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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2018)

Prostate cancer diagnosis and treatment options need to be improved as over- and under-treatment, as well as disease recurrence and resistance to current therapies, continue to be challenges. Prostate stem cell antigen (PSCA) is overexpressed in the majority of prostate cancers and correlates with grade, stage, and metastatic potential. Antibodies, which are highly specific for their target, can be labeled with radionuclides and fluorophores for molecular imaging and therapy. This dissertation describes the use of humanized anti-PSCA antibody fragments for dual-modality immuno-positron emission tomography (immunoPET)/fluorescence imaging and for radioimmunotherapy of prostate cancer in preclinical models.

Prostate cancer visualization could be improved by using immunoPET for preoperative non-invasive disease detection, and fluorescence imaging for intraoperative guidance of tumor resection; the power of both imaging modalities can be combined on a single agent. In this work, the dual-labeled humanized anti-PSCA A11 cys-minibody (A11 cMb) was evaluated for successive immunoPET/fluorescence imaging in subcutaneous and orthotopic prostate cancer models. A11 cMb was site-specifically conjugated with the near-infrared fluorophore Cy5.5 and radiolabeled with 124I or 89Zr. 124I- and 89Zr-A11 cMb-Cy5.5 were used to detect subcutaneous and intraprostatic PSCA-positive tumors. High contrast immunoPET/fluorescence imaging with 124I-A11 cMb-Cy5.5 identified both high PSCA-expression and moderate PSCA-expression subcutaneous tumors. 89Zr-A11 cMb-Cy5.5 immunoPET showed uptake in the prostate without interfering signal in the bladder, and ex vivo fluorescence imaging clearly showed signal in the tumor and not the surrounding seminal vesicles. These studies support the potential for dual-modality A11 cMb to be translated for preoperative whole-body disease detection and real-time surgical guidance in prostate cancer patients.

Prostate cancer that metastasizes often becomes resistant to current therapies and eventually progresses, and alternative therapy options include radioimmunotherapy which delivers a high radiation dose to the tumor with the aim of minimizing dose to normal organs. Anti-PSCA antibody fragments radiolabeled with a cytotoxic radionuclide, such as the beta-emitter 131I or 177Lu, may be effective for radioimmunotherapy with lower toxicity compared to intact antibodies. 124I- and 89Zr-A11 minibody (A11 Mb) immunoPET was used to guide dosimetry studies, and 131I- and 177Lu-A11 Mb was administered to 22Rv1-PSCA s.c. tumor-bearing nude mice to complete dosimetry estimation. 131I-A11 Mb had a higher projected therapeutic index, or tumor-to-radiosensitive tissue dose, and was used in subsequent therapy studies. 131I-A11 Mb inhibited PSCA-positive tumor growth in a dose-dependent manner and extended survival with minimal off-target toxicity. Additionally, preliminary biodistribution studies in knock-in transgenic mice that express human-PSCA were similar to that of nude mice. Therefore, radioimmunotherapy will likely not be toxic to organs with normal PSCA expression (stomach, bladder), and A11 Mb may be suitable for human translation.

The results of this work demonstrate the potential of anti-PSCA minibodies as theranostic agents for disease diagnosis, surgical guidance, and treatment. Additionally, the success of anti-PSCA dual-modality imaging and radioimmunotherapy in preclinical models support further evaluation in the clinic.

