ABSTRACT
In human celiac disease (CeD) HLA-DQ2.5 presents gluten peptides to antigen-specific CD4+ T cells, thereby instigating immune activation and enteropathy. Targeting HLA-DQ2.5 with neutralizing antibody for treating CeD may be plausible, yet using pan-HLA-DQ antibody risks affecting systemic immunity, while targeting selected gluten peptide:HLA-DQ2.5 complex (pHLA-DQ2.5) may be insufficient. Here we generate a TCR-like, neutralizing antibody (DONQ52) that broadly recognizes more than twenty-five distinct gluten pHLA-DQ2.5 through rabbit immunization with multi-epitope gluten pHLA-DQ2.5 and multidimensional optimization. Structural analyses show that the proline-rich and glutamine-rich motif of gluten epitopes critical for pathogenesis is flexibly recognized by multiple tyrosine residues present in the antibody paratope, implicating the mechanisms for the broad reactivity. In HLA-DQ2.5 transgenic mice, DONQ52 demonstrates favorable pharmacokinetics with high subcutaneous bioavailability, and blocks immunity to gluten while not affecting systemic immunity. Our results thus provide a rationale for clinical testing of DONQ52 in CeD.
Subject(s)
Celiac Disease , Glutens , Mice , Animals , Humans , Rabbits , Glutens/chemistry , Antibodies, Neutralizing , HLA-DQ Antigens , Peptides/chemistry , Epitopes/chemistry , Mice, TransgenicABSTRACT
To combat infectious diseases, vaccines are considered the best prophylactic strategy for a wide range of the population, but even when vaccines are effective, the administration of therapeutic antibodies against viruses could provide further treatment options, particularly for vulnerable groups whose immunity against the viruses is compromised. Therapeutic antibodies against dengue are ideally engineered to abrogate binding to Fcγ receptors (FcγRs), which can induce antibody-dependent enhancement (ADE). However, the Fc effector functions of neutralizing antibodies against SARS-CoV-2 have recently been reported to improve post-exposure therapy, while they are dispensable when administered as prophylaxis. Hence, in this report, we investigated the influence of Fc engineering on anti-virus efficacy using the anti-dengue/Zika human antibody SIgN-3C and found it affected the viremia clearance efficacy against dengue in a mouse model. Furthermore, we demonstrated that complement activation through antibody binding to C1q could play a role in anti-dengue efficacy. We also generated a novel Fc variant, which displayed the ability for complement activation but showed very low FcγR binding and an undetectable level of the risk of ADE in a cell-based assay. This Fc engineering approach could make effective and safe anti-virus antibodies against dengue, Zika and other viruses.
ABSTRACT
A conventional antibody targeting a soluble antigen in circulation typically requires a huge dosage and frequent intravenous administration to neutralize the antigen. This is because antigen degradation is reduced by the formation of antigen-antibody immune complexes, which escape from lysosomal degradation using neonatal Fc receptor (FcRn)-mediated recycling. To address this, we developed an antigen-sweeping antibody that combines pH-dependent antigen binding and Fc engineering to enhance Fc receptor binding. The sweeping antibody actively eliminates the plasma antigens by increasing the cellular uptake of the immune complex and dissociating the antigens in the acidic endosome for degradation. Strong antigen sweeping can reduce the dosage, potentially achieve higher efficacy, and expand the scope of antigen space available for targeting by antibodies. In this study, to further improve the sweeping efficacy, we developed a novel antibody Fc variant by enhancing Fcγ receptor IIb (FcγRIIb) binding and modulating charge characteristics for increased cellular uptake of the immune complex, together with enhancing FcRn binding for efficient salvage of the antigen-free antibodies. Our Fc variant achieved strong antigen sweeping in cynomolgus monkeys with antibody pharmacokinetics comparable to a wild-type human IgG1 antibody. The positive-charge substitutions enhanced uptake of the immune complex by FcγRIIb-expressing cells in vitro, which was completely inhibited by an anti-FcγRIIb antibody. This suggests that the strong in vivo sweeping efficacy improved by the charge engineering is more likely achieved by FcγRIIb-dependent uptake of the immune complex rather than nonspecific uptake. We expect this novel Fc engineering can maximize the antigen sweeping efficacy even in humans and create novel therapeutic antibodies that meet unmet medical needs for patients.
