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1.
Int J Mol Sci ; 22(8)2021 Apr 17.
Article in English | MEDLINE | ID: mdl-33920699

ABSTRACT

Immune checkpoint inhibitors (ICIs) have demonstrated remarkable efficacy in a growing number of malignancies. However, overcoming primary or secondary resistances is difficult due to pharmacokinetics issues and side effects associated with high systemic exposure. Local or regional expression of monoclonal antibodies (mAbs) using gene therapy vectors can alleviate this problem. In this work, we describe a high-capacity adenoviral vector (HCA-EFZP-aPDL1) equipped with a mifepristone-inducible system for the controlled expression of an anti-programmed death ligand 1 (PD-L1) blocking antibody. The vector was tested in an immune-competent mouse model of colorectal cancer based on implantation of MC38 cells. A single local administration of HCA-EFZP-aPDL1 in subcutaneous lesions led to a significant reduction in tumor growth with minimal release of the antibody in the circulation. When the vector was tested in a more stringent setting (rapidly progressing peritoneal carcinomatosis), the antitumor effect was marginal even in combination with other immune-stimulatory agents such as polyinosinic-polycytidylic acid (pI:C), blocking mAbs for T cell immunoglobulin, mucin-domain containing-3 (TIM-3) or agonistic mAbs for 4-1BB (CD137). In contrast, macrophage depletion by clodronate liposomes enhanced the efficacy of HCA-EFZP-aPDL1. These results highlight the importance of addressing macrophage-associated immunoregulatory mechanisms to overcome resistance to ICIs in the context of colorectal cancer.


Subject(s)
Antibodies, Blocking/genetics , B7-H1 Antigen/metabolism , Carcinoma/therapy , Genetic Therapy/methods , Immunotherapy/methods , Macrophages/immunology , Peritoneal Neoplasms/therapy , Adenoviridae/genetics , Animals , Antibodies, Blocking/immunology , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/immunology , Cell Line , Female , Genetic Vectors/genetics , Immune Checkpoint Inhibitors/immunology , Immune Checkpoint Inhibitors/therapeutic use , Immunologic Factors/therapeutic use , Mice , Mice, Inbred C57BL , Poly I-C/therapeutic use
2.
J Allergy Clin Immunol ; 143(6): 2227-2237.e10, 2019 06.
Article in English | MEDLINE | ID: mdl-30543818

ABSTRACT

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec) 8 is selectively expressed on eosinophils, mast cells, and basophils and, when engaged on eosinophils, can cause cell death. OBJECTIVE: We sought to characterize surface and soluble Siglec-8 (sSiglec-8) levels in normal donors (NDs) and eosinophilic donors (EOs) and assess the efficacy of anti-Siglec-8 antibodies in inducing eosinophil cell death in vitro. METHODS: Eosinophil expression of Siglec-8 was assessed by using flow cytometry and quantitative PCR. Serum sSiglec-8 levels were measured by means of ELISA. Induction of eosinophil death by IgG4 (chimeric 2E2 IgG4) and afucosylated IgG1 (chimeric 2E2 IgG1 [c2E2 IgG1]) anti-Siglec-8 antibodies was evaluated in vitro by using flow cytometry and in vivo in humanized mice. RESULTS: Siglec-8 was consistently expressed on eosinophils from NDs and EOs and did not correlate with absolute eosinophil count or disease activity. sSiglec-8 levels were measurable in sera from most donors unrelated to absolute eosinophil counts or Siglec-8 surface expression. c2E2 IgG1 and chimeric 2E2 IgG4 were equally effective at inducing cell death (Annexin-V positivity) of purified eosinophils from NDs and EOs after overnight IL-5 priming. In contrast, killing of purified eosinophils without IL-5 was only seen in EOs, and natural killer cell-mediated eosinophil killing was seen only with c2E2 IgG1. Finally, treatment of humanized mice with anti-Siglec antibody led to robust depletion of IL-5-induced eosinophilia in vivo. CONCLUSIONS: Siglec-8 is highly expressed on blood eosinophils from EOs and NDs and represents a potential therapeutic target for eosinophilic disorders. Enhanced killing of eosinophils in the presence of IL-5 might lead to increased efficacy in patients with IL-5-driven eosinophilia.


Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, B-Lymphocyte/metabolism , Eosinophilia/immunology , Eosinophils/immunology , Killer Cells, Natural/immunology , Lectins/metabolism , Animals , Antibodies, Blocking/genetics , Antigens, CD/genetics , Antigens, CD/immunology , Antigens, Differentiation, B-Lymphocyte/genetics , Antigens, Differentiation, B-Lymphocyte/immunology , Cell Death , Cells, Cultured , Cytotoxicity, Immunologic , Eosinophilia/therapy , Humans , Immunoglobulin G/genetics , Interleukin-5/metabolism , Lectins/genetics , Lectins/immunology , Leukocyte Count , Mice , Mice, SCID , Molecular Targeted Therapy , Recombinant Fusion Proteins/genetics , Transcriptome
3.
J Comput Biol ; 26(9): 962-974, 2019 09.
Article in English | MEDLINE | ID: mdl-30570348

ABSTRACT

Norovirus is the causing agent of acute gastroenteritis disease globally. Efforts in developing therapeutics against virus infection mostly fail due to emergence of drug resistance that is a consequence of presence of high mutation rates in virus genome during virus' life cycle. In this study, we computationally analyzed the affinity of a drug target, wild type VP1 envelope protein and its three variants to a therapeutic antibody FAB5I2. We have found that mutations break important hydrogen bonds and cause high fluctuations in residues that form VP1-FAB5I2 complex interface. In addition to changes in dynamics, we also revealed that the affinity of FAB5I2 to VP1 protein drops significantly upon mutations in terms of relative binding free energy.


