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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2013)

Prostate stem cell antigen (PSCA) is a cell surface glycoprotein that has low levels of expression in normal prostate, bladder, and stomach, but is expressed in 83-100%, and overexpressed in 40-100%, of prostate cancers. PSCA expression has been shown to correlate with the Gleason score, seminal vesicle and capsular invasion, advanced clinical stage, androgen independence, MYC gene-amplification, and a poor prognosis. PSCA is also highly overexpressed in the majority of prostate cancer bone metastases (87- 100%) and many metastases to other sites (67% liver, 67-95% lymph node). ImmunoPET is a highly flexible imaging technique that can be utilized to image virtually any cell surface protein using radiolabeled antibodies or antibody fragments. This dissertation describes the use of a radiolabeled anti-PSCA A11 minibody (scFv-CH3 dimer, 80 kDa) for immunoPET imaging of PSCA expression in mouse models of prostate cancer and demonstrates the potential of the A11 minibody for highly specific imaging of local prostate cancer and metastases in the clinic.

In this work, 124I and 89Zr-DFO radiolabeling of the anti-PSCA minibody are compared. Imaging with both radionuclides showed specific tumor targeting in 22rv1�PSCA and LAPC-9 tumors and achieved high contrast imaging in both tumor models. However, 124I- labeled A11 minibody achieved superior tumor:soft tissue contrast and was determined to have higher clinical potential. A quantitative method of μPET imaging of both 124I and 89Zr radionuclides using recovery coefficient based partial volume correction was also established and validated.

The PSCA promoter contains an androgen response element and expression of PSCA has been shown to be regulated by androgens in the mouse prostate and in human prostate cancer biopsy tissue. In the present work, treatment with the second generation anti-androgen MDV-3100 was demonstrated to cause downregulation of PSCA in LAPC-9 xenografts. In addition, 124I-labeled A11 minibody showed decreased uptake in LAPC-9 xenografts treated with MDV-3100 compared to vehicle controls suggesting a potential use for the A11 minibody in imaging response to androgen deprivation therapy.

The highest percentage of prostate cancer metastases are osteoblastic bone metastases and most clinical staging of prostate cancer metastases utilizes 99mTc-MDP or 18F- Fluoride bone scans to image increases in bone turnover in response to these metastases. In this work we demonstrate that 124I-labeled A11 minibody has higher sensitivity and specificity for detecting LAPC-9 intratibial xenografts than 18F-Fluoride bone scans.

The A11 minibody binds only to human PSCA (hPSCA) and does not cross-react with the murine homologue mPSCA. In order to address the background uptake caused by endoge- nous hPSCA expression, the creation of an hPSCA knock-in mouse and imaging using the A11 minibody in this model is described. We found a pattern of hPSCA expression in these mice similar to the pattern of normal expression in humans. Uptake of 124I-labeled A11 minibody correlated strongly with the expression of hPSCA, but demonstrated that the overall increase in background uptake of the A11 minibody due to endogenous expression of human PSCA is minimal.

The results presented in this work demonstrate robust targeting of PSCA expression by the A11 minibody and clinical investigation of the A11 minibody for imaging local prostate cancer and prostate cancer metastases is warranted.