    Cover page: ImmunoPET/fluorescence imaging and radioimmunotherapy of PSCA-positive prostate cancer
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2019)
    Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence for visualization of superficial tissue layers for image-guided surgery. Methods: We developed a universal dual-modality linker (DML) that facilitates site-specific conjugation to a cysteine residue-bearing antibody fragment, introduction of a commercially available fluorescent dye (via an amine-reactive prosthetic group), and rapid and efficient radiolabeling via click chemistry with 18F-labeled trans-cyclooctene (18F-TCO). To generate a dual-modality antibody fragment-based imaging agent, the DML was labeled with the far-red dye sulfonate cyanine 5 (sCy5), site-specifically conjugated to the C-terminal cysteine of the anti-prostate stem cell antigen (PSCA) cys-diabody A2, and subsequently radiolabeled by click chemistry with 18F-TCO. The new imaging probe was evaluated in a human PSCA-positive prostate cancer xenograft model by sequential immuno-PET and optical imaging. Uptake in target tissues was confirmed by ex vivo biodistribution. Results: We successfully synthesized a DML for conjugation of a fluorescent dye and 18F. The anti-PSCA cys-diabody A2 was site-specifically conjugated with either DML or sCy5 and radiolabeled via click chemistry with 18F-TCO. Immuno-PET imaging confirmed in vivo antigen-specific targeting of prostate cancer xenografts as early as 1 h after injection. Rapid renal clearance of the 50-kDa antibody fragment enables same-day imaging. Optical imaging showed antigen-specific fluorescent signal in PSCA-positive xenografts and high contrast to surrounding tissue and PSCA-negative xenografts. Conclusion: The DML enables site-specific conjugation away from the antigen-binding site of antibody fragments, with a controlled linker-to-protein ratio, and combines signaling moieties for 2 imaging systems into 1 molecule. Dual-modality imaging could provide both noninvasive whole-body imaging with organ-level biodistribution and fluorescence image-guided identification of tumor margins during surgery.
      Cover page: A Dual-Modality Linker Enables Site-Specific Conjugation of Antibody Fragments for 18F-Immuno-PET and Fluorescence Imaging
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2020)
      Positron emission tomography (PET) molecular imaging is a powerful tool for interrogating physiological and biochemical processes to understand the biology of disease and advance therapeutic developments. Near-infrared fluorescence (NIRF) optical imaging has become increasingly popular for intraoperative staging to enable cellular resolution imaging of tumor margins during surgical resection. In addition, engineered antibody fragments have emerged as promising molecular imaging agents given their exquisite target selectivity, rapid systemic clearance and site-selective chemical modification. We report a tri-functional platform for construction of a modular antibody fragment that can rapidly be labeled with radionuclides or fluorophores for PET or NIRF molecular imaging of prostate stem cell antigen (PSCA).
        Cover page: Tri-functional platform for construction of modular antibody fragments for in vivo 18 F-PET or NIRF molecular imaging
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2019)

        Purpose

        Metabolic imaging using [18F]FDG is the current standard for clinical PET; however, some malignancies (e.g., indolent lymphomas) show low avidity for FDG. The majority of B cell lymphomas express CD20, making it a valuable target both for antibody-based therapy and imaging. We previously developed PET tracers based on the humanised anti-CD20 antibody obinutuzumab (GA101). Preclinical studies showed that the smallest bivalent fragment, the cys-diabody (GAcDb, 54.5 kDa) with a peak uptake at 1-2 h post-injection and a biological half-life of 2-5 h, is compatible with short-lived positron emitters such as fluorine-18 (18F, t1/2 110 min), enabling same-day imaging.

        Methods

        GAcDb was radiolabeled using amine-reactive N-succinimidyl 4-[18F]-fluorobenzoate ([18F]SFB), or thiol-reactive N-[2-(4-[18F]-fluorobenzamido)ethyl]maleimide ([18F]FBEM) for site-specific conjugation to C-terminal cysteine residues. Both tracers were used for immunoPET imaging of the B cell compartment in human CD20 transgenic mice (hCD20TM). [18F]FB-GAcDb immunoPET was further evaluated in a disseminated lymphoma (A20-hCD20) syngeneic for hCD20TM and compared to [18F]FDG PET. Tracer uptake was confirmed by ex vivo biodistribution.

        Results

        The GAcDb was successfully 18F-radiolabeled using two different conjugation methods resulting in similar specific activities and without impairing immunoreactivity. Both tracers ([18F]FB-GAcDb and [18F]FBEM-GAcDb) specifically target human CD20-expressing B cells in transgenic mice. Fast blood clearance results in high contrast PET images as early as 1 h post injection enabling same-day imaging. [18F]FB-GAcDb immunoPET detects disseminated lymphoma disease in the context of normal tissue expression of hCD20, with comparable sensitivity as [18F]FDG PET but with added specificity for the therapeutic target.