Subject(s)
Antigen-Antibody Complex , Antigens , Animals , Humans , Hydrogen-Ion Concentration , Immunoglobulin Fc Fragments , Macaca fascicularisABSTRACT
Myostatin, a member of the transforming growth factor-ß superfamily, is an attractive target for muscle disease therapy because of its role as a negative regulator of muscle growth and strength. Here, we describe a novel antibody therapeutic approach that maximizes the potential of myostatin-targeted therapy. We generated an antibody, GYM329, that specifically binds the latent form of myostatin and inhibits its activation. Additionally, via "sweeping antibody technology", GYM329 reduces or "sweeps" myostatin in the muscle and plasma. Compared with conventional anti-myostatin agents, GYM329 and its surrogate antibody exhibit superior muscle strength-improvement effects in three different mouse disease models. We also demonstrate that the superior efficacy of GYM329 is due to its myostatin specificity and sweeping capability. Furthermore, we show that a GYM329 surrogate increases muscle mass in normal cynomolgus monkeys without any obvious toxicity. Our findings indicate the potential of GYM329 to improve muscle strength in patients with muscular disorders.
Subject(s)
Antibodies, Monoclonal/pharmacology , Muscle Strength/drug effects , Muscular Diseases/physiopathology , Myostatin/immunology , Animals , Bone Morphogenetic Proteins/metabolism , Disease Models, Animal , Female , Growth Differentiation Factors/metabolism , Macaca fascicularis , Male , Mice, Inbred C57BL , Muscle, Skeletal/pathology , Muscle, Skeletal/physiopathology , Muscular Atrophy/pathology , Muscular Atrophy/physiopathology , Organ Size , Signal TransductionABSTRACT
BACKGROUND: The complement system usually helps protect against microbial infection, but it could also be involved in the onset of various diseases. Inhibition of complement component 5 (C5) with eculizumab has resulted in a significant reduction of hemolysis, reduction of thromboembolic events, and increased survival in patients with Paroxysmal Nocturnal Hemoglobinuria (PNH). However, eculizumab requires frequent intravenous infusions due to the abundance of C5 in plasma and some patients may still experience breakthrough hemolysis. This review introduces the recent body of knowledge on recycling technology and discusses the likely therapeutic benefits of SKY59, a novel recycling antibody, for PNH and complement-mediated disorders. METHODS: By using recycling technology, we created a novel anti-C5 antibody, SKY59, capable of binding to C5 pH-dependently. RESULTS: In cynomolgus monkeys, SKY59 robustly inhibited C5 and complement activity for significantly longer than a conventional antibody. SKY59 also showed an inhibitory effect on C5 variant p.Arg885His, whereas eculizumab does not suppress complement activity in patients with this type of mutation. CONCLUSION: SKY59 is a promising anti-C5 biologic agent that has significant advantages over current therapies such as long duration of action and efficacy against C5 variants.
Subject(s)
Antibodies/immunology , Complement C5 , Hemoglobinuria, Paroxysmal , Hemolysis , HumansABSTRACT
The complex molecular formats of recent therapeutic antibodies, including bispecific antibodies, antibody fragments, and other fusion proteins, makes the task of purifying the desired molecules in a limited number of purification steps more and more challenging. Manufacturing these complicated biologics can be substantially improved in the affinity capture stage if the simple bind-and-elute mode is accompanied by targeted removal of the impurities, such as mis-paired antibodies and oligomers or aggregates. Here, we report a method, based on the binding valency to Protein L resin, of separating proteins during the elution step by simply controlling the conductivity at low pH. We show that the method efficiently separated targeted antibodies from mis-paired and aggregated species. Notably, the number of Protein L binding sites can be built into the molecule by design to facilitate the purification. This method may be useful for purifying various antibody formats at laboratory and manufacturing scales.