    Cover page: Anti-PSCA minibody for immunoPET imaging of prostate cancer in preclinical models
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2013)
    Individual ventilated cages (IVC) are increasing in popularity. Although mice avoid IVC in preference testing, they show no aversion when provided additional nesting material or the cage is not ventilated. Given the high ventilation rate in IVC, we developed 3 hypotheses: that mice housed in IVC experience more cold stress than do mice housed in static cages; that IVC-induced cold stress affects the results of experiments using mice; and that, when provided shelters, mice behaviorally thermoregulate and thereby rescue the cold-stress effects of IVC. To test these hypotheses, we housed mice in IVC, IVC with shelters, and static cages maintained at 20 to 21 °C. We quantified the cold stress of each housing system on mice by assessing nonshivering thermogenesis and brown adipose vacuolation. To test housing effects in a common, murine model of human disease, we implanted mice with subcutaneous epidermoid carcinoma cells and quantified tumor growth, tumor metabolism, and adrenal weight. Mice housed in IVC had histologic signs of cold stress and significantly higher nonshivering thermogenesis, smaller subcutaneous tumors, lower tumor metabolism, and larger adrenal weights than did mice in static cages. Shelters rescued IVC-induced nonshivering thermogenesis, adrenal enlargement, and phenotype-dependent cold-mediated histologic changes in brown adipose tissue and tumor size. IVC impose chronic cold stress on mice, alter experimental results, and are a source of systemic confounders throughout rodent-dependent research. Allowing mice to exhibit behavioral thermoregulation through seeking shelter markedly rescues the experiment-altering effects of housing-imposed cold stress, improves physiologic uniformity, and increases experimental reproducibility across housing systems.
      Cover page: Individually ventilated cages impose cold stress on laboratory mice: a source of systemic experimental variability.
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2017)
      Purpose: The B-cell antigen CD20 provides a target for antibody-based positron emission tomography (immunoPET). We engineered antibody fragments targeting human CD20 and studied their potential as immunoPET tracers in transgenic mice (huCD20TM) and in a murine lymphoma model expressing human CD20.Experimental Design: Anti-CD20 cys-diabody (cDb) and cys-minibody (cMb) based on rituximab and obinutuzumab (GA101) were radioiodinated and used for immunoPET imaging of a murine lymphoma model. Pairwise comparison of obinutuzumab-based antibody fragments labeled with residualizing (89Zr) versus non-residualizing (124I) radionuclides by region of interest analysis of serial PET images was conducted both in the murine lymphoma model and in huCD20TM to assess antigen modulation in vivoResults:124I-GAcDb and 124I-GAcMb produced high-contrast immunoPET images of B-cell lymphoma and outperformed the respective rituximab-based tracers. ImmunoPET imaging of huCD20TM showed specific uptake in lymphoid tissues. The use of the radiometal 89Zr as alternative label for GAcDb and GAcMb yielded greater target-specific uptake and retention compared with 124I-labeled tracers. Pairwise comparison of 89Zr- and 124I-labeled GAcDb and GAcMb allowed assessment of in vivo internalization of CD20/antibody complexes and revealed that CD20 internalization differs between malignant and endogenous B cells.Conclusions: These obinutuzumab-based PET tracers have the ability to noninvasively and quantitatively monitor CD20-expression and have revealed insights into CD20 internalization upon antibody binding in vivo Because they are based on a humanized mAb they have the potential for direct clinical translation and could improve patient selection for targeted therapy, dosimetry prior to radioimmunotherapy, and prediction of response to therapy. Clin Cancer Res; 23(23); 7242-52. ©2017 AACR.
        Cover page: ImmunoPET of Malignant and Normal B Cells with 89Zr- and 124I-Labeled Obinutuzumab Antibody Fragments Reveals Differential CD20 Internalization In Vivo
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2014)

        Purpose

        Intact antibodies have a long serum persistence resulting in high background signal that inhibits their direct translation as imaging agents. Engineering of antibody fragments through the introduction of mutations in the fragment crystallizable (Fc) region can dramatically reduce serum persistence. We sought to develop a Fc-mutated, anti-CA19-9 antibody fragment (anti-CA 19-9 scFv-Fc H310A) to provide micro-positron emission tomography (microPET) imaging of pancreatic cancer xenografts.

        Procedures

        The anti-CA19-9 scFv-Fc H310A was successfully expressed and purified. Biochemical characterization included size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and flow cytometry. The antibody fragment was labeled with iodine-124 ((124)I) and injected into mice containing human pancreatic cancer xenografts. MicroPET/CT images were then obtained. Blood, organ, and tumor radioactivity was measured and expressed as the percent of injected dose per gram of tissue (%ID/g).

        Results

        Biochemical characterization was consistent with the creation of a 105 kD dimer containing a human Fc region. Flow cytometry demonstrated antigen-specific binding, and cell-based ELISA further established a dissociation constant (K D) of 10.7 nM. (124)I-labeled scFv-Fc H310A localized to the antigen-positive tumor xenografts as detected by microPET. Objective confirmation of targeting was demonstrated by higher %ID/g in the antigen-positive tumor compared to the blood, antigen-negative tumor, and liver.

