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1.
MAbs ; 16(1): 2362432, 2024.
Article in English | MEDLINE | ID: mdl-38849989

ABSTRACT

In contrast to natural antibodies that rely mainly on the heavy chain to establish contacts with their cognate antigen, we have developed a bispecific antibody format in which the light chain (LC) drives antigen binding and specificity. To better understand epitope-paratope interactions in this context, we determined the X-ray crystallographic structures of an antigen binding fragment (Fab) in complex with human CD47 and another Fab in complex with human PD-L1. These Fabs contain a κ-LC and a λ-LC, respectively, which are paired with an identical heavy chain (HC). The structural analysis of these complexes revealed the dominant contribution of the LCs to antigen binding, but also that the common HC provides some contacts in both CD47 and PD-L1 Fab complexes. The anti-CD47 Fab was affinity optimized by diversifying complementary-determining regions of the LC followed by phage display selections. Using homology modeling, the contributions of the amino acid modification to the affinity increase were analyzed. Our results demonstrate that, despite a less prominent role in natural antibodies, the LC can mediate high affinity binding to different antigens and neutralize their biological function. Importantly, Fabs containing a common variable heavy (VH) domain enable the generation of bispecific antibodies retaining a truly native structure, maximizing their therapeutic potential.


Subject(s)
Antibodies, Bispecific , B7-H1 Antigen , CD47 Antigen , Immunoglobulin Fab Fragments , Antibodies, Bispecific/chemistry , Antibodies, Bispecific/immunology , Humans , CD47 Antigen/immunology , CD47 Antigen/chemistry , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/immunology , B7-H1 Antigen/immunology , B7-H1 Antigen/chemistry , B7-H1 Antigen/antagonists & inhibitors , Crystallography, X-Ray , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/immunology , Models, Molecular
2.
J Hematol Oncol ; 16(1): 117, 2023 12 12.
Article in English | MEDLINE | ID: mdl-38087365

ABSTRACT

BACKGROUND: T-cell retargeting to eliminate CEACAM5-expressing cancer cells via CEACAM5xCD3 bispecific antibodies (BsAbs) showed limited clinical activity so far, mostly due to insufficient T-cell activation, dose-limiting toxicities, and formation of anti-drug antibodies (ADA). METHODS: We present here the generation and preclinical development of NILK-2301, a BsAb composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format). RESULTS: NILK-2301 binds CD3ɛ on T-cells with its lambda light chain arm with an affinity of ≈100 nM, and the CEACAM5 A2 domain on tumor cells by its kappa light chain arm with an affinity of ≈5 nM. FcγR-binding is abrogated by the "LALAPA" mutation (Leu234Ala, Leu235Ala, Pro329Ala). NILK-2301 induced T-cell activation, proliferation, cytokine release, and T-cell dependent cellular cytotoxicity of CEACAM5-positive tumor cell lines (5/5 colorectal, 2/2 gastric, 2/2 lung), e.g., SK-CO-1 (Emax = 89%), MKN-45 (Emax = 84%), and H2122 (Emax = 97%), with EC50 ranging from 0.02 to 0.14 nM. NILK-2301 binds neither to CEACAM5-negative or primary colon epithelial cells nor to other CEACAM family members. NILK-2301 alone or in combination with checkpoint inhibition showed activity in organotypic tumor tissue slices and colorectal cancer organoid models. In vivo, NILK-2301 at 10 mg/kg significantly delayed tumor progression in colon- and a pancreatic adenocarcinoma model. Single-dose pharmacokinetics (PK) and tolerability in cynomolgus monkeys at 0.5 or 10 mg/kg intravenously or 20 mg subcutaneously showed dose-proportional PK, bioavailability ≈100%, and a projected half-life in humans of 13.1 days. NILK-2301 was well-tolerated. Data were confirmed in human FcRn TG32 mice. CONCLUSIONS: In summary, NILK-2301 combines promising preclinical activity and safety with lower probability of ADA-generation due to its format compared to other molecules and is scheduled to enter clinical testing at the end of 2023.