Subject(s)
Antibodies, Bispecific/isolation & purification , Antibodies, Monoclonal/isolation & purification , Bacterial Proteins/metabolism , Chromatography, Affinity/methods , Single-Chain Antibodies/isolation & purification , Antibodies, Bispecific/metabolism , Antibodies, Monoclonal/metabolism , CD3 Complex/immunology , Electric Conductivity , HLA-A2 Antigen/immunology , Humans , Hydrogen-Ion Concentration , Ion Exchange Resins , Protein Binding , Protein Engineering , Receptor, ErbB-2/immunology , Single-Chain Antibodies/metabolismABSTRACT
The antibody drug market is rapidly expanding, and various antibody engineering technologies are being developed to create antibodies that can provide better benefit to patients. Although bispecific antibody drugs have been researched for more than 30â¯years, currently only a limited number of bispecific antibodies have achieved regulatory approval. Of the few successful examples of industrially manufacturing a bispecific antibody, the "common light chain format" is an elegant technology that simplifies the purification of a whole IgG-type bispecific antibody. Using this IgG format, the bispecific function can be introduced while maintaining the natural molecular shape of the antibody. In this article, we will first introduce the outline, prospects, and limitations of the common light chain format. Then, we will describe the identification and optimization process for ERY974, an anti-glypican-3â¯×â¯anti-CD3ε T cell-redirecting bispecific antibody with a common light chain. This format includes one of Chugai's proprietary technologies, termed ART-Ig technology, which consists of a method to identify a common light chain, isoelectric point (pI) engineering to purify the desired bispecific IgG antibody from byproducts, and Fc heterodimerization by an electrostatic steering effect. Furthermore, we describe some tips for de-risking the antibody when engineering a T cell redirecting antibody.
Subject(s)
Antibodies, Bispecific , Immunoglobulin G , Immunoglobulin Light Chains , Protein Engineering/methods , Animals , CD3 Complex/immunology , Glypicans/immunology , Humans , MiceABSTRACT
Modulating the complement system is a promising strategy in drug discovery for disorders with uncontrolled complement activation. Although some of these disorders can be effectively treated with an antibody that inhibits complement C5, the high plasma concentration of C5 requires a huge dosage and frequent intravenous administration. Moreover, a conventional anti-C5 antibody can cause C5 to accumulate in plasma by reducing C5 clearance when C5 forms an immune complex (IC) with the antibody, which can be salvaged from endosomal vesicles by neonatal Fc receptor (FcRn)-mediated recycling. In order to neutralize the increased C5, an even higher dosage of the antibody would be required. This antigen accumulation can be suppressed by giving the antibody a pH-dependent C5-binding property so that C5 is released from the antibody in the acidic endosome and then trafficked to the lysosome for degradation, while the C5-free antibody returns back to plasma. We recently demonstrated that a pH-dependent C5-binding antibody, SKY59, exhibited long-lasting neutralization of C5 in cynomolgus monkeys, showing potential for subcutaneous delivery or less frequent administration. Here we report the details of the antibody engineering involved in generating SKY59, from humanizing a rabbit antibody to improving the C5-binding property. Moreover, because the pH-dependent C5-binding antibodies that we first generated still accumulated C5, we hypothesized that the surface charges of the ICs partially contributed to a slow uptake rate of the C5-antibody ICs. This idea motivated us to engineer the surface charges of the antibody. Our surface-charge engineered antibody consequently exhibited a high capacity to sweep C5 and suppressed the C5 accumulation in vivo by accelerating the cycle of sweeping: uptake of ICs into cells, release of C5 from the antibody in endosomes, and salvage of the antigen-free antibody. Thus, our engineered anti-C5 antibody, SKY59, is expected to provide significant benefits for patients with complement-mediated disorders.
Subject(s)
Antibodies, Monoclonal/genetics , Complement Activation/drug effects , Complement C5/antagonists & inhibitors , Protein Engineering/methods , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibody Affinity , Complement Activation/immunology , Complement C5/immunology , Complement C5/isolation & purification , Computer Simulation , Drug Discovery/methods , Endosomes/immunology , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Humans , Hydrogen-Ion Concentration , Immune System Diseases/drug therapy , Immune System Diseases/immunology , Macaca fascicularis , Mice , Mice, Transgenic , Mutagenesis , Receptors, Fc/genetics , Receptors, Fc/immunology , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Time FactorsABSTRACT
This corrects the article DOI: 10.1038/srep45839.
