ABSTRACT
Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Asthma/therapy , Mast Cells/enzymology , Mast Cells/immunology , Tryptases/antagonists & inhibitors , Tryptases/immunology , Adolescent , Allosteric Regulation/immunology , Animals , Cell Line , Female , Humans , Macaca fascicularis , Male , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Mice, SCID , RabbitsABSTRACT
The folding and insertion of integral ß-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect ß-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize a monoclonal antibody that selectively inhibits an essential component of the Escherichia coli ß-barrel assembly machine, BamA. In the absence of complement or other immune factors, the unmodified antibody MAB1 demonstrates bactericidal activity against an E. coli strain with truncated LPS. Direct binding of MAB1 to an extracellular BamA epitope inhibits its ß-barrel folding activity, induces periplasmic stress, disrupts outer membrane integrity, and kills bacteria. Notably, resistance to MAB1-mediated killing reveals a link between outer membrane fluidity and protein folding by BamA in vivo, underscoring the utility of this antibody for studying ß-barrel membrane protein folding within a living cell. Identification of this BamA antagonist highlights the potential for new mechanisms of antibiotics to inhibit Gram-negative bacterial growth by targeting extracellular epitopes.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antibodies, Bacterial/pharmacology , Antibodies, Monoclonal/pharmacology , Bacterial Outer Membrane Proteins/antagonists & inhibitors , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli/drug effects , Membrane Fluidity/drug effects , Bacterial Outer Membrane Proteins/immunology , Bacterial Outer Membrane Proteins/metabolism , Cell Membrane/drug effects , Cell Membrane/immunology , Cell Membrane/metabolism , Escherichia coli/immunology , Escherichia coli/metabolism , Escherichia coli Proteins/immunology , Escherichia coli Proteins/metabolism , Models, Molecular , Protein Conformation , Protein FoldingABSTRACT
Integral ß-barrel membrane proteins are folded and inserted into the Gram-negative bacterial outer membrane by the ß-barrel assembly machine (BAM). This essential complex, composed of a ß-barrel protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE, resides in the outer membrane, a unique asymmetrical lipid bilayer that is difficult to recapitulate in vitro Thus, the probing of BAM function in living cells is critical to fully understand the mechanism of ß-barrel folding. We recently identified an anti-BamA monoclonal antibody, MAB1, that is a specific and potent inhibitor of BamA function. Here, we show that the inhibitory effect of MAB1 is enhanced when BAM function is perturbed by either lowering the level of BamA or removing the nonessential BAM lipoproteins, BamB, BamC, or BamE. The disruption of BAM reduces BamA activity, increases outer membrane (OM) fluidity, and activates the σE stress response, suggesting the OM environment and BAM function are interconnected. Consistent with this idea, an increase in the membrane fluidity through changes in the growth environment or alterations to the lipopolysaccharide in the outer membrane is sufficient to provide resistance to MAB1 and enable the BAM to tolerate these perturbations. Amino acid substitutions in BamA at positions in the outer membrane spanning region or the periplasmic space remote from the extracellular MAB1 binding site also provide resistance to the inhibitory antibody. Our data highlight that the outer membrane environment is a critical determinant in the efficient and productive folding of ß-barrel membrane proteins by BamA.IMPORTANCE BamA is an essential component of the ß-barrel assembly machine (BAM) in the outer membranes of Gram-negative bacteria. We have used a recently described inhibitory anti-BamA antibody, MAB1, to identify the molecular requirements for BAM function. Resistance to this antibody can be achieved through changes to the outer membrane or by amino acid substitutions in BamA that allosterically affect the response to MAB1. Sensitivity to MAB1 is achieved by perturbing BAM function. By using MAB1 activity and functional assays as proxies for BAM function, we link outer membrane fluidity to BamA activity, demonstrating that an increase in membrane fluidity sensitizes the cells to BAM perturbations. Thus, the search for potential inhibitors of BamA function must consider the membrane environment in which ß-barrel folding occurs.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Lipoproteins/metabolism , Membrane Fluidity , Protein Folding , Protein Multimerization , Amino Acid Substitution , Bacterial Outer Membrane Proteins/genetics , DNA Mutational Analysis , Escherichia coli/genetics , Escherichia coli Proteins/geneticsABSTRACT