Subject(s)
Antibodies, Blocking/chemistry , Antibodies, Monoclonal/chemistry , Antigens, Viral/chemistry , Molecular Dynamics Simulation , Mutation , Norovirus/chemistry , Viral Envelope Proteins/chemistry , Antibodies, Blocking/genetics , Antigens, Viral/genetics , Humans , Norovirus/genetics , Viral Envelope Proteins/genetics
4.
Front Immunol ; 9: 881, 2018.
Article in English | MEDLINE | ID: mdl-29887861

ABSTRACT

IL-10 is a potent immunosuppressive cytokine that promotes the differentiation of tolerogenic dendritic cells (DC-10), and the subsequent induction of antigen-specific T regulatory type 1 (Tr1) cells, which suppress immune responses. However, IL-10 acts on multiple cell types and its effects are not solely inhibitory, therefore, limiting its use as immunomodulant. APVO210 is a bispecific fusion protein composed of an anti-CD86 antibody fused with monomeric IL-10 (ADAPTIR™ from Aptevo Therapeutics). APVO210 specifically induces IL-10R signaling in CD86+ antigen-presenting cells, but not in T and B cells. In this study, we tested whether APVO210 promotes the differentiation of tolerogenic DC-10 and the differentiation of antigen-specific CD4+ Tr1 cells in vitro. We compared the effect of APVO210 with that of recombinant human (rh) IL-10 on the in vitro differentiation of DC-10, induction of alloantigen-specific anergic CD4+ T cells, enrichment in CD49b+LAG3+ Tr1 cells mediating antigen-specific suppression, and stability upon exposure to inflammatory cytokines. APVO210 induced the differentiation of tolerogenic DC (DC-A210) that produced high levels of IL-10, expressed CD86, HLA-G, and intermediate levels of CD14 and CD16. These DC-A210 induced alloantigen-specific anergic T-cell cultures (T-alloA210) that were enriched in CD49b+ LAG3+ Tr1 cells, produced high levels of IL-10, and had suppressive properties. The phenotype and high IL-10 production by DC-A210, and the alloantigen-specific anergy of T-alloA210 were preserved upon exposure to the inflammatory cytokines IL-1ß, IL-6, and TNF-α. The effects of APVO210 were comparable to that of dimeric rh IL-10. In conclusion, our data demonstrate that APVO210 drives the differentiation of tolerogenic DC and functional alloantigen-specific Tr1 cells in vitro. Since APVO210 specifically targets CD86+ cells, we hypothesize that it will specifically target CD86+ DC to induce Tr1 cells in vivo, and mediate antigen-specific immunological tolerance by induction of tolerogenic DC and Tr1 cells.


Subject(s)
Antibodies, Blocking/immunology , Cell Differentiation/drug effects , Immunoconjugates/pharmacology , Interleukin-10/immunology , T-Lymphocytes, Regulatory/immunology , Antibodies, Blocking/genetics , B7-2 Antigen/immunology , Blood Buffy Coat/cytology , Cell Differentiation/immunology , Cells, Cultured , Dendritic Cells/drug effects , Dendritic Cells/immunology , Healthy Volunteers , Humans , Immune System Diseases/drug therapy , Immune System Diseases/immunology , Immune Tolerance/drug effects , Immune Tolerance/immunology , Immunoconjugates/genetics , Immunoconjugates/immunology , Immunoconjugates/therapeutic use , Interleukin-10/genetics , Primary Cell Culture , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/therapeutic use , T-Lymphocytes, Regulatory/drug effects
5.
J Biol Chem ; 293(8): 2815-2828, 2018 02 23.
Article in English | MEDLINE | ID: mdl-29321208

ABSTRACT

Conformational changes in proteins due to ligand binding are ubiquitous in biological processes and are integral to many biological systems. However, it is often challenging to link ligand-induced conformational changes to a resulting biological function because it is difficult to distinguish between the energetic components associated with ligand binding and those due to structural rearrangements. Here, we used a unique approach exploiting conformation-specific and regio-specific synthetic antibodies (sABs) to probe the energetic contributions of ligand binding to conformation changes. Using maltose-binding protein (MBP) as a model system, customized phage-display selections were performed to generate sABs that stabilize MBP in different conformational states, modulating ligand-binding affinity in competitive, allosteric, or peristeric manners. We determined that the binding of a closed conformation-specific sAB (sAB-11M) to MBP in the absence of maltose is entropically driven, providing new insight into designing antibody-stabilized protein interactions. Crystal structures of sABs bound to MBP, together with biophysical data, delineate the basis of free energy differences between different conformational states and confirm the use of the sABs as energy probes for dissecting enthalpic and entropic contributions to conformational transitions. Our work provides a foundation for investigating the energetic contributions of distinct conformational dynamics to specific biological outputs. We anticipate that our approach also may be valuable for analyzing the energy landscapes of regulatory proteins controlling biological responses to environmental changes.