ABSTRACT
Cancer care is being revolutionized by immunotherapies such as immune checkpoint inhibitors, engineered T cell transfer, and cell vaccines. The bispecific T cell-redirecting antibody (TRAB) is one such promising immunotherapy, which can redirect T cells to tumor cells by engaging CD3 on a T cell and an antigen on a tumor cell. Because T cells can be redirected to tumor cells regardless of the specificity of T cell receptors, TRAB is considered efficacious for less immunogenic tumors lacking enough neoantigens. Its clinical efficacy has been exemplified by blinatumomab, a bispecific T cell engager targeting CD19 and CD3, which has shown marked clinical responses against hematological malignancies. However, the success of TRAB in solid tumors has been hampered by the lack of a target molecule with sufficient tumor selectivity to avoid "on-target off-tumor" toxicity. Glypican 3 (GPC3) is a highly tumor-specific antigen that is expressed during fetal development but is strictly suppressed in normal adult tissues. We developed ERY974, a whole humanized immunoglobulin G-structured TRAB harboring a common light chain, which bispecifically binds to GPC3 and CD3. Using a mouse model with reconstituted human immune cells, we revealed that ERY974 is highly effective in killing various types of tumors that have GPC3 expression comparable to that in clinical tumors. ERY974 also induced a robust antitumor efficacy even against tumors with nonimmunogenic features, which are difficult to treat by inhibiting immune checkpoints such as PD-1 (programmed cell death protein-1) and CTLA-4 (cytotoxic T lymphocyte-associated protein-4). Immune monitoring revealed that ERY974 converted the poorly inflamed tumor microenvironment to a highly inflamed microenvironment. Toxicology studies in cynomolgus monkeys showed transient cytokine elevation, but this was manageable and reversible. No organ toxicity was evident. These data provide a rationale for clinical testing of ERY974 for the treatment of patients with GPC3-positive solid tumors.
Subject(s)
Antibodies, Bispecific/therapeutic use , Glypicans/immunology , Neoplasms/immunology , Neoplasms/pathology , T-Lymphocytes/immunology , Animals , Antibodies, Bispecific/administration & dosage , Antibodies, Bispecific/pharmacokinetics , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , CD3 Complex/metabolism , Cytokines/metabolism , Humans , Immunocompetence/drug effects , Injections, Intravenous , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Macaca fascicularis , Mice, Transgenic , Steroids/pharmacology , Steroids/therapeutic use , T-Lymphocytes/drug effectsABSTRACT
Prediction of the plasma/serum mAb concentration-time profile in human is important to determine the required dose regime. This study proposes an approach for predicting the plasma/serum mAb concentration-time profile after intravenous and subcutaneous injection in human based on comprehensive analysis of reported pharmacokinetic data. Optimal scaling exponents from cynomolgus monkey to human for CL, Q, Vc, and Vp were estimated as 0.8, 0.75, 1.0, and 0.95, respectively. The estimated exponents were used to predict plasma/serum mAb concentration-time profile in human from pharmacokinetic data in cynomolgus monkey, and the results had reasonable accuracy with symmetric variability of prediction. Then, data reported for pharmacokinetics in human were used to estimate optimal ka and F after subcutaneous injection. The geometric mean of ka was suitable to predict Tmax, and F which was estimated from CL was suitable to predict Cmax. Our approach is useful for predicting the plasma/serum mAb concentration-time profile after intravenous and subcutaneous injection in human. Moreover, the study also investigated the possibility of predicting pharmacokinetic parameters of mAbs with increased FcRn binding mutations in human and found that our approach of prediction based on reported pharmacokinetic data may also be applicable to mAbs with these mutations.
Subject(s)
Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/blood , Humans , Injections, Intravenous , Injections, Subcutaneous , Receptors, Fc/genetics , Receptors, Fc/metabolismABSTRACT
T cell-mediated immunotherapy is an attractive strategy for treatment in various disease areas. In this therapeutic approach, the CD3 complex is one of the key molecules to modulate T cell functions; however, in many cases, we cannot evaluate the drug candidates in animal experiments because the therapeutics, usually monoclonal antibodies specific to human CD3, cannot react to mouse endogenous Cd3. Although immunodeficient mice transfused with human hematopoietic stem or precursor cells, known as humanized mice, are available for these studies, mice humanized in this manner are not completely immune competent. In this study we have succeeded in establishing a novel mouse strain in which all the three components of the Cd3 complex - Cd3ε, Cd3δ, and Cd3γ - are replaced by their human counterparts, CD3E, CD3D, and CD3G. Basic immunological assessments have confirmed that this strain of human CD3 EDG-replaced mice are entirely immune competent, and we have also demonstrated that a bispecific antibody that simultaneously binds to human CD3 and a tumor-associated antigen (e.g. ERBB2 or GPC3) can be evaluated in human CD3 EDG-replaced mice engrafted with tumors. Our mouse model provides a novel means to evaluate the in vivo efficacy of human CD3-mediated therapy.