The proto-oncogenes ETV1, ETV4 and ETV5 encode transcription factors in the E26 transformation-specific (ETS) family, which includes the most frequently rearranged and overexpressed genes in prostate cancer. Despite being critical regulators of development, little is known about their post-translational regulation. Here we identify the ubiquitin ligase COP1 (also known as RFWD2) as a tumour suppressor that negatively regulates ETV1, ETV4 and ETV5. ETV1, which is mutated in prostate cancer more often, was degraded after being ubiquitinated by COP1. Truncated ETV1 encoded by prostate cancer translocation TMPRSS2:ETV1 lacks the critical COP1 binding motifs and was 50-fold more stable than wild-type ETV1. Almost all patient translocations render ETV1 insensitive to COP1, implying that this confers a selective advantage to prostate epithelial cells. Indeed, COP1 deficiency in mouse prostate elevated ETV1 and produced increased cell proliferation, hyperplasia, and early prostate intraepithelial neoplasia. Combined loss of COP1 and PTEN enhanced the invasiveness of mouse prostate adenocarcinomas. Finally, rare human prostate cancer samples showed hemizygous loss of the COP1 gene, loss of COP1 protein, and elevated ETV1 protein while lacking a translocation event. These findings identify COP1 as a tumour suppressor whose downregulation promotes prostatic epithelial cell proliferation and tumorigenesis.
Subject(s)
Nuclear Proteins/metabolism , Proto-Oncogene Proteins c-ets/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Amino Acid Motifs , Animals , Carrier Proteins/metabolism , Cell Line , Cell Line, Tumor , Cell Proliferation , Cell Transformation, Neoplastic , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Male , Mice , Nuclear Proteins/deficiency , PTEN Phosphohydrolase/deficiency , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Protein Binding , Transcription Factors/genetics , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/genetics , UbiquitinationABSTRACT
Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2-1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV) glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta) to fetal infection (fetus and amniotic fluid). Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans.
Subject(s)
Antibodies, Monoclonal, Murine-Derived/pharmacology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , Cytomegalovirus Infections/congenital , Cytomegalovirus Infections/drug therapy , Cytomegalovirus/immunology , Animals , Antibodies, Monoclonal, Murine-Derived/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Cytomegalovirus Infections/immunology , Cytomegalovirus Infections/pathology , Disease Models, Animal , Guinea Pigs , HEK293 Cells , HumansABSTRACT
Early success with brentuximab vedotin in treating classical Hodgkin lymphoma spurred an influx of at least 20 monomethyl auristatin E (MMAE) antibody-drug conjugates (ADCs) into clinical trials. While three MMAE-ADCs have been approved, most of these conjugates are no longer being investigated in clinical trials. Some auristatin conjugates show limited or no efficacy at tolerated doses, but even for drugs driving initial remissions, tumor regrowth and metastasis often rapidly occur. Here we describe the development of second-generation therapeutic ADCs targeting Lymphocyte antigen 6E (Ly6E) where the tubulin polymerization inhibitor MMAE (Compound 1) is replaced with DNA-damaging agents intended to drive increased durability of response. Comparison of a seco-cyclopropyl benzoindol-4-one (CBI)-dimer (compound 2) to MMAE showed increased potency, activity across more cell lines, and resistance to efflux by P-glycoprotein, a drug transporter commonly upregulated in tumors. Both anti-Ly6E-CBI and -MMAE conjugates drove single-dose efficacy in xenograft and patient-derived xenograft models, but seco-CBI-dimer conjugates showed reduced tumor outgrowth following multiple weeks of treatment, suggesting that they are less susceptible to developing resistance. In parallel, we explored approaches to optimize the targeting antibody. In contrast to immunization with recombinant Ly6E or Ly6E DNA, immunization with virus-like particles generated a high-affinity anti-Ly6E antibody. Conjugates to this antibody improve efficacy versus a previous clinical candidate both in vitro and in vivo with multiple cytotoxics. Conjugation of compound 2 to the second-generation antibody results in a substantially improved ADC with promising preclinical efficacy.