Subject(s)
Antibodies, Blocking/metabolism , Escherichia coli K12/enzymology , Escherichia coli Proteins/metabolism , Maltose-Binding Proteins/metabolism , Maltose/metabolism , Models, Molecular , Molecular Probes/metabolism , Amino Acid Substitution , Antibodies, Blocking/chemistry , Antibodies, Blocking/genetics , Antibody Affinity , Apoproteins/chemistry , Apoproteins/metabolism , Biotinylation , Crystallography, X-Ray , Escherichia coli K12/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Ligands , Maltose/chemistry , Maltose-Binding Proteins/chemistry , Maltose-Binding Proteins/genetics , Molecular Probes/chemistry , Molecular Probes/genetics , Mutation , Peptide Library , Protein Conformation , Protein Engineering , Protein Processing, Post-Translational , Protein Stability , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Thermodynamics
6.
Trends Immunol ; 35(9): 403-5, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25131302

ABSTRACT

HIV rapidly mutates to escape antibody detection, and B cells counter this mutation by continual evolution to restore recognition, serendipitously resulting in the evolution of neutralizing activity in a fraction of infected individuals. A recent Cell paper describes how antibody repertoires stochastically collaborated, shaping the viral swarm and utilizing viral immune evasion to their advantage.


Subject(s)
Antibodies, Blocking/immunology , Antibodies, Viral/immunology , B-Lymphocytes/immunology , HIV Infections/immunology , HIV/immunology , Animals , Antibodies, Blocking/genetics , Antibodies, Viral/genetics , Antibody Affinity , Antigenic Modulation , Antigens, Viral/immunology , B-Lymphocytes/virology , Cell Differentiation , HIV/genetics , Humans , Immune Evasion , Immunity, Humoral , Mutation/genetics
7.
Int Immunopharmacol ; 21(1): 119-27, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24800661

ABSTRACT

Interleukin-1ß (IL-1ß) and interleukin-17A (IL-17A) are inducible factors and important cytokines in the pathogenesis of rheumatoid arthritis (RA). In the present study, three bispecific and neutralizing antibodies (BsAB-1, BsAB-2 and BsAB-3) against both hIL-1ß and hIL-17A were constructed, their therapeutic efficacy was compared on collagen induced arthritis (CIA) model mice. In vitro assays demonstrated that the three antibodies could simultaneously bind to target both hIL-1ß and hIL-17A. Mice with CIA were subcutaneously administered with one of three antibodies every two days for 29 days, we noticed that, compared with the BsAB-2 and BsAB-3, BsAB-1 antibody therapy resulted in more significant effect on alleviating the severity of arthritis by preventing bone damage and cartilage destruction and substantially decreasing production of CII-specific antibodies. In addition, BsAB-1 antibody was more potent in the inhibition of mRNA expression of IL-2, IL-1ß, IL-17A, TNF-α and MMP-3 in the spleen of CIA mice compared to the other two. In summary, BsAB-1 is superior over BsAB-2 and BsAB-3 for the treatment of RA model mice, and may be chosen as an ideal candidate for further development of therapeutic drugs for treatment of RA.


Subject(s)
Antibodies, Bispecific/administration & dosage , Antibodies, Blocking/administration & dosage , Arthritis, Experimental/therapy , Arthritis, Rheumatoid/therapy , Bone and Bones/drug effects , Animals , Antibodies, Bispecific/genetics , Antibodies, Blocking/genetics , Antibody Formation/drug effects , Arthritis, Experimental/immunology , Arthritis, Rheumatoid/immunology , Bone and Bones/pathology , Cells, Cultured , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Down-Regulation , Humans , Interleukin-17/immunology , Interleukin-1beta/immunology , Male , Mice , Mice, Inbred Strains
8.
Biochem Biophys Res Commun ; 446(1): 132-6, 2014 Mar 28.
Article in English | MEDLINE | ID: mdl-24569074

ABSTRACT

Compelling evidence suggests that vascular endothelial growth factor (VEGF), due to its essential role in angiogenesis, is a critical target for cancer treatment. Neutralizing monoclonal antibodies against VEGF are important class of drugs used in cancer therapy. However, the cost of production, large size, and immunogenicity are main drawbacks of conventional monoclonal therapy. Nanobodies are the smallest antigen-binding antibody fragments, which occur naturally in camelidae. Because of their remarkable features, we decided to use an immune library of nanobody to direct phage display to recognition of novel functional epitopes on VEGF. Four rounds of selection were performed and six phage-displayed nanobodies were obtained from an immune phage library. The most reactive clone in whole-cell ELISA experiments, was purified and assessed in proliferation inhibition assay. Purified ZFR-5 not only blocked interaction of VEGF with its receptor in cell ELISA experiments, but also was able to significantly inhibit proliferation response of human umbilical vein endothelial cells to VEGF in a dose-dependent manner. Taken together, our study demonstrates that by using whole-cell ELISA experiments, nanobodies against antigenic regions included in interaction of VEGF with its receptors can be directed. Because of unique and intrinsic properties of a nanobody and the ability of selected nanobody for blocking the epitope that is important for biological function of VEGF, it represents novel potential drug candidate.


Subject(s)
Neoplasms/therapy , Single-Domain Antibodies/immunology , Vascular Endothelial Growth Factor A/immunology , Antibodies, Blocking/genetics , Antibodies, Blocking/immunology , Antibodies, Blocking/therapeutic use , Cell Proliferation , Cell Surface Display Techniques , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Human Umbilical Vein Endothelial Cells , Humans , Neoplasms/blood supply , Neoplasms/immunology , Neovascularization, Pathologic/immunology , Peptide Library , Single-Domain Antibodies/genetics , Single-Domain Antibodies/therapeutic use , Vascular Endothelial Growth Factor A/antagonists & inhibitors
9.
MAbs ; 6(2): 437-45, 2014.
Article in English | MEDLINE | ID: mdl-24492299

