2014 — 2017 |
Mulchandani, Ashok (co-PI) [⬀] Ge, Xin |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Spore-Based Designer Enzyme Cascade Biocatalysts @ University of California-Riverside
Abstract: The creation of value-added products such as fine chemicals and pharmaceuticals by chemical transformations has resulted in significant improvements in the quality of life we have been enjoying. Many of these chemical transformation processes use catalysts. These catalysts may be inorganic or biological in nature. Enzyme catalysts would be widely utilized to perform these chemical transformation processes, as they frequently offer advantages of high yield, high selectivity, high product purity, along with operation at ambient temperature and pressure in aqueous environment at moderate pH. However, many biocatalytic reactions involve expensive co-enzymes or co-factors and their recycling is essential for the processes to be cost-effective. This turns out to be a difficult or expensive process step, thereby limiting the ability to gain the advantages of using enzyme catalysts.
Principal investigators Xin Ge and Ashok Mulchandani from the University of California Riverside looked to cellular reactions in nature to develop an approach to circumvent this issue. Inspired by the substrate channeling phenomena seen in multi-enzyme cascades in nature for circumventing unfavorable thermodynamics and kinetics, the PIs will explore the development of a modular designer biocatalyst platform on the surface of spores, where enzyme cascade is spatially organized with tunable stoichiometry to achieve highly efficient cofactor regeneration. The enzyme system is easy to produce and reuse, and has high stability. The modular nature of the system will allow easy insertion of the genes of the desired enzymes and control of the stoichiometric ratios on the surface.
This collaborative research project is significant as it will lead to development of a novel robust modular platform for designer biocatalysts to address the needs of chemicals and pharmaceuticals manufacturing. A number of applications are readily envisioned. The improved catalysts and processes will increase US technological competitiveness. Collectively, the benefits from this research will support efficient, economical and green engineering production of many fine chemicals and pharmaceuticals. In addition, the PIs plan activities which will develop a globally competitive and divergent STEM workforce through the increased participation of women and underrepresented minorities. UC Riverside is the minority serving institution with the largest Hispanic student population among all UC campuses. The investigators plan to hire minority graduate and undergraduate students as research assistants for this project. The investigators also plan new curriculum efforts and are collaborating with a local middle school to establish an interactive science program titled Bio- catalysis for clean fuels.
Most oxidoreductase enzymes involved in specialty chemical synthesis utilize expensive pyridine nucleotides as cofactors for catalysis. These enzyme catalytic processes have shortcomings in terms of cofactor recycling that limit total turnover number and productivity yields. The goal of the proposed research is to develop a modular designer biocatalyst platform for highly efficient cofactor regeneration. Inspired by the substrate channeling phenomena observed in nature and other studies of engineered multienzyme cascades and mini-cellulosomes, the scaffoldin- cohesin - dockerin system will be used to build a spatially organized multienzyme complex designed for highly efficient cofactor regeneration. This enzyme complex will consist of proximally located producing and regenerating dehydrogenases in desired stoichiometry on a selected surface to allow channeling of oxidized cofactor from the producing dehydrogenase to the regenerating dehydrogenases and vice versa, solving the regeneration problems. Because of their formidable resistance to extremes of temperatures, pH, solvents, humidity and radiations, bacterial spores will serve as the surface display for the enzyme cascade. Various control and reference experiments will be carried out for the typical synthesis reaction of ketone reduction to alcohol, and the enzyme coupled regeneration of the cofactor will be demonstrated in both aqueous and nonaqueous media. Extensive characterization and catalytic performance assessments are planned by the PIs. This information will be published and available for investigators of other biocatalytic applications.
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0.954 |
2015 — 2020 |
Ge, Xin |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Career: Generation of Highly Selective Inhibitory Antibodies by Novel Paratope Design, Function-Based Screening, and Deep Sequencing @ University of California-Riverside
1453645 Ge, Xin
This research will significantly advance our understanding on the mechanisms of enzymatic inhibition and virus neutralization, and thus will lead to development of a panel of novel methods for the generation of highly potent inhibitory antibodies for a broad range of applications in pharmaceutical and biotechnological industries. Specifically, the work aims to develop antibodies that interfere and inhibit the binding of protein cleaving enzymes or proteases. The project will (i) increase US technological competitiveness; (ii) develop a globally competitive STEM workforce; (iii) increase participation of women and underrepresented minorities; and (iv) contribute to undergraduate and graduate STEM education. UC Riverside is the minority serving institution with the largest Hispanic student population among all UC campuses. More specifically, the PI plans to first prepare UCR STEM undergraduates for a global marketplace and dynamic scientific communities through collaboration with two Chinese universities; and second increase university/college enrollment of high school graduates in Riverside and San Bernardino Counties, especially those from minority and other disadvantaged socio-economic backgrounds, by inspiring their interests in science and engineering programs through lectures, workshops and interacting events.