        Conclusions

        We successfully engineered and produced an anti-CA19-9 scFv-Fc H310A antibody fragment that retains similar affinity when compared to the parental intact murine antibody. Additionally, our engineered and mutated fragment exhibited antigen-specific microPET imaging of both subcutaneous and orthotopic pancreatic cancer xenografts at early time points secondary to decreased serum half-life.
          Cover page: A Mutated Anti-CA19-9 scFv-Fc for Positron Emission Tomography of Human Pancreatic Cancer Xenografts
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2014)
          The noninvasive detection and quantification of CD8(+) T cells in vivo are important for both the detection and staging of CD8(+) lymphomas and for the monitoring of successful cancer immunotherapies, such as adoptive cell transfer and antibody-based immunotherapeutics. Here, antibody fragments are constructed to target murine CD8 to obtain rapid, high-contrast immuno-positron emission tomography (immuno-PET) images for the detection of CD8 expression in vivo. The variable regions of two anti-murine CD8-depleting antibodies (clones 2.43 and YTS169.4.2.1) were sequenced and reformatted into minibody (Mb) fragments (scFv-CH3). After production and purification, the Mbs retained their antigen specificity and bound primary CD8(+) T cells from the thymus, spleen, lymph nodes, and peripheral blood. Importantly, engineering of the parental antibodies into Mbs abolished the ability to deplete CD8(+) T cells in vivo. The Mbs were subsequently conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid for (64)Cu radiolabeling. The radiotracers were injected i.v. into antigen-positive, antigen-negative, immunodeficient, antigen-blocked, and antigen-depleted mice to evaluate specificity of uptake in lymphoid tissues by immuno-PET imaging and ex vivo biodistribution. Both (64)Cu-radiolabeled Mbs produced high-contrast immuno-PET images 4 h postinjection and showed specific uptake in the spleen and lymph nodes of antigen-positive mice.
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2014)

            Purpose

            Prostate stem cell antigen (PSCA) is highly expressed in local prostate cancers and prostate cancer bone metastases and its expression correlates with androgen receptor activation and a poor prognosis. In this study, we investigate the potential clinical applications of immunoPET with the anti-PSCA A11 minibody, an antibody fragment optimized for use as an imaging agent. We compare A11 minibody immunoPET to (18)F-Fluoride PET bone scans for detecting prostate cancer bone tumors and evaluate the ability of the A11 minibody to image tumor response to androgen deprivation.

            Experimental design

            Osteoblastic, PSCA-expressing, LAPC-9 intratibial xenografts were imaged with serial (124)I-anti-PSCA A11 minibody immunoPET and (18)F-Fluoride bone scans. Mice bearing LAPC-9 subcutaneous xenografts were treated with either vehicle or MDV-3100 and imaged with A11 minibody immunoPET/CT scans pre- and posttreatment. Ex vivo flow cytometry measured the change in PSCA expression in response to androgen deprivation.

            Results

            A11 minibody demonstrated improved sensitivity and specificity over (18)F-Fluoride bone scans for detecting LAPC-9 intratibial xenografts at all time points. LAPC-9 subcutaneous xenografts showed downregulation of PSCA when treated with MDV-3100 which A11 minibody immunoPET was able to detect in vivo.

            Conclusions

            A11 minibody immunoPET has the potential to improve the sensitivity and specificity of clinical prostate cancer metastasis detection over bone scans, which are the current clinical standard-of-care. A11 minibody immunoPET additionally has the potential to image the activity of the androgen signaling axis in vivo which may help evaluate the clinical response to androgen deprivation and the development of castration resistance.
              Cover page: Applications of ImmunoPET: Using 124I-Anti-PSCA A11 Minibody for Imaging Disease Progression and Response to Therapy in Mouse Xenograft Models of Prostate Cancer
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2017)
              Currently, there is no curative treatment for advanced metastatic prostate cancer, and options, such as chemotherapy, are often nonspecific, harming healthy cells and resulting in severe side effects. Attaching targeting ligands to agents used in anticancer therapies has been shown to improve efficacy and reduce nonspecific toxicity. Furthermore, the use of triggered therapies can enable spatial and temporal control over the treatment. Here, we combined an engineered prostate cancer-specific targeting ligand, the A11 minibody, with a novel photothermal therapy agent, polypeptide-based gold nanoshells, which generate heat in response to near-infrared light. We show that the A11 minibody strongly binds to the prostate stem cell antigen that is overexpressed on the surface of metastatic prostate cancer cells. Compared to nonconjugated gold nanoshells, our A11 minibody-conjugated gold nanoshell exhibited significant laser-induced, localized killing of prostate cancer cells in vitro. In addition, we improved upon a comprehensive heat transfer mathematical model that was previously developed by our laboratory. By relaxing some of the assumptions of our earlier model, we were able to generate more accurate predictions for this particular study. Our experimental and theoretical results demonstrate the potential of our novel minibody-conjugated gold nanoshells for metastatic prostate cancer therapy.
                Cover page: Engineering A11 Minibody-Conjugated, Polypeptide-Based Gold Nanoshells for Prostate Stem Cell Antigen (PSCA)–Targeted Photothermal Therapy
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