Subject(s)
CD3 Complex/immunology , T-Lymphocytes/immunology , Animals , Antibodies, Bispecific/immunology , Antibodies, Monoclonal/immunology , Hematopoietic Stem Cells/immunology , Humans , MiceABSTRACT
Dysregulation of the complement system is linked to the pathogenesis of a variety of hematological disorders. Eculizumab, an anti-complement C5 monoclonal antibody, is the current standard of care for paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). However, because of high levels of C5 in plasma, eculizumab has to be administered biweekly by intravenous infusion. By applying recycling technology through pH-dependent binding to C5, we generated a novel humanized antibody against C5, SKY59, which has long-lasting neutralization of C5. In cynomolgus monkeys, SKY59 suppressed C5 function and complement activity for a significantly longer duration compared to a conventional antibody. Furthermore, epitope mapping by X-ray crystal structure analysis showed that a histidine cluster located on C5 is crucial for the pH-dependent interaction with SKY59. This indicates that the recycling effect of SKY59 is driven by a novel mechanism of interaction with its antigen and is distinct from other known pH-dependent antibodies. Finally, SKY59 showed neutralizing effect on C5 variant p.Arg885His, while eculizumab does not inhibit complement activity in patients carrying this mutation. Collectively, these results suggest that SKY59 is a promising new anti-C5 agent for patients with PNH and other complement-mediated disorders.
Subject(s)
Antibodies, Neutralizing/immunology , Complement C5/antagonists & inhibitors , Complement C5/immunology , Animals , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/chemistry , Complement C5/chemistry , Crystallography, X-Ray , Hemoglobinuria, Paroxysmal/drug therapy , Humans , Macaca fascicularis , Protein Binding , Protein ConformationABSTRACT
1. The number of developed therapeutic monoclonal antibodies (mAbs) has increased in this decade. This study aims to predict their pharmacokinetic profiles after intravenous (iv) injection using only the data taken after subcutaneous (sc) injection in cynomolgus monkey. 2. Two-compartment model parameters, Q, Vc and Vp, were collected from the published data after iv injection in cynomolgus monkey for 21 mAbs (Group A). Bioavailability after sc injection (F), CL and serum/plasma concentration after iv injection of other published 19 mAbs (Group B) were predicted using the estimated geometric means of Q, Vc and Vp in Group A and the serum/plasma concentration after sc injection in Group B. 3. F and CL of 18 out of 19 mAbs in Group B were successfully predicted within 30% difference of observed value. Moreover, most of the observed serum/plasma concentrations after iv injection of mAbs in Group B were successfully predicted within 2-fold difference. Our approach suggests that iv injection might not be required to evaluate absorption of mAbs after sc injection in cynomolgus monkey. Therefore, our approach might reduce the time and cost of drug development, reduce the burden on resources, and also contribute to animal welfare.
Subject(s)
Antibodies, Monoclonal/pharmacokinetics , Injections, Intravenous , Injections, Subcutaneous , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/therapeutic use , Biological Availability , Humans , Macaca fascicularis , Models, BiologicalABSTRACT
Previously, we have reported novel engineered antibody with pH-dependent antigen-binding (recycling antibody), and with both pH-dependent antigen-binding and increased FcRn-binding at neutral pH (sweeping antibody). The purpose of this study is to perform PK/PD predictions to better understand the potential applications of the antibodies as therapeutics. To demonstrate the applicability of recycling and sweeping antibodies over conventional antibodies, PK/PD analyses were performed. PK/PD parameters for antibody and antigen dynamics were estimated from the results of a pharmacokinetic study in human FcRn transgenic mice. A simulation study was performed using the estimated PK/PD parameters with various target antigen profiles. In comparison to conventional antibody, recycling antibody enhanced antibody-antigen complex clearance by 3 folds, while sweeping antibody accelerated antigen clearance by 10 folds in a pharmacokinetic study. Simulation results showed that recycling and sweeping antibodies can improve dosage frequency and reduce the required dose for target antigens with various clearances, plasma concentrations or binding kinetics. Moreover, importance of the association rate constant to enhance the beneficial effect of antibodies was shown. These results support the conclusion that recycling and sweeping antibodies can be applied to various target antigens with different profiles, and expand the number of antigens that antibodies can target.
Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antigen-Antibody Reactions , Antigens/chemistry , Antigens/immunology , Animals , Antibody Specificity , Binding Sites, Antibody , Humans , Hydrogen-Ion Concentration , Mice , Mice, Transgenic , Models, Animal , Protein BindingABSTRACT
In this study, we describe novel tetravalent, bispecific antibody derivatives that bind two different epitopes on the HIV coreceptor CCR5. The basic protein formats that we applied were derived from Morrison-type bispecific antibodies: whole IgGs to which we connected single-chain antibodies (scFvs) via (Gly4Ser)n sequences at either the C or N terminus of the light chain or heavy chain. By design optimization, including disulfide stabilization of scFvs or introduction of 30-amino-acid linkers, stable molecules could be obtained in amounts that were within the same range as or no less than 4-fold lower than those observed with monoclonal antibodies in transient expression assays. In contrast to monospecific CCR5 antibodies, bispecific antibody derivatives block two alternative docking sites of CCR5-tropic HIV strains on the CCR5 coreceptor. Consequently, these molecules showed 18- to 57-fold increased antiviral activities compared to the parent antibodies. Most importantly, one prototypic tetravalent CCR5 antibody had antiviral activity against virus strains resistant to the single parental antibodies. In summary, physical linkage of two CCR5 antibodies targeting different epitopes on the HIV coreceptor CCR5 resulted in tetravalent, bispecific antibodies with enhanced antiviral potency against wild-type and CCR5 antibody-resistant HIV-1 strains.
Subject(s)
Antibodies, Bispecific/pharmacology , HIV-1/drug effects , Receptors, CCR5/immunology , Cell Line , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , HIV-1/immunology , HumansABSTRACT
Glypican 3 (GPC3), a GPI-anchored heparan sulfate proteoglycan, is expressed in the majority of hepatocellular carcinoma (HCC) tissues. Using MRL/lpr mice, we successfully generated a series of anti-GPC3 monoclonal antibodies (mAbs). GPC3 was partially cleaved between Arg358 and Ser359, generating a C-terminal 30-kDa fragment and an N-terminal 40-kDa fragment. All mAbs that induced antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against cells expressing GPC3 recognized the 30-kDa fragment, indicating that the C-terminal region of GPC3 serves as an epitope for mAb with ADCC and/or CDC inducing activities. Chimeric mAbs with Fc replaced by human IgG1 were created from GC33, one of the mAbs that reacted with the C-terminal 30-kDa fragment. Chimeric GC33 induced not only ADCC against GPC3-positive human HCC cells but also was efficacious against the Huh-7 human HCC xenograft. Thus, mAbs against the C-terminal 30-kDa fragment such as GC33 are useful in therapy targeting HCC.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antibody-Dependent Cell Cytotoxicity , Carcinoma, Hepatocellular/drug therapy , Glypicans/antagonists & inhibitors , Liver Neoplasms/drug therapy , Neoplasm Proteins/antagonists & inhibitors , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , CHO Cells , Carcinoma, Hepatocellular/immunology , Cell Line, Tumor , Cricetinae , Cricetulus , Glypicans/immunology , Humans , Immunodominant Epitopes/immunology , Liver Neoplasms/immunology , Mice , Neoplasm Proteins/immunology , Xenograft Model Antitumor AssaysABSTRACT
Carnitine and acetylcarnitine are important for the acquisition of motility and maturation of spermatozoa in the epididymis. In this study, we examined the involvement of carnitine/organic cation transporter (OCTN) in carnitine and acetylcarnitine transport in epididymal spermatozoa of mice. Uptake of both compounds by epididymal spermatozoa was time-dependent and partially Na(+)-dependent. Kinetic analyses revealed the presence of a high-affinity transport system in the spermatozoa, with K(m) values of 23.6 and 6.57 muM for carnitine and acetylcarnitine respectively in the presence of Na(+). Expression of OCTN2 and OCTN3 in epididymal spermatozoa was confirmed by immunofluorescence analysis. The involvement of these two transporters in carnitine and acetylcarnitine transport was supported by a selective inhibition study. We conclude that both Na(+)-dependent and -independent carnitine transporters, OCTN2 and OCTN3, mediate the supply of carnitine and acetylcarnitine to epididymal spermatozoa in mice.
