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010518 N.Long Full Thesis.pdf (7.37 MB)

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Biophysical Enhancement of Protein Therapeutics and Diagnostics Through Engineered Linkers

Long, Nicholas E
http://rave.ohiolink.edu/etdc/view?acc_num=osu151515985462363

Abstract Details

2018, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
Proteins play a major role in virtually every biological process. Thus, proteins are an ideal platform for the next generation of therapeutics. Over the last few decades, technological and scientific advances in protein production and engineering have led to a new wave of protein-based biologics used in clinical settings. In this body of work, we have engineered both a protein-based cancer diagnostic and an immunotherapeutic. Antibody-based biologics are becoming one of the most widely approved drug platforms and owe their success to their versatility in binding targets, high stability, and low toxicity. The anti-TAG-72 cancer-targeting antibody, 3E8, is one such molecule that shows great potential as a diagnostic. We have designed and biophysically characterized a library of 3E8 single chain antibody fragments (scFV) with varying linker composition and length as well as domain orientations. In this library, we have found substantial variation in protein stability, binding affinity, and oligomeric states. Surprisingly, a drastic difference in the oligomeric state of these constructs was seen between conventional IMAC purification and Protein L purification. Therefore, the literature rules for scFV linker design must be updated to include the dependencies on purification method. A single antibody construct with optimal biophysical properties (3E8.G4S) was further characterized and subjected to in vivo pharmacokinetic studies. Due to its multimeric composition, 3E8.G4S showed a longer and more favorable clearance time compared to that of a fast clearing scFV. Xenograft mouse imaging and biodistribution studies revealed successful targeting of a colorectal tumor by 3E8.G4S with little accumulation in normal tissues. To determine the versatility of 3E8-based diagnostics and therapeutic agents, an expansive immunohistochemical analysis of TAG-72 expression was performed in over 1,500 tumors spanning 18 different cancer types. The results of this study showed enhanced staining of engineered 3E8.scFV.FLAG compared to commercially available anti-TAG-72 IgG, B72.3. We found statistically significant TAG-72 expression differences in colon, prostate, lung, cervical, ovarian, pancreatic, gastrointestinal, rectal, and breast cancers when compared to corresponding normal tissues. The intense staining of these diseased tissues by 3E8.scFV.FLAG suggests wide applicability for its clinical use as a cancer detection agent and consequently the viability of 3E8.G4S as a state of the art cancer imaging agent. Manipulation of the Notch signaling pathway is another promising therapeutic strategy for a variety of diseases. Specifically, activation of the Notch1 receptor by a clustered DLL1 ligand has been shown to enhance T-cell maturation and promote cancer cell death. This makes the engineering of a multimeric and discrete form of DLL1 a viable cancer therapy. We have identified and validated the critical binding domain of the DLL1 ligand through in vitro cell based and in vivo Notch inhibition studies. Additionally, the minimal multivalency for Notch activation was determined to be four DLL1 repeats. Through E. coli transgenic expression and protein refolding, we have successfully created a 4x and 6x tandem repeat of the DLL1 ligand capable of in vitro cell activation of the Notch1 pathway. Through the activation of host immune cells, this novel Notch activator has the potential to be a broadly used cancer immunotherapeutic.
Thomas Magliery (Advisor)
Christopher Jaroniec (Committee Member)
Edward Martin, Jr. (Committee Member)
Richard Swenson (Committee Member)
166 p.
Biochemistry; Biomedical Research; Biophysics; Immunology; Medicine; Molecular Biology
Antibody fragments; scFv; linker; protein engineering; drug design; notch; dll1

Recommended Citations

Citations

  • Long, N. E. (2018). Biophysical Enhancement of Protein Therapeutics and Diagnostics Through Engineered Linkers [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu151515985462363

    APA Style (7th edition)

  • Long, Nicholas. Biophysical Enhancement of Protein Therapeutics and Diagnostics Through Engineered Linkers. 2018. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu151515985462363.

    MLA Style (8th edition)

  • Long, Nicholas. "Biophysical Enhancement of Protein Therapeutics and Diagnostics Through Engineered Linkers." Doctoral dissertation, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu151515985462363

    Chicago Manual of Style (17th edition)

osu151515985462363
273
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This open access ETD is published by The Ohio State University and OhioLINK.