As extremely important signaling molecules, proteases precisely control a wide variety of physiological processes, and thus represent one of the largest families of potential pharmaceutical targets. Considering that ~2% of the human genome is estimated to encode proteases, specificity is highly desired for any protease inhibition therapy. However, proteases share high amino acid similarity among the same class of proteases and their active sites are extensively conserved. It has been a challenging task to develop small molecule inhibitors that can deliver required specificities. Therefore, antibodies are emerging as a very attractive alternative for highly selective inhibition. To date, at least three obstacles make the routine discovery of protease-inhibiting monoclonal antibodies (mAbs) considerably difficult: (i) low antigenicity of the proteolytic active sites, (ii) lack of a function-based selection method, and (iii) loss of beneficial clones during the selection. The long-term goal of this CAREER award is to develop therapeutic monoclonal antibodies (mAbs) or biologics that inhibit specific proteases for biomedical applications. The objective of this research is to overcome the technical hurdles and establish general methodologies that facilitate the identification of inhibitory antibodies. The hypothesis is that convex antigen-binding sites (paratopes) are inhibition-prone. This central hypothesis will be tested by the following three specific approaches: (1) Clearly verify inhibition mechanisms by design, construction and optimization of synthetic human antibody libraries enriched with convex paratopes; (2) Efficiently identify inhibitory antibodies by developing a function-based high-throughput screening method; (3) Systematically understand sequence-inhibition landscapes by deep sequencing and data mining.
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0.954 |
2016 — 2020 |
Ge, Xin |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structure-Based Design of Camel-Like Human Selective Mabs Against Mmps in Disease @ University of California Riverside
? DESCRIPTION (provided by applicant): Evidence suggests that matrix metalloproteinase (MMPs) play important, albeit frequently paradoxical, roles in multiple pathologies, including cancer, neuropathic pain, chronic wounds, hypertension, and inflammatory diseases. It is an urgent need to develop selective and efficient inhibitors of individual MMPs for biomedical research and disease therapies. However, the catalytic domains of MMPs share a high degree of sequence and fold homology, and thus distinguishing among MMPs using small molecule inhibitors is exceedingly difficult. Because of their exquisite specificity, antibody-based inhibitrs are emerging as promising MMP- blocking agents. Unfortunately, to date, at least two major obstacles make the routine discovery of MMP- inhibiting mAbs difficult: (1) low antigenicity of the MMP active site, and (2) lack of function-based selection methods. Our long-term goal is to develop therapeutic mAbs that would inhibit specific MMPs in disease. The objectives of this project are to (1) overcome these obstacles and establish general, streamlined methodologies for the discovery and engineering of inhibitory antibodies; and (2) use these MMP inhibitory mAbs to advance our understandings on mechanisms of cancer cell migration and test their therapeutic potentials in xenograft models. Our central hypotheses are (1) convex antigen-binding sites (paratopes) are inhibitory; (2) development of a quantitative, function-based high-throughput screening (HTS) greatly accelerates the discovery of inhibitory mAbs; and (3) blocking the specific MMP activities by highly selective functional mAbs inhibits cancer cell migration in vitro and invasion in vivo. Building on our team's expertise in protein engineering, biophysics, cell biology and cancer biology, we will, Aim 1: design, synthesis and optimize human antibody libraries carrying convex paratopes; Aim 2: isolate inhibitory antibodies by function-based high-throughput screening; Aim 3: characterize inhibitory mAbs and elucidate inhibition mechanisms; and Aim 4: test the ability of inhibitory mAbs to alter cancer cell migration in vitro and invasion in vivo. The approaches are innovative, because it will (1) create synthetic human antibody libraries carrying inhibitory paratopes; (2) develop a groundbreaking function-based (rather than binding-based) HTS method for facile discovery of mAbs inhibiting the individual MMPs; (3) uncover how the structure and mechanics of the extracellular matrix controls MMP activity during cancer cell migration and invasion; (4) transform novel antibody-based selective MMP inhibitors into drug leads applicable for biopharmaceutical developments. The proposed research is significant because it will (1) establish a pipeline technology can be readily applied for many biomedically important proteases, one of the largest families of pharmaceutical targets; (2) advance our understanding of the molecular mechanisms by which cancer cells migrate through the extracellular matrix; and (3) initialize the development toward novel immunotherapeutic agents blocking cancer metastasis. Title: Structure-based design of camel-like human selective mAbs against MMPs in disease Keywords: matrix metalloproteinase; inhibitory antibody; synthetic antibody library; camelid antibody; high-throughput screening; complementarity determining region.