ABSTRACT

Antibodies isolated from human donors are increasingly being developed for anti-infective therapeutics. These antibodies undergo affinity maturation in vivo, minimizing the need for engineering of therapeutic leads for affinity. However, the affinities required for some therapeutic applications may be higher than the affinities of the leads obtained, requiring further affinity maturation in vitro. To improve the neutralization potency of natural human antibody MSL-109 targeting human cytomegalovirus (CMV), we affinity matured the antibody against the gH/gL glycoprotein complex. A phage display library where most of the six complementary-determining regions (CDRs) were allowed to vary in only one amino acid residue at a time was used to scan for mutations that improve binding affinity. A T55R mutation and multiple mutations in position 53 of the heavy chain were identified that, when present individually or in combination, resulted in higher apparent affinities to gH/gL and improved CMV neutralization potency of Fab fragments expressed in bacterial cells. Three of these mutations in position 53 introduced glycosylation sites in heavy chain CDR 2 (CDR H2) that impaired binding of antibodies expressed in mammalian cells. One high affinity (KD<10 pM) variant was identified that combined the D53N and T55R mutations while avoiding glycosylation of CDR H2. However, all the amino acid substitutions identified by phage display that improved binding affinity without introducing glycosylation sites required between two and four simultaneous nucleotide mutations to avoid glycosylation. These results indicate that the natural human antibody MSL-109 is close to a local affinity optimum. We show that affinity maturation by phage display can be used to identify and bypass barriers to in vivo affinity maturation of antibodies imposed by glycosylation and codon usage. These constraints may be relatively prevalent in human antibodies due to the codon usage and the amino acid sequence encoded by the natural human repertoire.


Subject(s)
Antibodies, Blocking/metabolism , Antibodies, Monoclonal/metabolism , Cytomegalovirus/immunology , Animals , Antibodies, Blocking/genetics , Antibodies, Monoclonal/genetics , Antibody Affinity , CHO Cells , Cell Surface Display Techniques , Cricetulus , DNA Mutational Analysis , Glycosylation , Humans , Mutagenesis , Mutation/genetics , Transgenes/genetics
10.
MAbs ; 6(2): 446-59, 2014.
Article in English | MEDLINE | ID: mdl-24492304

ABSTRACT

Botulinum toxins (BoNTs) are among the most toxic substances on earth, with serotype A toxin being the most toxic substance known. They are responsible for human botulism, a disease characterized by flaccid muscle paralysis that occurs naturally through food poisoning or the colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNT has been classified as a category A agent by the Centers for Disease Control, and it is one of six agents with the highest potential risk of use as bioweapons. Human or human-like neutralizing antibodies are thus required for the development of anti-botulinum toxin drugs to deal with this possibility. In this study, Macaca fascicularis was hyperimmunized with a recombinant light chain of BoNT/A. An immune phage display library was constructed and, after multistep panning, several scFv with nanomolar affinities that inhibited the endopeptidase activity of BoNT/A1 in vitro as scFv-Fc, with a molar ratio (ab binding site:toxin) of up to 1:1, were isolated. The neutralization of BoNT/A-induced paralysis by the SEM120-IID5, SEM120-IIIC1 and SEM120-IIIC4 antibodies was demonstrated in mouse phrenic nerve-hemidiaphragm preparations with the holotoxin. The neutralization observed is the strongest ever measured in the phrenic nerve-hemidiaphragm assay for BoNT/A1 for a monoclonal antibody. Several scFv-Fc inhibiting the endopeptidase activity of botulinum neurotoxin A were isolated. For SEM120-IID5, SEM120-IIIC1, and SEM120-IIIC4, inhibitory effects in vitro and protection against the toxin ex vivo were observed. The human-like nature of these antibodies makes them promising lead candidates for further development of immunotherapeutics for this disease.


Subject(s)
Antibodies, Blocking/metabolism , Botulinum Toxins, Type A/immunology , Botulism/therapy , Clostridium botulinum type A/immunology , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin Light Chains, Surrogate/metabolism , Immunotherapy/methods , Paralysis/prevention & control , Phrenic Nerve/drug effects , Single-Chain Antibodies/metabolism , Animals , Antibodies, Blocking/administration & dosage , Antibodies, Blocking/genetics , Botulinum Toxins, Type A/adverse effects , Botulism/complications , Botulism/immunology , Cell Surface Display Techniques , Epitope Mapping , Humans , Immunity/genetics , Immunization , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Light Chains, Surrogate/administration & dosage , Immunoglobulin Light Chains, Surrogate/genetics , Macaca fascicularis , Male , Mice , Mice, Inbred BALB C , Paralysis/etiology , Paralysis/immunology , Phrenic Nerve/immunology , Single-Chain Antibodies/genetics
11.
Thromb Haemost ; 111(5): 824-32, 2014 May 05.
Article in English | MEDLINE | ID: mdl-24402608

ABSTRACT

One of the main disadvantages of current t-PA thrombolytic treatment is the increased bleeding risk. Upon activation, thrombin activatable fibrinolysis inhibitor (TAFI) is a very powerful antifibrinolytic enzyme. Therefore, co-administration of a TAFI inhibitor during thrombolysis could reduce the required t-PA dose without compromising the thrombolytic efficacy. In this study we generated and characterised a nanobody that is inhibitory towards rat TAFI and evaluated its profibrinolytic property in vitro and in vivo. Nanobody VHH-rTAFI-i81 inhibits (at a 16-fold molar ratio nanobody over TAFI) the thrombin/thrombomodulin (T/TM)-mediated activation of rat TAFI (rTAFI) by 83 ± 1.8% with an IC50 of 0.46 (molar ratio nanobody over TAFI). The affinity (KA) of VHH-rTAFI-i81 for rTAFI, as determined by surface plasmon resonance (Biacore®), is 2.5 ± 0.2 x 10(10) M(-1) and illustrates a very strong binding. In an in vitro clot lysis assay, administration of VHH-rTAFI-i81 strongly enhances the degree of lysis and reduces time to reach full lysis of t-PA-mediated clot lysis. Epitope mapping discloses that Lys392 is of primary importance for the nanobody/rTAFI interaction besides minor contributions of Tyr175 and Glu183. In vivo application of VHH-rTAFI-i81 in a tissue factor-induced mouse thromboembolism model significantly decreases fibrin deposition in the lungs in the absence of exogenous administered t-PA. Nanobody VHH-rTAFI-i81 is a very potent inhibitor of T/TM-mediated TAFI activation. Co-administration of this nanobody and t-PA enhances the fibrinolytic efficacy. In an in vivo mouse thromboembolism model, VHH-rTAFI-i81 reduces fibrin deposition in the lungs.