Subject(s)
Acetylcarnitine/pharmacokinetics , Carnitine/pharmacokinetics , Membrane Proteins/metabolism , Organic Cation Transport Proteins/metabolism , Spermatozoa/metabolism , Animals , Betaine/analogs & derivatives , Betaine/pharmacology , Biological Transport, Active , Carnitine/pharmacology , Epididymis , Fluorescent Antibody Technique , Male , Membrane Proteins/analysis , Membrane Proteins/antagonists & inhibitors , Mice , Organic Cation Transport Proteins/analysis , Organic Cation Transport Proteins/antagonists & inhibitors , Pyrilamine/pharmacology , Solute Carrier Family 22 Member 5 , Sperm Maturation/physiology , Sperm Motility/physiologyABSTRACT
We have been investigating the functional display of multipass membrane protein such as transporter or G-protein coupled receptor on the budded baculovirus (BV). We tested the use of a viral envelope protein gp64 transgenic mouse for the direct immunization of these membrane proteins displayed on BVs. The gp64 transgenic mice showed only a weak response to virus compared to wild type BALB/c mice. Immunizing gp64 transgenic mice with the BV expressing peptide transporter PepT1, we obtained 47 monoclonal antibodies (mAbs). These mAbs were specific to the PepT1 on the pancreatic cancer cells AsPC-1 by fluorocytometric analysis and exhibited antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity to AsPC-1. We also generated 7 mAbs by immunizing gp64 transgenic mice on a CCR2-deficient background with the BV expressing chemokine receptor CCR2 together with partially purified CCR2. These mAbs possessed specific binding to CCR2 in CHO cells on fluorocytometric analysis, and exhibited neutralizing activities for ligand-dependent inhibition of cyclic AMP production. This method provides a powerful tool for the generation of therapeutic/diagnostic mAbs against membrane proteins.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Baculoviridae/genetics , Cell Adhesion Molecules/genetics , Membrane Glycoproteins/genetics , Membrane Proteins/immunology , Peptide Library , Viral Envelope Proteins/genetics , Viral Proteins/genetics , Animals , Baculoviridae/metabolism , CHO Cells , Cell Line, Tumor , Cricetinae , Cricetulus , Immunization , Membrane Proteins/genetics , Mice , Mice, Transgenic , Peptide Transporter 1 , Receptors, CCR2 , Receptors, Chemokine/immunology , Symporters/immunology , Viral Envelope Proteins/immunologyABSTRACT
Arsenic is an established human carcinogen. The role of aquaglyroporins (AQPs) in arsenic disposition was recently identified. In order to examine whether organic anion transporting polypeptide-C (OATP-C) also plays a role in arsenic transport, OATP-C cDNA was transfected into cells of a human embryonic kidney cell line (HEK-293). Transfection increased uptake of the model OATP-C substrate, estradiol-17beta-D-glucuronide, by 10-fold. In addition, we measured uptake and cytotoxicity of arsenate, arsenite, monomethylarsonate(MMA(V)), and dimethylarsinate (DMA(V)). Transfection of OATP-C increased uptake and cytotoxicity of arsenate and arsenite, but not of MMA(V) or DMA(V). Rifampin and taurocholic acid (a substrate of OATP-C) reversed the increased toxicity of arsenate and arsenite seen in OATP-C-transfected cells. The increase in uptake of inorganic arsenic was not as great as that of estradiol-17beta-D-glucuronide. Our results suggest that OATP-C can transport inorganic arsenic in a (GSH)-dependent manner. However, this may not be the major pathway for arsenic transport.