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Open Access Publications from the University of California

Scholarly Works (16 results)

  • Article
  • Peer Reviewed
UCLA Previously Published Works (2009)
Abstract Background Antibody Directed Enzyme Prodrug Therapy (ADEPT) can be used to generate cytotoxic agents at the tumor site. To date non-human enzymes have mainly been utilized in ADEPT. However, these non-human enzymes are immunogenic limiting the number of times that ADEPT can be administered. To overcome the problem of immunogenicity, a fully human enzyme, capable of converting a non-toxic prodrug to cytotoxic drug was developed and joined to a human tumor specific scFv yielding a fully human targeting agent. Methods A double mutant of human purine nucleoside phosphorylase (hDM) was developed which unlike the human enzyme can cleave adenosine-based prodrugs. For tumor-specific targeting, hDM was fused to the human anti-HER2/neu single chain Fv (scFv), C6 MH3B1. Enzymatic activity of hDM with its natural substrates and prodrugs was determined using spectrophotomeric approaches. A cell proliferation assay was used to assess the cytotoxicity generated following conversion of prodrug to drug as a result of enzymatic activity of hDM. Affinity of the targeting scFv, C6 MH3B1 fused to hDM to Her2/neu was confirmed using affinity chromatography, surface plasmon resonance, and flow-cytometry. Results In vitro hDM-C6 MH3B1 binds specifically to HER2/neu expressing tumor cells and localizes hDM to tumor cells, where the enzymatic activity of hDM-C6 MH3B1, but not the wild type enzyme, results in phosphorolysis of the prodrug, 2-fluoro-2'-deoxyadenosine to the cytotoxic drug 2-fluoroadenine (F-Ade) causing inhibition of tumor cell proliferation. Significantly, the toxic small drug diffuses through the cell membrane of HER2/neu expressing cells as well as cells that lack the expression of HER2/neu, causing a bystander effect. F-Ade is toxic to cells irrespective of their growth rate; therefore, both the slowly dividing tumor cells and the non-dividing neighboring stromal cells that support tumor growth should be killed. Analysis of potential novel MHCII binding peptides resulting from fusion of hDM to C6 MH3B1 and the two mutations in hDM, and of the structure of hDM compared to the wild-type enzyme suggests that hDM-C6 MH3B1 should exhibit minimal immunogenicity in humans. Conclusion hDM-C6 MH3B1 constitutes a novel human based protein that addresses some of the limitations of ADEPT that currently preclude its successful use in the clinic.
    Cover page: Characterization of an engineered human purine nucleoside phosphorylase fused to an anti-her2/neu single chain Fv for use in ADEPT
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2009)

    Background

    Purity, yield, speed and cost are important considerations in plasmid purification, but it is difficult to achieve all of these at the same time. Currently, there are many protocols and kits for DNA purification, however none maximize all four considerations.

    Methodology/principal findings

    We now describe a fast, efficient and economic in-house protocol for plasmid preparation using glass syringe filters. Plasmid yield and quality as determined by enzyme digestion and transfection efficiency were equivalent to the expensive commercial kits. Importantly, the time required for purification was much less than that required using a commercial kit.