Subject(s)
Antibodies, Blocking/metabolism , Fibrinolytic Agents/therapeutic use , Lung/drug effects , Single-Domain Antibodies/administration & dosage , Thromboembolism/drug therapy , Animals , Antibodies, Blocking/genetics , Carboxypeptidase B2/metabolism , Disease Models, Animal , Drug Therapy, Combination , Epitope Mapping , Female , Fibrin/metabolism , Humans , In Vitro Techniques , Lung/metabolism , Mice , Protein Binding , Rats , Single-Domain Antibodies/adverse effects , Single-Domain Antibodies/genetics , Single-Domain Antibodies/metabolism , Surface Plasmon Resonance , Thrombin/metabolism , Thrombomodulin/metabolism , Tissue Plasminogen Activator/therapeutic use
12.
PLoS One ; 8(11): e80712, 2013.
Article in English | MEDLINE | ID: mdl-24312238

ABSTRACT

Shiga toxin 1 (Stx1) is a virulence factor of enterohemorrhagic Escherichia coli, such as the O157:H7 strain. In the intestines, secretory IgA (SIgA) is a major component of the immune defense against pathogens and toxins. To form SIgA, the production of dimeric IgA that retains biological activity is an important step. We previously established hybrid-IgG/IgA having variable regions of the IgG specific for the binding subunit of Stx1 (Stx1B) and the heavy chain constant region of IgA. If hybrid-IgG/IgA cDNAs can be expressed in plants, therapeutic or preventive effects may be expected in people eating those plants containing a "plantibody". Here, we established transgenic Arabidopsis thaliana expressing dimeric hybrid-IgG/IgA. The heavy and light chain genes were placed under the control of a bidirectional promoter and terminator of the chlorophyll a/b-binding protein of Arabidopsis thaliana (expression cassette). This expression cassette and the J chain gene were subcloned into a single binary vector, which was then introduced into A. thaliana by means of the Agrobacterium method. Expression and assembly of the dimeric hybrid-IgG/IgA in plants were revealed by ELISA and immunoblotting. The hybrid-IgG/IgA bound to Stx1B and inhibited Stx1B binding to Gb3, as demonstrated by ELISA. When Stx1 holotoxin was pre-treated with the resulting plantibody, the cytotoxicity of Stx1 was inhibited. The toxin neutralization was also demonstrated by means of several assays including Stx1-induced phosphatidylserine translocation on the plasma membrane, caspase-3 activation and 180 base-pair DNA ladder formation due to inter-nucleosomal cleavage. These results indicate that edible plants containing hybrid-IgG/IgA against Stx1B have the potential to be used for immunotherapy against Stx1-caused food poisoning.


Subject(s)
Immunoglobulin A/immunology , Immunoglobulin G/immunology , Plantibodies/immunology , Shiga Toxin 1/immunology , Antibodies, Blocking/genetics , Antibodies, Blocking/immunology , Antigens, Tumor-Associated, Carbohydrate/immunology , Antigens, Tumor-Associated, Carbohydrate/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Cell Death/immunology , Gene Expression , Gene Order , Immunoglobulin A/genetics , Immunoglobulin G/genetics , Plantibodies/genetics , Plants, Genetically Modified , Protein Binding/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Shiga Toxin 1/metabolism
13.
J Dermatol Sci ; 72(1): 25-31, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23827201

ABSTRACT

BACKGROUND: Pathogenic autoantibodies in bullous pemphigoid (BP) recognize the non-collagenous 16A domain (NC16A) of collagen XVII (COL17), a hemidesmosomal component at the skin membrane. This immune inflammation involves activation of the complement cascade via the classical pathway. With similar antigen binding activity, Fab and single-chain variable fragments (scFv) of pathogenic anti-COL17 antibodies can interfere with COL17 binding of autoantibodies, blocking subsequent complement activation and granulocyte activation. OBJECTIVE: To characterize the biological functions of human anti-COL17 scFv antibody. METHODS: We constructed scFv antibodies against the corresponding antigen from parental Fab by expression in Escherichia coli. IgG autoantibodies against COL17 were purified by affinity chromatography from serum of BP patients. The inhibitory effects of anti-COL17 scFv on binding of BP autoantibodies to the NC16A domain of human COL17 antigen were observed by inhibition ELISA, immunofluorescence, and inhibition of complement activation. Reactive oxygen production assay and BP cryosection model were performed to assess the inhibitory effect of scFv on granulocyte activation and then the dermal-epidermal separation. RESULTS: ELISA and Western blot showed specific binding of scFv to COL17. We found that anti-COL17 scFv can inhibit the binding of intact IgG purified from BP parents to the corresponding COL17 antigen and then subsequent C1q and C3 activation and granulocyte activation in vitro. Most importantly, we confirmed that recombinant scFv can inhibit BP-IgG induced dermal-epidermal separation by BP cryosection model. CONCLUSION: The anti-COL17 scFv antibody can inhibit the binding of BP-IgG autoantibodies to COL17, thereby affecting subsequent complement activation and granulocyte activation in vitro. Our results suggest that blocking pathogenic epitopes using engineered scFv is an efficient BP therapy.


