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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2014)

Exploiting antibodies' highly specific binding affinities to various biomarkers, immuno-positron emission tomography (immunoPET) provides a non-invasive way to obtain whole body information on in vivo molecular events that may be crucial for diagnosis and prognosis, which is increasingly important for diagnostics and prognostics. Small, fully human or humanized antibody fragments are preferred for immunoPET imaging probe development due to their favorable pharmacokinetic properties and low risk of immunogenicity. Phage display technology provides a convenient approach to rapidly generate fully human antibody candidates, showing the potential to accelerate and streamline the development process of antibody fragment based immunoPET imaging probes, which is strongly needed as increasing numbers of targeted therapies are being developed every year. Chapter 2 and Chapter 3 describe the use of a traditional fully human scFv phage display library for developing anti-MET antibody fragments for both immunoPET imaging and potential therapeutic application. Multiple fully human anti-MET scFv clones were isolated and reformatted. Three cys-diabody clones showed high affinities (0.7 to 5.1 nM) and inhibitory effect on MET over-expressing cells. The H2 cys-diabody and minibody were able to distinguish a MET over-expressing gefitinib resistant tumor from the parental tumor at as early as 4 hours post injection, indicating the potential of same day imaging for patient stratification. To further exploit the benefit of the phage display technology and streamline the antibody fragment devlopment, as described in Chapter 4 and Chapter 5, two novel fully human scFv phage display libraries with customized linkers and restriction sites were constructed to simplify the antibody fragment reformatting process. The libraries were used for selections against several different targets and successfully generated multiple positive clones. ScFvs from these libraries were rapidly reformatted into diabodies using the restriction sites in the customized linkers. Size exclusion chromatography analysis of these antibody fragments proved the shortened linkers can successfully induce dimerization for the majority of the diabody clones. Together, the studies presented in this dissertation highlight a successful example of the use of phage display technology for developing fully human antibody fragments for immunoPET imaging and how phage display libraries can be improved to further streamline this process.

    Cover page: Streamlined Development of Fully Human Antibody Fragments for ImmunoPET Imaging Using Phage Display Technology
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2014)
    MET, the receptor of hepatocyte growth factor, plays important roles in tumorigenesis and drug resistance in numerous cancers, including non-small cell lung cancer (NSCLC). As increasing numbers of MET inhibitors are being developed for clinical applications, antibody fragment-based immunopositron emission tomography (immunoPET) has the potential to rapidly quantify in vivo MET expression levels for drug response evaluation and patient stratification for these targeted therapies. Here, fully human single-chain variable fragments (scFvs) isolated from a phage display library were reformatted into bivalent cys-diabodies (scFv-cys dimers) with affinities to MET ranging from 0.7 to 5.1 nmol/L. The candidate with the highest affinity, H2, was radiolabeled with (89)Zr for immunoPET studies targeting NSCLC xenografts: low MET-expressing Hcc827 and the gefitinib-resistant Hcc827-GR6 with 4-fold MET overexpression. ImmunoPET at as early as 4 hours after injection produced high-contrast images, and ex vivo biodistribution analysis at 20 hours after injection showed about 2-fold difference in tracer uptake levels between the parental and resistant tumors (P < 0.01). Further immunoPET studies using a larger fragment, the H2 minibody (scFv-CH3 dimer), produced similar results at later time points. Two of the antibody clones (H2 and H5) showed in vitro growth inhibitory effects on MET-dependent gefitinib-resistant cell lines, whereas no effects were observed on resistant lines lacking MET activation. In conclusion, these fully human antibody fragments inhibit MET-dependent cancer cells and enable rapid immunoPET imaging to assess MET expression levels, showing potential for both therapeutic and diagnostic applications.
      Cover page: Anti-MET ImmunoPET for Non–Small Cell Lung Cancer Using Novel Fully Human Antibody Fragments
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2015)
      Phage display libraries of human single-chain variable fragments (scFvs) are a reliable source of fully human antibodies for scientific and clinical applications. Frequently, scFvs form the basis of larger, bivalent formats to increase valency and avidity. A small and versatile bivalent antibody fragment is the diabody, a cross-paired scFv dimer (∼55 kDa). However, generation of diabodies from selected scFvs requires decreasing the length of the interdomain scFv linker, typically by overlap PCR. To simplify this process, we designed two scFv linkers with integrated restriction sites for easy linker length reduction (17-residue to 7-residue or 18-residue to 5-residue, respectively) and generated two fully human scFv phage display libraries. The larger library (9 × 10(9) functional members) was employed for selection against a model antigen, human N-cadherin, yielding novel scFv clones with low nanomolar monovalent affinities. ScFv clones from both libraries were reformatted into diabodies by restriction enzyme digestion and re-ligation. Size-exclusion chromatography analysis confirmed the proper dimerization of most of the diabodies. In conclusion, these specially designed scFv phage display libraries allow us to rapidly reformat the selected scFvs into diabodies, which can greatly accelerate early stage antibody development when bivalent fragments are needed for candidate screening.