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0.954 |
2021 |
Ge, Xin Urayama, Akihiko |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Protease Activated Bi-Specific Antibody Prodrugs For Efficient Transportation Across Blood-Brain Barrier @ University of Texas Hlth Sci Ctr Houston
PROJECT SUMMARY The blood-brain barrier (BBB) poses the greatest challenge for developing effective therapies for neurological diseases. Inspired by receptor-mediated transcytosis, bi-specific antibodies (bsAbs) against transferrin receptor (TfR) have demonstrated significant improvements of CNS delivery. However, the overall brain penetration was still modest, with large majority of administrated bsAbs remain in blood. As transcytosis at BBB is a bi-directional process and inevitably leads anti-TfR bsAb reaching a concentration equilibrium between the blood and the brain sides, we hypothesize that by minimizing abluminal-to-luminal efflux, the concentration equilibrium can be shifted toward BBB penetration. Our design principle is to fuse the variable fragment (Fv) of anti-TfR to the N-terminal of a therapeutic IgG, via cleavable linker(s) specific to disease-associated protease present in the brain. Once delivered to the brain by TfR-mediated transcytosis, therapeutic IgG will be activated and stays at the brain side because it loses binding ability to TfR. Released anti-TfR Fv will transport back to the blood side then be eliminated by renal clearance. TfR-bound prodrugs will be further transcytosed and thus forming a net flow of therapeutic Ab penetration from blood to brain. Our long-term goal is to develop a highly efficient BBB delivery approach that enables effective treatments of neurological disorders such as brain cancer and neurodegenerative diseases. The objective of this MPI R21 project is to prove the concept of this novel BBB delivery technology based on protease-activated prodrug designs. We will use cathepsin S activated anti-amyloid ? (A?) for Alzheimer?s disease (AD) as the model system in this study. Building on our collective expertise on protein engineering, protease biochemistry, BBB transportation and AD, we will, Aim 1: design, construct and optimize protease-activated bi-specific antibody prodrugs; and Aim 2: validate BBB penetration and therapeutic efficacy of antibody prodrugs using mouse models of cerebral amyloid angiopathy (CAA). The approaches are innovative, because the protease-cleavable prodrug designs can prevent the reverse transcytosis, shift the concentration equilibrium, and thus promote therapeutics penetration from blood to brain. The proposed research is significant because it develops a platform technology enabling to (1) improve BBB penetration of biologics including monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs), (2) greatly reduce off-site on- target side effects by in situ activation in brain, and (3) treat a variety of neurological disorders currently non- targetable.