Subject(s)
Antibodies, Blocking/administration & dosage , Autoantigens/immunology , Non-Fibrillar Collagens/antagonists & inhibitors , Non-Fibrillar Collagens/immunology , Pemphigoid, Bullous/immunology , Pemphigoid, Bullous/therapy , Single-Chain Antibodies/administration & dosage , Antibodies, Blocking/genetics , Antigen-Antibody Reactions , Autoantibodies/immunology , Autoimmunity , Complement Activation , Granulocytes/immunology , Humans , Immunoglobulin G/immunology , Immunotherapy , Protein Engineering , Single-Chain Antibodies/genetics , Collagen Type XVII
14.
Biochem Biophys Res Commun ; 430(1): 14-9, 2013 Jan 04.
Article in English | MEDLINE | ID: mdl-23159621

ABSTRACT

A previous study showed that the minimal epitope recognised by the PLY-5 mAb in the conserved undecapeptide Trp-rich loop of bacterial CDCs should consist of WEWWRT (Jacobs et al., 1999) [5]. Now, through immunoscreening of amino acid substitution analogues, it is concluded that the second Trp and the Arg residues are essential in the PLY-5 epitope. The E residue is an auxiliary epitope contributor. Antibody modelling and docking simulations provided support for these findings. For recognition by the antibody, the Trp-rich loop flipped out, mimicking the mechanism of membrane insertion. The displaced second Trp was seen to establish aromatic stacking interactions with aromatic residues of the antibody paratope and the notably extruded guanidium tip of the arginine residue mediated electrostatic interactions with well-exposed carboxylic groups of glutamic residues on the surface of the paratope. Thus, the epitope/paratope interaction is mainly mediated by aromatic and by ionic interactions.


Subject(s)
Antibodies, Blocking/immunology , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Cholesterol/immunology , Cytotoxins/immunology , Epitopes/immunology , Streptolysins/immunology , Amino Acid Sequence , Amino Acid Substitution , Animals , Antibodies, Blocking/genetics , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Neutralizing/genetics , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/immunology , Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/immunology , Conserved Sequence , Cytotoxins/antagonists & inhibitors , Epitope Mapping , Epitopes/chemistry , Epitopes/genetics , Hemolysin Proteins/antagonists & inhibitors , Hemolysin Proteins/immunology , Hemolysis/immunology , Mice , Molecular Sequence Data , Oligopeptides/immunology , Streptolysins/antagonists & inhibitors , Tryptophan/immunology
15.
J Immunol ; 190(1): 240-9, 2013 Jan 01.
Article in English | MEDLINE | ID: mdl-23209327

ABSTRACT

Cerebral Plasmodium falciparum malaria is characterized by adhesion of infected erythrocytes (IEs) to the cerebral microvasculature. This has been linked to parasites expressing the structurally related group A subset of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of IE adhesion ligands and to IEs with affinity for ICAM-1. However, recent evidence has cast doubt on both these associations, tempering hopes of the feasibility of developing a vaccine based on ICAM-1-binding PfEMP1. In this study, we report the identification of a domain cassette (DC) present in group A var genes from six genetically distinct P. falciparum parasites. The three domains in the cassette, which we call DC4, had a high level of sequence identity and cluster together phylogenetically. Erythrocytes infected by these parasites and selected in vitro for expression of DC4 adhered specifically to ICAM-1. The ICAM-1-binding capacity of DC4 was mapped to the C-terminal third of its Duffy-binding-like ß3 domain. DC4 was the target of broadly cross-reactive and adhesion-inhibitory IgG Abs, and levels of DC4-specific and adhesion-inhibitory IgG increased with age among P. falciparum-exposed children. Our study challenges earlier conclusions that group A PfEMP1 proteins are not central to ICAM-1-specific IE adhesion and support the feasibility of developing a vaccine preventing cerebral malaria by inhibiting cerebral IE sequestration.


Subject(s)
Antibodies, Blocking/metabolism , Antigens, Protozoan/metabolism , Erythrocyte Membrane/metabolism , Intercellular Adhesion Molecule-1/metabolism , Mutagenesis, Insertional/immunology , Plasmodium falciparum/immunology , Protozoan Proteins/metabolism , Animals , Antibodies, Blocking/genetics , Antigens, Protozoan/classification , Antigens, Protozoan/genetics , Binding Sites, Antibody/genetics , Cell Adhesion/genetics , Cell Adhesion/immunology , Conserved Sequence/genetics , Conserved Sequence/immunology , Cross Reactions/immunology , Erythrocyte Membrane/genetics , Erythrocyte Membrane/immunology , Genomics/methods , HEK293 Cells , Humans , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/immunology , Mutagenesis, Insertional/genetics , Plasmodium falciparum/genetics , Protein Binding/genetics , Protein Binding/immunology , Protein Structure, Tertiary/genetics , Protozoan Proteins/classification , Protozoan Proteins/genetics , Rats
16.
J Immunol ; 189(9): 4592-601, 2012 Nov 01.
Article in English | MEDLINE | ID: mdl-23028056

ABSTRACT

VCAM-1 plays a key role in leukocyte trafficking during inflammatory responses. However, molecular mechanisms underlying this function have not been clearly elucidated. In this study, using phage display technology, we developed a rabbit/human chimeric VCAM-1 Ab, termed VCAM-1 domain 6 (VCAM-1-D6), which specifically recognizes aa 511-599 within the sixth Ig-like domain. We report that the VCAM-1-D6 Ab blocked U937 cell transmigration across activated HUVECs but did not alter adhesion of U937 cells to the HUVECs. We also demonstrate that VCAM-1-D6 does not alter TNF-α-stimulated endothelial cell chemokine or cytokine production. Furthermore, through in vivo efficacy testing using a mouse islet allograft model, we demonstrate that VCAM-1-D6 significantly alleviates allograft rejection by blocking leukocyte infiltration to the grafted islets. Taken together, our results suggest that the VCAM-1-D6 Ab may block VCAM-1-mediated inflammation and could be a useful tool in treating inflammatory diseases.