Subject(s)
Antibodies, Monoclonal/immunology , Antigens, Surface/immunology , Antineoplastic Agents/immunology , Immunoconjugates/immunology , Oligopeptides/immunology , Xenograft Model Antitumor Assays/methods , Animals , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Antineoplastic Agents, Immunological/pharmacokinetics , Antineoplastic Agents, Immunological/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/immunology , Female , GPI-Linked Proteins/immunology , HEK293 Cells , Humans , Immunoconjugates/pharmacokinetics , Immunoconjugates/pharmacology , Mice, SCID , Rats, Sprague-Dawley , Tumor Burden/drug effects , Tumor Burden/immunologyABSTRACT
Aim: Tryptase is a tetrameric trypsin-like serine protease contained within the secretory granules of mast cells and is an important mediator of allergic inflammatory responses in respiratory diseases. Detection of active tryptase in the airway may provide important information about asthma and other respiratory diseases. Materials & Methods: An activity based probe has been incorported within an immunoassay to allow for measurement of active tryptase in human tissues. Results: A specific Simoa immunoassay to measure active tryptase in nasosorption samples was developed and qualified using an activity-based probe label and a specific antitryptase capture antibody. Conclusion: The assay was capable of measuring active tryptase in human samples, which will enable evaluation of the role of tryptase proteolytic activity in human disease.
Subject(s)
Immunoassay/methods , Immunologic Tests/methods , Mast Cells/pathology , Tryptases/metabolism , HumansABSTRACT
Human ß-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of allergic inflammatory responses in asthma. Antibodies generally inhibit proteases by blocking substrate access by binding to active sites or exosites or by allosteric modulation. The bivalency of IgG antibodies can increase potency via avidity, but has never been described as essential for activity. Here we report an inhibitory anti-tryptase IgG antibody with a bivalency-driven mechanism of action. Using biochemical and structural data, we determine that four Fabs simultaneously occupy four exosites on the ß-tryptase tetramer, inducing allosteric changes at the small interface. In the presence of heparin, the monovalent Fab shows essentially no inhibition, whereas the bivalent IgG fully inhibits ß-tryptase activity in a hinge-dependent manner. Our results suggest a model where the bivalent IgG acts akin to molecular pliers, pulling the tetramer apart into inactive ß-tryptase monomers, and may provide an alternative strategy for antibody engineering.
Subject(s)
Antibodies, Monoclonal/metabolism , Immunoglobulin G/metabolism , Tryptases/metabolism , Allosteric Regulation/drug effects , Amino Acid Sequence , Heparin/pharmacology , Humans , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin G/chemistry , Models, Molecular , Mutant Proteins/chemistry , Protein Binding/drug effects , Protein Multimerization , Tryptases/chemistryABSTRACT
PURPOSE: The treatment of acute myeloid leukemia (AML) has not significantly changed in 40 years. Cytarabine- and anthracycline-based chemotherapy induction regimens (7 + 3) remain the standard of care, and most patients have poor long-term survival. The reapproval of Mylotarg, an anti-CD33-calicheamicin antibody-drug conjugate (ADC), has demonstrated ADCs as a clinically validated option to enhance the effectiveness of induction therapy. We are interested in developing a next-generation ADC for AML to improve upon the initial success of Mylotarg. EXPERIMENTAL DESIGN: The expression pattern of CLL-1 and its hematopoietic potential were investigated. A novel anti-CLL-1-ADC, with a highly potent pyrrolobenzodiazepine (PBD) dimer conjugated through a self-immolative disulfide linker, was developed. The efficacy and safety profiles of this ADC were evaluated in mouse xenograft models and in cynomolgus monkeys. RESULTS: We demonstrate that CLL-1 shares similar prevalence and trafficking properties that make CD33 an excellent ADC target for AML, but lacks expression on hematopoietic stem cells that hampers current CD33-targeted ADCs. Our anti-CLL-1-ADC is highly effective at depleting tumor cells in AML xenograft models and lacks target independent toxicities at doses that depleted target monocytes and neutrophils in cynomolgus monkeys. CONCLUSIONS: Collectively, our data suggest that an anti-CLL-1-ADC has the potential to become an effective and safer treatment for AML in humans, by reducing and allowing for faster recovery from initial cytopenias than the current generation of ADCs for AML.
