demonstrated that leverages synergy in tumor cell death identified for the combination of anti-Epidermal Growth Factor Receptor (EGFR) antibodies cetuximab and matuzumab to create a cross-masked antibody design. Specifically, the antibodies' epitope (domain III of the EGFR) was fused N-terminally to a single chain variable fragment (scFv) construct of cetuximab and 425 (matuzumab's murine precursor) through an MMP-9 site containing linker. In each mask, mutations were introduced into EGFR domain III (EGFRdIII) to reduce the intramolecular affinity—S460P/G461N for 425 as determined herein and mutations from the literature for cetuximab. These mask-antibody fusions were mixed to form a heterodimer or cross-masked structure. MMP-9 exposure increased the affinity 8-fold in surface plasmon resonance (SPR) analysis using immobilized EGFRdIII. Flow cytometry confirmed higher binding after MMP-9 exposure. We also explored small peptides as masks to replace the EGFRdIII. Using diffraction methods, it was shown that previously described (cetuximab binding) peptides bind to a novel framework binding site, but not the expected complementarity determining regions (CDRs). Size exclusion chromatography and SPR confirmed simultaneous binding of cetuximab, EGFRdIII and the mimotope. Further, the mimotope bound the fragment antigen binding [F(ab)'], but not a scFv construct of cetuximab that places a linker across the peptide binding site. While the intention was to use these peptides to occlude the CDRs, these peptides could also be used as an anchor to sterically occlude tumor-derived antigen and thus create a non-covalent mask. Initial studies define critical parameters in implementing this concept. Taken together, modulation of antibody affinity through covalent/non-covalent masking represents a novel and potentially powerful method to improve the targeting of therapeutic antibodies to the tumor.

Data sharing can accelerate scientific discovery while increasing return on investment beyond the researcher or group that produced them. Data repositories enable data sharing and preservation over the long term, but little is known about scientists’ perceptions of them and their perspectives on data management and sharing practices. Using focus groups with scientists from five disciplines (atmospheric and earth science, computer science, chemistry, ecology, and neuroscience), we asked questions about data management to lead into a discussion of what features they think are necessary to include in data repository systems and services to help them implement the data sharing and preservation parts of their data management plans. Participants identified metadata quality control and training as problem areas in data management. Additionally, participants discussed several desired repository features, including: metadata control, data traceability, security, stable infrastructure, and data use restrictions. We present their desired repository features as a rubric for the research community to encourage repository utilization. Future directions for research are discussed.

A chimpanzee adenovirus-based vaccination approach elicits acute and long-term protection against ebolavirus challenge in nonhuman primates. Ebolavirus disease causes high mortality, and the current outbreak has spread unabated through West Africa. Human adenovirus type 5 vectors (rAd5) encoding ebolavirus glycoprotein (GP) generate protective immunity against acute lethal Zaire ebolavirus (EBOV) challenge in macaques, but fail to protect animals immune to Ad5, suggesting natural Ad5 exposure may limit vaccine efficacy in humans. Here we show that a chimpanzee-derived replication-defective adenovirus (ChAd) vaccine also rapidly induced uniform protection against acute lethal EBOV challenge in macaques. Because protection waned over several months, we boosted ChAd3 with modified vaccinia Ankara (MVA) and generated, for the first time, durable protection against lethal EBOV challenge.

Using data from the Diviner Lunar Radiometer Experiment, we show that four regions of the Moon previously described as \"red spots\" exhibit mid-infrared spectra best explained by quartz, silica-rich glass, or alkali feldspar. These lithologies are consistent with evolved rocks similar to lunar granites in the Apollo samples. The spectral character of these spots is distinct from surrounding mare and highlands material and from regions composed of pure plagioclase feldspar. The variety of landforms associated with the silicic spectral character suggests that both extrusive and intrusive silicic magmatism occurred on the Moon. Basaltic underplating is the preferred mechanism for silicic magma generation, leading to the formation of extrusive landforms. This mechanism or silicate liquid immiscibility could lead to the formation of intrusive bodies.

We obtained direct global measurements of the lunar surface using multispectral thermal emission mapping with the Lunar Reconnaissance Orbiter Diviner Lunar Radiometer Experiment. Most lunar terrains have spectral signatures that are consistent with known lunar anorthosite and basalt compositions. However, the data have also revealed the presence of highly evolved, silica-rich lunar soils in kilometer-scale and larger exposures, expanded the compositional range of the anorthosites that dominate the lunar crust, and shown that pristine lunar mantle is not exposed at the lunar surface at the kilometer scale. Together, these observations provide compelling evidence that the Moon is a complex body that has experienced a diverse set of igneous processes.

Carbonaceous chondrite meteorites, which include the sulfide-rich “Yamato-type” chondrites (CYs), have undergone a complex history of aqueous and thermal alteration and offer crucial insights into early outer solar system conditions. In this study, we evaluate thermal infrared (TIR) reflectance spectra of three CY chondrites. We observe a broad spectral plateau near 10 μm, a spectral signature that has been observed in remote observations of some primitive, low-albedo asteroids, including Jupiter Trojans. We compare our data to CY emissivity spectra, spectra of Fe-sulfide and olivine mixtures, and remote Jupiter Trojan observations and establish the plateau and low albedo are a result of a high content of fine-particulate Fe-sulfide of these meteorites. We therefore suggest that D- and P-type asteroids, like Jupiter’s Trojan asteroids, could have a high abundance of Fe sulfide on their surfaces as a potential result of aqueous alteration followed by dehydration, shedding light on the processes shaping the outer solar system.