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0.954 |
2021 |
Galan, Jacob Anthony [⬀] Ge, Xin Sanchez, Elda E. |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Rational Structure-Based Design of Broad Neutralizing Humanized Svmp Mabs @ Texas a&M University-Kingsville
PROJECT SUMMARY In this R21 award, we aim to develop a novel and broad neutralizing human monoclonal antibody for treating snakebite envenoming by rational structure-based design in order to produce a more effective and safer next generation antivenom. Snake envenomation is a serious global public health concern and ranked on the Wor ld Health Organization?s list of neglected tropical diseases, killing on average 125,000 people per year and leaving another 400,000 permanently disabled. The majority of snake envenomation in the US, inflicted by members of the snake family Viperidae, causes local tissue damage (such as myonecrosis, blisters, and local inflammation and pain) and systemic effects, including hemorrhage and coagulopathies which can lead to shock, renal failure and death. Snake venom metalloproteinases are major causative agents for spontaneous systemic bleeding and coagulopathies. Current antivenoms, produced by immunization of domestic animals, have limited efficacy in the prevention of both local and systemic effects of Viperidae envenomation as well as an associated risk of hypersensitivity reactions. Our long-term goal is to develop novel, effective humanized antivenom therapeutics for Viperidae envenomation. The objective of this project is to test the hypothesis that camelid-inspired inhibitory paratope synthetic human antibodies targeted to the active site of medically-relevant viperid venom metalloproteinases (svMPs) can provide broad antivenom protection without cross-reaction with human metalloproteinases and without the risk of hypersensitivity. This objective will be addressed through our established collaboration of complementary expertise between the snake venom toxinology team at National Natural Toxins Research Center (NNTRC) and the antibody discovery team at University of California Riverside (UCR). To test our hypothesis, we will address the following three Specific Aims. Aim 1: Qualitative and Quantitative Characterization of the hemorrhagic activity of viperid svMPs (Galan), Aim 2: Discovery of Broadly Neutralizing svMP-Specific Human mAbs (Ge). Aim 3: Evaluation of the antivenom efficacy of svMP inhibitory mAbs in vitro (Ge) and in vivo (Sanchez). The proposed research is significant because it will advance our understanding of the hemorrhagic aspects caused by snake envenomation at biochemical/cellular levels and develop effective humanized mAb antivenoms, which will be directly translatable for therapeutic use. The novelties of our project are (1) development and application of a novel Hemorrhage Score system to characterize svMPs; (2) isolation of humanized svMP-specific antivenom mAbs from libraries carrying novel convex paratopes; (3) development groundbreaking functional (rather than binding-based) HTS for facile discovery of mAbs inhibiting hemorrhagic snake toxins; and (4) potentially shifting the conventional antivenom production into specific neutralizing humanized mAb therapeutics.
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0.954 |
2021 |
Ge, Xin |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Therapeutic Monoclonal Antibodies Inhibiting Proteases of Biomedical Importance @ University of Texas Hlth Sci Ctr Houston
PROJECT SUMMARY As important signaling molecules, proteases precisely control a wide variety of physiological processes both in health and in diseases, and thus represent one of the largest families of pharmaceutical targets. Despite decades of intensive efforts, conventional drug discovery strategies have only achieved a limited success by targeting a small fraction of all therapeutically relevant proteases. It is because small-molecule inhibitors are often lack of specificity and/or appropriate pharmacokinetic properties required for effective and safe protease-based therapy. In these aspects, monoclonal antibodies (mAbs) are emerging as attractive alternatives with significant advantages such as high selectivity, long serum half-life, potential to cross the blood-brain barrier, and as inducible prodrugs. Since the invention of hybridoma technology, tremendous progress has been made in mAb discovery and engineering. However, routine discovery of protease-inhibiting mAbs is still a considerable challenge in general, due to the incompatibility of human antibody paratope for enzyme inhibition, and lack of functional high-throughput screening methods. My laboratory has been committed to the development of streamlined methodologies that facilitate the generation of therapeutic mAbs as safe and effective protease inhibitors. Over the past five years, we have made significant progress, and established a series of novel technologies, including camelid-inspired convex paratope human antibody libraries, and inhibition-based rather than binding-based selection/screening methods. Combining these enabling approaches, we discovered, characterized, and optimized panels of potent and specific mAbs inhibiting numerous proteases of biomedical importance. Furthermore, our protease inhibitory mAbs have shown significant therapeutic efficacy in mouse models of cancers, neuropathic pains, obesity, and stroke. By overcoming longstanding challenges, these achievements have opened the exciting opportunity. In the next five years, we will extend our powerful technologies to many other well-documented proteases, of which therapeutic inhibitors are urgently needed. Furthermore, we will develop additional technologies to achieve unique and therapy-desirable features: (1) function-specific (substrate-dependent) inhibition, (2) broad-spectrum inhibition on a group of proteases, and (3) epitope-specific inhibition by rational design. Overall, it has been estimated that proteases account for 5-10% of all drug targets have been studied for pharmaceutical development. The completion of proposed research will unambiguously advance therapeutic mAb developments targeting biomedically important proteases, e.g. against the present danger, SARS-CoV-2, by inhibiting TMPRSS2 (type II transmembrane serine protease) as a broad neutralization approach without the unwanted antibody-dependent enhancement.
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0.954 |