Subject(s)
Antibodies, Anti-Idiotypic/pharmacology , Immunoconjugates/pharmacology , Lectins, C-Type/immunology , Leukemia, Myeloid, Acute/drug therapy , Receptors, Mitogen/immunology , Animals , Flow Cytometry , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lectins, C-Type/antagonists & inhibitors , Lectins, C-Type/genetics , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/pathology , Mice , Receptors, Mitogen/antagonists & inhibitors , Receptors, Mitogen/genetics , Sialic Acid Binding Ig-like Lectin 3/genetics , Sialic Acid Binding Ig-like Lectin 3/immunology , Xenograft Model Antitumor AssaysABSTRACT
Outer membrane proteins (OMPs) in Gram-negative bacteria dictate permeability of metabolites, antibiotics, and toxins. Elucidating the structure-function relationships governing OMPs within native membrane environments remains challenging. We constructed a diverse library of >3000 monoclonal antibodies to assess the roles of extracellular loops (ECLs) in LptD, an essential OMP that inserts lipopolysaccharide into the outer membrane of Escherichia coli. Epitope binning and mapping experiments with LptD-loop-deletion mutants demonstrated that 7 of the 13 ECLs are targeted by antibodies. Only ECLs inaccessible to antibodies were required for the structure or function of LptD. Our results suggest that antibody-accessible loops evolved to protect key extracellular regions of LptD, but are themselves dispensable. Supporting this hypothesis, no α-LptD antibody interfered with essential functions of LptD. Our experimental workflow enables structure-function studies of OMPs in native cellular environments, provides unexpected insight into LptD, and presents a method to assess the therapeutic potential of antibody targeting.
Subject(s)
Antibodies, Monoclonal/metabolism , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Animals , Anti-Bacterial Agents/pharmacology , Binding Sites , Epitope Mapping , Epitopes/metabolism , Escherichia coli/growth & development , Escherichia coli/metabolism , Mice, Inbred BALB C , Protein Structure, Secondary , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
Monoclonal antibodies (mAbs) have enabled numerous basic research discoveries and therapeutic approaches for many protein classes. However, there still exist a number of target classes, such as multi-pass membrane proteins, for which antibody discovery is difficult, due in part to lack of high quality, recombinant protein. Here we describe the impact of several parameters on antigen expression and the development of mAbs against human claudin 4 (CLDN4), a potential multi-indication cancer target. Using gene gun-based DNA delivery and bioluminescence imaging, we optimize promoter type by comparing expression profiles of four robust in vivo promoters. In addition, we observe that most vectors rapidly lose expression, ultimately reaching almost background levels by three days post-delivery. Recognizing this limitation, we next explored skin pretreatment strategies as an orthogonal method to further boost the efficiency of mAb generation. We show that SDS pretreatment can boost antigen expression, but fails to significantly increase mAb discovery efficiency. In contrast, we find that sandpaper pretreatment yields 5-fold more FACS+ anti-CLDN4 hybridomas, without impacting antigen expression. Our findings coupled with other strategies to improve DNA immunizations should improve the success of mAb discovery against other challenging targets and enable the generation of critical research tools and therapeutic candidates.