Subject(s)
Antibodies, Blocking/physiology , Cell Adhesion/immunology , Cell Migration Inhibition/immunology , Endothelium, Vascular/cytology , Endothelium, Vascular/immunology , Immunoglobulin G/physiology , Leukocytes/immunology , Vascular Cell Adhesion Molecule-1/immunology , Animals , Antibodies, Blocking/genetics , Cell Adhesion/genetics , Cell Migration Inhibition/genetics , Endothelium, Vascular/chemistry , HEK293 Cells , Human Umbilical Vein Endothelial Cells , Humans , Immunoglobulin Fc Fragments/biosynthesis , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/physiology , Immunoglobulin G/biosynthesis , Immunoglobulin G/genetics , Leukocytes/cytology , Mice , Protein Structure, Tertiary/genetics , Rabbits , U937 Cells , Vascular Cell Adhesion Molecule-1/genetics
17.
Immunobiology ; 217(1): 17-27, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21962573

ABSTRACT

We recently mapped the regions on the heavy (H) chain of botulinum neurotoxin, type B (BoNT/B) recognized by blocking antibodies (Abs) from cervical dystonia (CD) patients who develop immunoresistance during toxin treatment. Since blocking could also be effected by Abs directed against regions on the light (L) chain, we have mapped here the L chain, using the same 30 CD antisera. We synthesized, purified and characterized 32 19-residue L chain peptides that overlapped successively by 5 residues (peptide L32 overlapped with peptide N1 of the H chain by 12 residues). In a given patient, Abs against the L chain seemed less intense than those against H chain. Most sera recognized a limited set of L chain peptides. The levels of Abs against a given region varied with the patient, consistent with immune responses to each epitope being under separate MHC control. The peptides most frequently recognized were: L13, by 30 of 30 antisera (100%); L22, by 23 of 30 (76.67%); L19, by 15 of 30 (50.00%); L26, by 11 of 30 (36.70%); and L14, by 12 of 30 (40.00%). The activity of L14 probably derives from its overlap with L13. The levels of Ab binding decreased in the following order: L13 (residues 169-187), L22 (295-313), L19 (253-271), and L26 (351-369). Peptides L12 (155-173), L18 (239-257), L15 (197-215), L1 (1-19) and L23 (309-327) exhibited very low Ab binding. The remaining peptides had little or no Ab-binding activity. The antigenic regions are analyzed in terms of their three-dimensional locations and the enzyme active site. With the previous localization of the antigenic regions on the BoNT/B H chain, the human Ab recognition of the entire BoNT/B molecule is presented and compared to the recognition of BoNT/A by human blocking Abs.


Subject(s)
Antibodies, Bacterial/immunology , Antibodies, Blocking/immunology , Botulinum Toxins/immunology , Immunity, Humoral , Neurotoxins/immunology , Peptide Fragments/immunology , Torticollis/immunology , Amino Acid Sequence , Animals , Antibodies, Bacterial/blood , Antibodies, Bacterial/chemistry , Antibodies, Blocking/blood , Antibodies, Blocking/genetics , Binding Sites, Antibody/genetics , Binding Sites, Antibody/immunology , Botulinum Toxins/administration & dosage , Botulinum Toxins/blood , Botulinum Toxins/chemistry , Botulinum Toxins, Type A/blood , Botulinum Toxins, Type A/chemistry , Botulinum Toxins, Type A/immunology , Clostridium botulinum/chemistry , Clostridium botulinum/immunology , Epitope Mapping , Humans , Immune Sera/immunology , Mice , Mice, Inbred ICR , Molecular Sequence Data , Neurotoxins/administration & dosage , Neurotoxins/blood , Neurotoxins/chemistry , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Binding/genetics , Protein Binding/immunology , Torticollis/blood , Torticollis/drug therapy , Torticollis/genetics , Treatment Failure
18.
Biotechnol Bioeng ; 109(6): 1461-70, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22170409

ABSTRACT

Sortase-mediated protein ligation is a biological covalent conjugation system developed from the enzymatic cell wall display mechanism found in Staphylococcus aureus. This three-component system requires: (i) purified Sortase A (SrtA) enzyme; (ii) a substrate containing the LPXTG peptide recognition sequence; and (iii) an oligo-glycine acceptor molecule. We describe cloning of the single-chain antibody sc528, which binds to the extracellular domain of the epidermal growth factor receptor (EGFR), from the parental monoclonal antibody and incorporation of a LPETGG tag sequence. Utilizing recombinant SrtA, we demonstrate successful incorporation of biotin from GGG-biotin onto sc528. EGFR is an important cancer target and is over-expressed in human tumor tissues and cancer lines, such as the A431 epithelial carcinoma cells. SrtA-biotinylated sc528 specifically bound EGFR expressed on A431 cells, but not negative control lines. Similarly, when sc528 was labeled with fluorescein we observed antigen-specific labeling. The ability to introduce functionality into recombinant antibodies in a controlled, site-specific manner has applications in experimental, diagnostic, and potentially clinical settings. For example, we demonstrate addition of all three reaction components in situ within a biosensor flow cell, resulting in oriented covalent capture and presentation of sc528, and determination of precise affinities for the antibody-receptor interaction.