        Conclusions

        [18F]FB-GAcDb and [18F]FBEM-GAcDb could monitor normal B cells and B cell malignancies non-invasively and quantitatively in vivo. In contrast to [18F]FDG PET, immunoPET provides not only information about the extent of disease but also about presence and localisation of the therapeutic target.
          Cover page: 18F-labeled anti-human CD20 cys-diabody for same-day immunoPET in a model of aggressive B cell lymphoma in human CD20 transgenic mice
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2018)
          Inflammatory bowel diseases (IBDs) in humans are characterized in part by aberrant CD4-positive (CD4+) T-cell responses. Currently, identification of foci of inflammation within the gut requires invasive procedures such as colonoscopy and biopsy. Molecular imaging with antibody fragment probes could be used to noninvasively monitor cell subsets causing intestinal inflammation. Here, GK1.5 cys-diabody (cDb), an antimouse CD4 antibody fragment derived from the GK1.5 hybridoma, was used as a PET probe for CD4+ T cells in the dextran sulfate sodium (DSS) mouse model of IBD. Methods: The DSS mouse model of IBD was validated by assessing changes in CD4+ T cells in the spleen and mesenteric lymph nodes (MLNs) using flow cytometry. Furthermore, CD4+ T cell infiltration in the colons of colitic mice was evaluated using immunohistochemistry. 89Zr-labeled GK1.5 cDb was used to image distribution of CD4+ T cells in the abdominal region and lymphoid organs of mice with DSS-induced colitis. Region-of-interest analysis was performed on specific regions of the gut to quantify probe uptake. Colons, ceca, and MLNs were removed and imaged ex vivo by PET. Imaging results were confirmed by ex vivo biodistribution analysis. Results: An increased number of CD4+ T cells in the colons of colitic mice was confirmed by anti-CD4 immunohistochemistry. Increased uptake of 89Zr-maleimide-deferoxamine (malDFO)-GK1.5 cDb in the distal colon of colitic mice was visible in vivo in PET scans, and region-of-interest analysis of the distal colon confirmed increased activity in DSS mice. MLNs from colitic mice were enlarged and visible in PET images. Ex vivo scans and biodistribution confirmed higher uptake in DSS-treated colons (DSS, 1.8 ± 0.40; control, 0.45 ± 0.12 percentage injected dose [%ID] per organ, respectively), ceca (DSS, 1.1 ± 0.38; control, 0.35 ± 0.09 %ID per organ), and MLNs (DSS, 1.1 ± 0.58; control, 0.37 ± 0.25 %ID per organ). Conclusion:89Zr-malDFO-GK1.5 cDb detected CD4+ T cells in the colons, ceca, and MLNs of colitic mice and may prove useful for further investigations of CD4+ T cells in preclinical models of IBD, with potential to guide development of antibody-based imaging in human IBD.
            Cover page: Immuno-PET in Inflammatory Bowel Disease: Imaging CD4-Positive T Cells in a Murine Model of Colitis
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2017)
            The importance of context in regulation of gene expression is now an accepted principle; yet the mechanism by which the microenvironment communicates with the nucleus and chromatin in healthy tissues is poorly understood. A functional role for nuclear and cytoskeletal architecture is suggested by the phenotypic differences observed between epithelial and mesenchymal cells. Capitalizing on recent advances in cryogenic techniques, volume electron microscopy and super-resolution light microscopy, we studied human mammary epithelial cells in three-dimensional (3D) cultures forming growth-arrested acini. Intriguingly, we found deep nuclear invaginations and tunnels traversing the nucleus, encasing cytoskeletal actin and/or intermediate filaments, which connect to the outer nuclear envelope. The cytoskeleton is also connected both to other cells through desmosome adhesion complexes and to the extracellular matrix through hemidesmosomes. This finding supports a physical and/or mechanical link from the desmosomes and hemidesmosomes to the nucleus, which had previously been hypothesized but now is visualized for the first time. These unique structures, including the nuclear invaginations and the cytoskeletal connectivity to the cell nucleus, are consistent with a dynamic reciprocity between the nucleus and the outside of epithelial cells and tissues.
              Cover page: Deep nuclear invaginations are linked to cytoskeletal filaments – integrated bioimaging of epithelial cells in 3D culture
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2019)

              Purpose

              The inability to intraoperatively distinguish primary tumor, as well as lymphatic spread, increases the probability of positive surgical margins, tumor recurrence, and surgical toxicity. The goal of this study was to develop a tumor-specific optical probe for real-time fluorescence-guided surgery.

              Experimental design

              A humanized antibody fragment against PSCA (A11 minibody, A11 Mb) was conjugated with a near-infrared fluorophore, IRDye800CW. The integrity and binding of the probe to PSCA were confirmed by gel electrophoresis, size-exclusion chromatography, and flow cytometry, respectively. The ability of the probe to detect tumor-infiltrated lymph nodes and metastatic lesions was evaluated in 2 xenograft models, as well as in transgenic mice expressing human PSCA (hPSCA). An invasive intramuscular model was utilized to evaluate the efficacy of the A11 Mb-IRDye800CW-guided surgery.

              Results

              A11 Mb was successfully conjugated with IRDye800CW and retained specific binding to PSCA. In vivo imaging showed maximal signal-to-background ratios at 48 hours. The A11 Mb-IRDye800CW specifically detected PSCA-positive primary tumors, tumor-infiltrated lymph nodes, and distant metastases with high contrast. Fluorescence guidance facilitated more complete tumor resection, reduced tumor recurrence, and improved overall survival, compared with conventional white light surgery. The probe successfully identified primary orthotopic tumors and metastatic lesions in hPSCA transgenic mice.

              Conclusions

              Real-time fluorescence image-guided surgery with A11 Mb-IRDye800CW enabled detection of lymph node metastases and positive surgical margins, facilitated more complete tumor removal, and improved survival, compared with white light surgery. These results may be translatable into clinical practice to improve surgical and patient outcomes.
                Cover page: Near-Infrared Dye-Labeled Anti-Prostate Stem Cell Antigen Minibody Enables Real-Time Fluorescence Imaging and Targeted Surgery in Translational Mouse Models
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