    Conclusions/significance

    This method provides DNA yield and quality similar to that obtained with commercial kits, but is more rapid and less costly.
      Cover page: A Rapid and Economic In-House DNA Purification Method Using Glass Syringe Filters
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2014)
      The true potential of cytokine therapies in cancer treatment is limited by the inability to deliver optimal concentrations into tumor sites due to dose-limiting systemic toxicities. To maximize the efficacy of cytokine therapy, recombinant antibody-cytokine fusion proteins have been constructed by a number of groups to harness the tumor-targeting ability of monoclonal antibodies. The aim is to guide cytokines specifically to tumor sites where they might stimulate more optimal anti-tumor immune responses while avoiding the systemic toxicities of free cytokine therapy. Antibody-cytokine fusion proteins containing interleukin (IL)-2, IL-12, IL-21, tumor necrosis factor (TNF)α, and interferons (IFNs) α, β, and γ have been constructed and have shown anti-tumor activity in preclinical and early-phase clinical studies. Future priorities for development of this technology include optimization of tumor targeting, bioactivity of the fused cytokine, and choice of appropriate agents for combination therapies. This review is intended to serve as a framework for engineering an ideal antibody-cytokine fusion protein, focusing on previously developed constructs and their clinical trial results.
        Cover page: Antibody-cytokine fusion proteins for treatment of cancer: engineering cytokines for improved efficacy and safety.
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2000)
        Human immunoglobulin (Ig)A exists in blood as two isotypes, IgA1 and IgA2, with IgA2 present as three allotypes: IgA2m(1), IgA2m(2), and IgA2m(n). We now demonstrate that recombinant, chimeric IgA1 and IgA2 differ in their pharmacokinetic properties. The major pathway for the clearance of all IgA2 allotypes is the liver. Liver-mediated uptake is through the asialoglycoprotein receptor (ASGR), since clearance can be blocked by injection of excess galactose-Ficoll ligand and suppressed in ASGR-deficient mice. In contrast, only a small percentage of IgA1 is cleared through this pathway. The clearance of IgA1 lacking the hinge region with its associated O-linked carbohydrate was more rapid than that of wild-type IgA1. IgA1 and IgA2 that are not rapidly eliminated by the ASGR are both removed through an undefined ASGR-independent pathway with half-lives of 14 and 10 h, respectively. The rapid clearance of IgA2 but not IgA1 through the liver may in part explain why the serum levels of IgA1 are greater than those of IgA2. In addition, dysfunction of the ASGR or altered N-linked glycosylation, but not O-glycans, that affects recognition by this receptor may account for the elevated serum IgA seen in liver disease and IgA nephropathy.
          Cover page: The N-Glycans Determine the Differential Blood Clearance and Hepatic Uptake of Human Immunoglobulin (Ig)a1 and Iga2 Isotypes
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2019)
          Aging results in increased myelopoiesis, which is linked to the increased incidence of myeloid leukemias and production of myeloid-derived suppressor cells. Here, we examined the contribution of plasma cells (PCs) to age-related increases in myelopoiesis, as PCs exhibit immune regulatory function and sequester in bone marrow (BM). PC number was increased in old BM, and they exhibited high expression of genes encoding inflammatory cytokines and pathogen sensors. Antibody-mediated depletion of PCs from old mice reduced the number of myeloid-biased hematopoietic stem cells and mature myeloid cells to levels in young animals, but lymphopoiesis was not rejuvenated, indicating that redundant mechanisms inhibit that process. PCs also regulated the production of inflammatory factors from BM stromal cells, and disruption of the PC-stromal cell circuitry with inhibitors of the cytokines IL-1 and TNF-α attenuated myelopoiesis in old mice. Thus, the age-related increase in myelopoiesis is driven by an inflammatory network orchestrated by PCs.
            Cover page: Plasma Cells Are Obligate Effectors of Enhanced Myelopoiesis in Aging Bone Marrow
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2016)
            Although recent advances have substantially improved the management of multiple myeloma, it remains an incurable malignancy. We now demonstrate that anti-CD138 molecules genetically fused to type I interferons (IFN) synergize with the approved therapeutic bortezomib in arresting the proliferation of human multiple myeloma cell lines both in vitro and in vivo. The anti-CD138-IFNα14 fusion protein was active in inducing increased expression of signal transducer and activator of transcription 1 (STAT1) and its phosphorylation while the cell death pathway induced by bortezomib included generation of reactive oxygen species. Interferon regulatory factor 4 (IRF4), an important survival factor for myeloma cells, was down regulated following combination treatment. Induction of cell death appeared to be caspase-independent because treatment with inhibitors of caspase activation did not decrease the level of cell death. The observed caspase-independent synergistic cell death involved mitochondrial membrane depolarization, and poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, and resulted in enhanced induction of apoptosis. Importantly, using 2 different in vivo xenograft models, we found that combination therapy of anti-CD138-IFNα14 and bortezomib was able to cure animals with established tumors (7 of 8 using OCI-My5 or 8 of 8 using NCI-H929). Thus, the combination of anti-CD138-IFNα with bortezomib shows great promise as a novel therapeutic approach for the treatment of multiple myeloma, a malignancy for which there are currently no cures.
              Cover page: Targeted immunotherapy using anti-CD138-interferon α fusion proteins and bortezomib results in synergistic protection against multiple myeloma
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2009)

              Background

              The events required to initiate host defenses against invading pathogens involve complex signaling cascades comprised of numerous adaptor molecules, kinases, and transcriptional elements, ultimately leading to the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha). How these signaling cascades are regulated, and the proteins and regulatory elements participating are still poorly understood.