        Cover page: A fully human scFv phage display library for rapid antibody fragment reformatting
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2016)
        Recently, single-cell molecular analysis has been leveraged to achieve unprecedented levels of biological investigation. However, a lack of simple, high-throughput single-cell methods has hindered in-depth population-wide studies with single-cell resolution. We report a microwell-based cytometric method for simultaneous measurements of gene and protein expression dynamics in thousands of single cells. We quantified the regulatory effects of transcriptional and translational inhibitors on cMET mRNA and cMET protein in cell populations. We studied the dynamic responses of individual cells to drug treatments, by measuring cMET overexpression levels in individual non-small cell lung cancer (NSCLC) cells with induced drug resistance. Across NSCLC cell lines with a given protein expression, distinct patterns of transcript-protein correlation emerged. We believe this platform is applicable for interrogating the dynamics of gene expression, protein expression, and translational kinetics at the single-cell level - a paradigm shift in life science and medicine toward discovering vital cell regulatory mechanisms.
          Cover page: Dual transcript and protein quantification in a massive single cell array
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2023)
          CAR (chimeric antigen receptor) T cell therapy has shown clinical success in treating hematological malignancies, but its treatment of solid tumors has been limited. One major challenge is on-target, off-tumor toxicity, where CAR T cells also damage normal tissues that express the targeted antigen. To reduce this detrimental side-effect, Boolean-logic gates like AND-NOT gates have utilized an inhibitory CAR (iCAR) to specifically curb CAR T cell activity at selected nonmalignant tissue sites. However, the strategy seems inefficient, requiring high levels of iCAR and its target antigen for inhibition. Using a TROP2-targeting iCAR with a single PD1 inhibitory domain to inhibit a CEACAM5-targeting CAR (CEACAR), we observed that the inefficiency was due to a kinetic delay in iCAR inhibition of cytotoxicity. To improve iCAR efficiency, we modified three features of the iCAR-the avidity, the affinity, and the intracellular signaling domains. Increasing the avidity but not the affinity of the iCAR led to significant reductions in the delay. iCARs containing twelve different inhibitory signaling domains were screened for improved inhibition, and three domains (BTLA, LAIR-1, and SIGLEC-9) each suppressed CAR T function but did not enhance inhibitory kinetics. When inhibitory domains of LAIR-1 or SIGLEC-9 were combined with PD-1 into a single dual-inhibitory domain iCAR (DiCARs) and tested with the CEACAR, inhibition efficiency improved as evidenced by a significant reduction in the inhibitory delay. These data indicate that a delicate balance between CAR and iCAR signaling strength and kinetics must be achieved to regulate AND-NOT gate CAR T cell selectivity.
            Cover page: Dual-inhibitory domain iCARs improve the efficiency of the AND-NOT gate CAR T strategy
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2022)
            BACKGROUND & AIMS: The stroma in pancreatic ductal adenocarcinoma (PDAC) contributes to its immunosuppressive nature and therapeutic resistance. Herein we sought to modify signaling and enhance immunotherapy efficacy by targeting multiple stromal components through both intracellular and extracellular mechanisms. METHODS: A murine liver metastasis syngeneic model of PDAC was treated with focal adhesion kinase inhibitor (FAKi), anti-programmed cell death protein 1 (PD-1) antibody, and stromal hyaluronan (HA) degradation by PEGylated recombinant human hyaluronidase (PEGPH20) to assess immune and stromal modulating effects of these agents and their combinations. RESULTS: The results showed that HA degradation by PEGPH20 and reduction in phosphorylated FAK expression by FAKi leads to improved survival in PDAC-bearing mice treated with anti-PD-1 antibody. HA degradation in combination with FAKi and anti-PD-1 antibody increases T-cell infiltration and alters T-cell phenotype toward effector memory T cells. FAKi alters the expression of T-cell modulating cytokines and leads to changes in T-cell metabolism and increases in effector T-cell signatures. HA degradation in combination with anti-PD-1 antibody and FAKi treatments reduces granulocytes, including granulocytic- myeloid-derived suppressor cells and decreases C-X-C chemokine receptor type 4 (CXCR4)-expressing myeloid cells, particularly the CXCR4-expressing granulocytes. Anti-CXCR4 antibody combined with FAKi and anti-PD-1 antibody significantly decreases metastatic rates in the PDAC liver metastasis model. CONCLUSIONS: This represents the first preclinical study to identify synergistic effects of targeting both intracellular and extracellular components within the PDAC stroma and supports testing anti-CXCR4 antibody in combination with FAKi as a PDAC treatment strategy.
              Cover page: Dual Stromal Targeting Sensitizes Pancreatic Adenocarcinoma for Anti-Programmed Cell Death Protein 1 Therapy.
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