Subject(s)
Aminoacyltransferases/metabolism , Bacterial Proteins/metabolism , Cysteine Endopeptidases/metabolism , ErbB Receptors/antagonists & inhibitors , Single-Chain Antibodies/metabolism , Staining and Labeling/methods , Aminoacyltransferases/genetics , Antibodies, Blocking/genetics , Antibodies, Blocking/immunology , Antibodies, Blocking/metabolism , Bacterial Proteins/genetics , Biotin/metabolism , Cell Line, Tumor , Cysteine Endopeptidases/genetics , ErbB Receptors/immunology , Humans , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology
19.
J Immunol ; 187(2): 1015-22, 2011 Jul 15.
Article in English | MEDLINE | ID: mdl-21690327

ABSTRACT

Multiple autoimmune diseases are characterized by the involvement of autoreactive Abs in pathogenesis. Problems associated with existing therapeutics such as the delivery of intravenous immunoglobulin have led to interest in developing alternative approaches using recombinant or synthetic methods. Toward this aim, in the current study, we demonstrate that the use of Fc-engineered Abs (Abs that enhance IgG degradation [Abdegs]) to block neonatal FcR (FcRn) through high-affinity, Fc region binding is an effective strategy for the treatment of Ab-mediated disease. Specifically, Abdegs can be used at low, single doses to treat disease in the K/B×N serum transfer model of arthritis using BALB/c mice as recipients. Similar therapeutic effects are induced by 25- to 50-fold higher doses of i.v. Ig. Importantly, we show that FcRn blockade is a primary contributing factor toward the observed reduction in disease severity. The levels of albumin, which is also recycled by FcRn, are not affected by Abdeg delivery. Consequently, Abdegs do not alter FcRn expression levels or subcellular trafficking behavior. The engineering of Ab Fc regions to generate potent FcRn blockers therefore holds promise for the therapy of Ab-mediated autoimmunity.


Subject(s)
Antibody Affinity , Arthritis, Experimental/immunology , Arthritis, Experimental/therapy , Immunoglobulin Fc Fragments/therapeutic use , Protein Engineering/methods , Receptors, Fc/antagonists & inhibitors , Animals , Anti-Inflammatory Agents, Non-Steroidal/metabolism , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Antibodies, Blocking/genetics , Antibodies, Blocking/metabolism , Antibodies, Blocking/therapeutic use , Antibody Affinity/genetics , Arthritis, Experimental/pathology , Glucose-6-Phosphate Isomerase/immunology , Histocompatibility Antigens Class I/metabolism , Humans , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin G/genetics , Immunoglobulin G/metabolism , Immunoglobulin G/therapeutic use , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Mice, Transgenic , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunology , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Receptors, Fc/deficiency , Receptors, Fc/metabolism , Recombinant Proteins/chemical synthesis , Recombinant Proteins/metabolism , Recombinant Proteins/therapeutic use , Severity of Illness Index
20.
Immunol Lett ; 135(1-2): 165-72, 2011 Mar 30.
Article in English | MEDLINE | ID: mdl-21078343

ABSTRACT

DC-SIGN is a C-type lectin of recognized importance in immunology and in the pathogenicity human pathogens. Monoclonal antibodies directed against DC-SIGN have been generated, but their systemic characterization for interfering with binding of the HIV-1 glycoprotein 120 has often been omitted. Moreover, so far, no anti-DC-SIGN monoclonal antibody has been described that recognizes its antigen after formalin fixation and paraffin embedding. In this study, we have generated new anti-DC-SIGN monoclonal antibodies using HeLa cells stably expressing DC-SIGN as immunogen. We have obtained 11 hybridoma clones producing antibodies that recognized DC-SIGN on monocyte-derived dendritic cells and on dermal-type macrophages. Seven monoclonal antibodies displayed a capacity to interfere with DC-SIGN binding to HIV-1 gp120. One recognized DC-SIGN on formalin-fixed dendritic cells and macrophages. Using this antibody we have obtained specific labelling of DC-SIGN and colocalisation with the dermal macrophage marker CD163 on human skin. The described monoclonal anti-human DC-SIGN antibodies will be of use to the scientific community to address fundamental immunology issues, in particular concerning macrophages and dendritic cells, and help elucidate infection events of pathogen targeting DC-SIGN as recognition receptor.


Subject(s)
Antibodies, Blocking/immunology , Antibodies, Monoclonal, Murine-Derived/immunology , Cell Adhesion Molecules/immunology , HIV Envelope Protein gp120/immunology , HIV-1/immunology , Lectins, C-Type/immunology , Receptors, Cell Surface/immunology , Animals , Antibodies, Blocking/genetics , Antibodies, Monoclonal, Murine-Derived/genetics , Antigens, CD/immunology , Antigens, Differentiation, Myelomonocytic/immunology , Cell Adhesion Molecules/genetics , Dendritic Cells/immunology , Dermis/immunology , Fixatives/chemistry , Formaldehyde/chemistry , HIV Envelope Protein gp120/genetics , HeLa Cells , Humans , Lectins, C-Type/genetics , Macrophages/immunology , Mice , Mice, Inbred BALB C , Monocytes/immunology , NIH 3T3 Cells , Receptors, Cell Surface/genetics
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