              Results

              We report here the development a completely random short-hairpin RNA (shRNA) library coupled with a novel forward genetic screening strategy to identify inhibitors of Toll-like receptor (TLR) dependent proinflammatory responses. We developed a murine macrophage reporter cell line stably transfected with a construct expressing diphtheria toxin-A (DT-A) under the control of the TNF-alpha-promoter. Stimulation of the reporter cell line with the TLR ligand lipopolysaccharide (LPS) resulted in DT-A induced cell death, which could be prevented by the addition of an shRNA targeting the TLR adaptor molecule MyD88. Utilizing this cell line, we screened a completely random lentiviral short hairpin RNA (shRNA) library for sequences that inhibited TLR-mediated TNF-alpha production. Recovery of shRNA sequences from surviving cells led to the identification of unique shRNA sequences that significantly inhibited TLR4-dependent TNF-alpha gene expression. Furthermore, these shRNA sequences specifically blocked TLR2 but not TLR3-dependent TNF-alpha production.

              Conclusions

              Thus, we describe the generation of novel tools to facilitate large-scale forward genetic screens in mammalian cells and the identification of potent shRNA inhibitors of TLR2 and TLR4- dependent proinflammatory responses.
                Cover page: Inhibitors of MyD88-Dependent Proinflammatory Cytokine Production Identified Utilizing a Novel RNA Interference Screening Approach
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2015)
                Treatment of patients with B-NHL with rituximab and CHOP has resulted in significant clinical responses. However, a subset of patients develops resistance to further treatments. The mechanism of unresponsiveness in vivo is not known. We have reported the development of rituximab-resistant clones derived from B-NHL cell lines as models to investigate the mechanism of resistance. The resistant clones exhibit hyper-activated survival/anti-apoptotic pathways and no longer respond to a combination of rituximab and drugs. Recent studies reported the therapeutic efficacy in mice bearing B-cell lymphoma xenografts following treatment with the anti-CD20-hIFNα fusion protein. We hypothesized that the fusion protein may bypass rituximab resistance and inhibit survival signaling pathways. Treatment of the rituximab-resistant clones with anti-CD20-hIFNα, but not with rituximab, IFNα, or rituximab+IFNα resulted in significant inhibition of cell proliferation and induction of cell death. Treatment with anti-CD20-hIFNα sensitized the cells to apoptosis by CDDP, doxorubicin and Treanda. Treatment with anti-CD20-hIFNα inhibited the NF-κB and p38 MAPK activities and induced the activation of PKC-δ and Stat-1. These effects were corroborated by the use of the inhibitors SB203580 (p38 MAPK) and Rottlerin (PKC-δ). Treatment with SB203580 enhanced the sensitization of the resistant clone by anti-CD20-hIFNα to CDDP apoptosis. In contrast, treatment with Rotterin inhibited significantly the sensitization induced by anti-CD20-hIFNα. Overall, the findings demonstrate that treatment with anti-CD20-hIFNα reverses resistance of B-NHL. These findings suggest the potential application of anti-CD20-hIFNα in combination with drugs in patients unresponsive to rituximab-containing regimens.
                  Cover page: Overcoming rituximab drug-resistance by the genetically engineered anti-CD20-hIFN-α fusion protein: Direct cytotoxicity and synergy with chemotherapy
                  • Article
                  • Peer Reviewed
                  UCLA Previously Published Works (2013)
                  Abstract Background Chimeric Antigen Receptors (CARs) consist of the antigen-recognition portion of a monoclonal antibody fused to an intracellular signaling domain capable of activating T-cells. CARs displayed on the surface of transduced cells perform non-MHC-restricted antigen recognition and activating intracellular signaling pathways for induction of target cytolysis, cytokine secretion and proliferation. Clinical trials are in progress assessing the use of mature T-lymphocytes transduced with CARs targeting CD19 antigen to treat B-lineage malignancies. CD19 is an attractive target for immunotherapy because of its consistent and specific expression in most of the stages of maturation and malignancies of B-lymphocyte origin, but not on hematopoietic stem cells. Antibodies against the extracellular domain of the CAR molecule (anti-Fab, Fc or idiotype) have been used for detection of CAR expression in research and clinical samples by flow cytometry, but may need development for each construct and present significant background in samples from xenograft models. Methods A specific reagent for the detection of anti-CD19 CAR expression was developed, a fusion protein consisting of human CD19 extracellular domains and the Fc region of human IgG1 (CD19sIg). Genes encoding CD19sIg fusion proteins were constructed by fusing either exons 1 to 3 (CD19sIg1-3) or exons 1 to 4 (CD19sIg1-4) of the human CD19 cDNA to a human IgG1Fc fragment. These fusion proteins are intended to work in similar fashion as the MHC Tetramers used for identification of antigen-specific T-cells, and may also have other applications in studies of activation of anti-CD19 CAR bearing cells. The CD19sIg proteins were produced from 293 T cells by stable lentiviral vector transduction and purification from culture medium. Results ELISA assays using several different monoclonal antibodies to CD19 demonstrated dose-related specific binding by the fusion molecule CD19sIg1-4, but no binding by CD19sIg1-3. Conjugation of the CD19sIg1-4 fusion protein to Alexa Fluor 488 allowed specific and sensitive staining of anti-CD19 CAR-bearing cells for flow cytometry assays, detecting as low as 0.5% of CAR-modified primary cells with minimal background staining. Conclusions This fusion molecule is a sensitive reagent for detection of anti-CD19 CAR derived from any monoclonal antibody present in CAR-modified T-cells.
                    Cover page: A CD19/Fc fusion protein for detection of anti-CD19 chimeric antigen receptors
                    • Article
                    • Peer Reviewed
                    UCLA Previously Published Works (2013)