Subject(s)
Antibodies, Viral/immunology , Antigens, Viral/genetics , Biolistics/methods , Immunization/methods , Promoter Regions, Genetic/genetics , Skin , Animals , Antibodies, Monoclonal/immunology , Antigens, Viral/immunology , Claudin-4/immunology , Gene Expression , HEK293 Cells , Humans , MiceABSTRACT
A primary barrier to the success of T cell-recruiting bispecific antibodies in the treatment of solid tumors is the lack of tumor-specific targets, resulting in on-target off-tumor adverse effects from T cell autoreactivity to target-expressing organs. To overcome this, we developed an anti-HER2/CD3 T cell-dependent bispecific (TDB) antibody that selectively targets HER2-overexpressing tumor cells with high potency, while sparing cells that express low amounts of HER2 found in normal human tissues. Selectivity is based on the avidity of two low-affinity anti-HER2 Fab arms to high target density on HER2-overexpressing cells. The increased selectivity to HER2-overexpressing cells is expected to mitigate the risk of adverse effects and increase the therapeutic index. Results included in this manuscript not only support the clinical development of anti-HER2/CD3 1Fab-immunoglobulin G TDB but also introduce a potentially widely applicable strategy for other T cell-directed therapies. The potential of this discovery has broad applications to further enable consideration of solid tumor targets that were previously limited by on-target, but off-tumor, autoimmunity.
Subject(s)
Antibody Affinity/immunology , CD3 Complex/immunology , Cytotoxicity, Immunologic , Receptor, ErbB-2/immunology , Antibodies, Bispecific/immunology , Cell Line, Tumor , Humans , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin G/metabolism , Lymphocyte Activation/immunology , Protein BindingABSTRACT
Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP ß-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a "targeted boost-and-sort" strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of ß-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins.
Subject(s)
Antibodies, Monoclonal/immunology , Bacterial Outer Membrane Proteins/genetics , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/isolation & purification , Bacterial Outer Membrane Proteins/immunology , Escherichia coli/immunology , Escherichia coli Proteins/immunology , Immunization , Protein Conformation , Protein Folding , Protein Transport/genetics , Protein Transport/immunology , VaccinationABSTRACT
Cancer stem cells (CSCs) are hypothesized to actively maintain tumors similarly to how their normal counterparts replenish differentiated cell types within tissues, making them an attractive therapeutic target for the treatment of cancer. Because most CSC markers also label normal tissue stem cells, it is unclear how to selectively target them without compromising normal tissue homeostasis. We evaluated a strategy that targets the cell surface leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a well-characterized tissue stem cell and CSC marker, with an antibody conjugated to distinct cytotoxic drugs. One antibody-drug conjugate (ADC) demonstrated potent tumor efficacy and safety in vivo. Furthermore, the ADC decreased tumor size and proliferation, translating to improved survival in a genetically engineered model of intestinal tumorigenesis. These data demonstrate that ADCs can be leveraged to exploit differences between normal and cancer stem cells to successfully target gastrointestinal cancers.