                    Chimeric Antigen Receptors (CARs) consist of the antigen-recognition portion of a monoclonal antibody fused to an intracellular signaling domain capable of activating T-cells. CARs displayed on the surface of transduced cells perform non-MHC-restricted antigen recognition and activating intracellular signaling pathways for induction of target cytolysis, cytokine secretion and proliferation. Clinical trials are in progress assessing the use of mature T-lymphocytes transduced with CARs targeting CD19 antigen to treat B-lineage malignancies. CD19 is an attractive target for immunotherapy because of its consistent and specific expression in most of the stages of maturation and malignancies of B-lymphocyte origin, but not on hematopoietic stem cells. Antibodies against the extracellular domain of the CAR molecule (anti-Fab, Fc or idiotype) have been used for detection of CAR expression in research and clinical samples by flow cytometry, but may need development for each construct and present significant background in samples from xenograft models.

                    A specific reagent for the detection of anti-CD19 CAR expression was developed, a fusion protein consisting of human CD19 extracellular domains and the Fc region of human IgG1 (CD19sIg). Genes encoding CD19sIg fusion proteins were constructed by fusing either exons 1 to 3 (CD19sIg1-3) or exons 1 to 4 (CD19sIg1-4) of the human CD19 cDNA to a human IgG1Fc fragment. These fusion proteins are intended to work in similar fashion as the MHC Tetramers used for identification of antigen-specific T-cells, and may also have other applications in studies of activation of anti-CD19 CAR bearing cells. The CD19sIg proteins were produced from 293 T cells by stable lentiviral vector transduction and purification from culture medium.

                    ELISA assays using several different monoclonal antibodies to CD19 demonstrated dose-related specific binding by the fusion molecule CD19sIg1-4, but no binding by CD19sIg1-3. Conjugation of the CD19sIg1-4 fusion protein to Alexa Fluor 488 allowed specific and sensitive staining of anti-CD19 CAR-bearing cells for flow cytometry assays, detecting as low as 0.5% of CAR-modified primary cells with minimal background staining.

                    This fusion molecule is a sensitive reagent for detection of anti-CD19 CAR derived from any monoclonal antibody present in CAR-modified T-cells.

                      Cover page: A CD19/Fc fusion protein for detection of anti-CD19 chimeric antigen receptors.
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