Subject(s)
Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Colonic Neoplasms/drug therapy , Immunotoxins/pharmacology , Neoplastic Stem Cells/drug effects , Receptors, G-Protein-Coupled/immunology , Animals , Antineoplastic Agents/immunology , Cell Line, Tumor , Cell Proliferation/drug effects , Colonic Neoplasms/genetics , Colonic Neoplasms/immunology , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Dose-Response Relationship, Drug , Feasibility Studies , Female , Gene Expression Regulation, Neoplastic , Genes, APC , Immunotoxins/immunology , Immunotoxins/metabolism , Inhibitory Concentration 50 , Male , Mice, Inbred C57BL , Mice, Nude , Mice, Transgenic , Neoplastic Stem Cells/immunology , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Rats, Sprague-Dawley , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Time Factors , Xenograft Model Antitumor AssaysABSTRACT
Multi-transmembrane proteins are especially difficult targets for antibody generation largely due to the challenge of producing a protein that maintains its native conformation in the absence of a stabilizing membrane. Here, we describe an immunization strategy that successfully resulted in the identification of monoclonal antibodies that bind specifically to extracellular epitopes of a 12 transmembrane protein, multi-drug resistant protein 4 (MRP4). These monoclonal antibodies were developed following hydrodynamic tail vein immunization with a cytomegalovirus (CMV) promoter-based plasmid expressing MRP4 cDNA and were characterized by flow cytometry. As expected, the use of the immune modulators fetal liver tyrosine kinase 3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor positively enhanced the immune response against MRP4. Imaging studies using CMV-based plasmids expressing luciferase showed that the in vivo half-life of the target antigen was less than 48 h using CMV-based plasmids, thus necessitating frequent boosting with DNA to achieve an adequate immune response. We also describe a comparison of plasmids, which contained MRP4 cDNA with either the CMV or CAG promoters, used for immunizations. The observed luciferase activity in this comparison demonstrated that the CAG promoter-containing plasmid pCAGGS induced prolonged constitutive expression of MRP4 and an increased anti-MRP4 specific immune response even when the plasmid was injected less frequently. The method described here is one that can be broadly applicable as a general immunization strategy to develop antibodies against multi-transmembrane proteins, as well as target antigens that are difficult to express or purify in native and functionally active conformation.
Subject(s)
Antibodies/immunology , Immunization , Multidrug Resistance-Associated Proteins/immunology , Plasmids , Vaccines, DNA , Animals , Cell Line , DNA, Complementary/immunology , DNA, Complementary/pharmacology , Humans , Mice, Inbred BALB C , Mice, Knockout , Multidrug Resistance-Associated Proteins/biosynthesis , Multidrug Resistance-Associated Proteins/genetics , Plasmids/immunology , Plasmids/pharmacology , Protein Structure, Secondary , Vaccines, DNA/immunology , Vaccines, DNA/pharmacologyABSTRACT
Hepsin, a type II transmembrane serine protease, is strongly up-regulated in prostate cancer. Hepsin overexpression in a mouse prostate cancer model resulted in tumor progression and metastasis, associated with basement membrane disorganization. We investigated whether hepsin enzymatic activity was linked to the basement membrane defects by examining its ability to initiate the plasminogen/plasmin proteolytic pathway. Because plasminogen is not processed by hepsin, we investigated the upstream activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator. Enzymatic assays with a recombinant soluble form of hepsin demonstrated that hepsin did not cleave pro-tissue-type plasminogen activator but efficiently converted pro-uPA into high molecular weight uPA by cleavage at the Lys158-Ile159 (P1-P1') peptide bond. uPA generated by hepsin displayed enzymatic activity toward small synthetic and macromolecular substrates indistinguishable from uPA produced by plasmin. The catalytic efficiency of pro-uPA activation by hepsin (kcat/Km 4.8 x 10(5) m(-1) s(-1)) was similar to that of plasmin, which is considered the most potent pro-uPA activator and was about 6-fold higher than that of matriptase. Conversion of pro-uPA was also demonstrated with cell surface-expressed full-length hepsin. A stable hepsinoverexpressing LnCaP cell line converted pro-uPA into high molecular weight uPA at a rate of 6.6 +/- 1.9 nm uPA h(-1), which was about 3-fold higher than LnCaP cells expressing lower hepsin levels on their surface. In conclusion, the ability of hepsin to efficiently activate pro-uPA suggests that it may initiate plasmin-mediated proteolytic pathways at the tumor/stroma interface that lead to basement membrane disruption and tumor progression.