Subject(s)
Adenocarcinoma , Antibodies, Bispecific , Pancreatic Neoplasms , Humans , Animals , Mice , Antibodies, Bispecific/pharmacology , Antibodies, Bispecific/therapeutic use , Adenocarcinoma/drug therapy , Pancreatic Neoplasms/drug therapy , Cell Line, Tumor , Immunotherapy , CD3 Complex , Carcinoembryonic Antigen , GPI-Linked Proteins
3.
Cell Rep ; 38(5): 110303, 2022 02 01.
Article in English | MEDLINE | ID: mdl-35108544

ABSTRACT

Across the animal kingdom, multivalency discriminates antibodies from all other immunoglobulin superfamily members. The evolutionary forces conserving multivalency above other structural hallmarks of antibodies remain, however, incompletely defined. Here, we engineer monovalent either Fc-competent or -deficient antibody formats to investigate mechanisms of protection of neutralizing antibodies (nAbs) and non-neutralizing antibodies (nnAbs) in virus-infected mice. Antibody bivalency enables the tethering of virions to the infected cell surface, inhibits the release of virions in cell culture, and suppresses viral loads in vivo independently of Fc gamma receptor (FcγR) interactions. In return, monovalent antibody formats either do not inhibit virion release and fail to protect in vivo or their protective efficacy is largely FcγR dependent. Protection in mice correlates with virus-release-inhibiting activity of nAb and nnAb rather than with their neutralizing capacity. These observations provide mechanistic insights into the evolutionary conservation of antibody bivalency and help refining correlates of nnAb protection for vaccine development.


Subject(s)
Antibodies, Viral/pharmacology , Antiviral Agents/pharmacology , HIV Antibodies/pharmacology , Receptors, Fc/drug effects , Animals , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/immunology , Epitopes/drug effects , Epitopes/immunology , HIV Antibodies/immunology , Immunoglobulin G/drug effects , Immunoglobulin G/immunology , Mice, Inbred C57BL , Receptors, IgG/drug effects , Receptors, IgG/immunology
4.
J Immunol Methods ; 500: 113182, 2022 01.
Article in English | MEDLINE | ID: mdl-34762914

ABSTRACT

Serology tests for SARS-CoV-2 have proven to be important tools to fight against the COVID-19 pandemic. These serological tests can be used in low-income and remote areas for patient contact tracing, epidemiologic studies and vaccine efficacy evaluations. In this study, we used a semi-stable mammalian episomal expression system to produce high quantities of the receptor-binding domain-RBD of SARS-CoV-2 in a simple and very economical way. The recombinant antigen was tested in an in-house IgG ELISA for COVID-19 with a panel of human sera. A performance comparison of this serology test with a commercial test based on the full-length spike protein showed 100% of concordance between tests. Thus, this serological test can be an attractive and inexpensive option in scenarios of limited resources to face the COVID-19 pandemic.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/economics , COVID-19 Serological Testing/economics , Costs and Cost Analysis , Enzyme-Linked Immunosorbent Assay , Genetic Engineering , Humans , Immunoglobulin G/genetics , Immunoglobulin G/metabolism , Protein Binding , Protein Interaction Domains and Motifs/genetics , Spike Glycoprotein, Coronavirus/genetics
5.
J Vet Res ; 65(3): 315-321, 2021 Sep.
Article in English | MEDLINE | ID: mdl-34917844

ABSTRACT

INTRODUCTION: Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) are cytokines widely used in ex vivo monocyte differentiation experiments, vaccine formulations and disease treatment. The aim of this study was to produce recombinant bovine GM-CSF and IL-4 in an episomal expression system that conserves the postransductional modification of the native proteins and to use the products to differentiate bovine monocytes into dendritic cells. MATERIAL AND METHODS: The recombinant proteins rGM-CSF and rIL-4 were expressed in PEAKrapid CRL-2828 human kidney cells, ATCC CRL-2828. The functional activity of the recombinant cytokines was monitored by registering morphological changes in bovine monocytes and assessing the expression of CD14 upon incubation with them. RESULTS: Both recombinant proteins were detected in the cell culture supernatant of transfected cells. Culture supernatants of transfected cells induced in bovine monocytes morphological changes that resemble macrophages or dendritic cells. In addition, bovine cells treated with rGM-CSF and rIL-4 showed reduced expression of the macrophage surface marker CD14 compared with untreated cells. This effect indicates the expected differentiation. The expression of the cytokines was stable after many successive cell passages and a freeze/thaw cycle. CONCLUSIONS: The semi-stable mammalian episomal expression system used in this study allowed us to easily produce functional bovine rGM-CSF and rIL-4 without the need for protein purification steps.

6.
Antibodies (Basel) ; 7(3)2018 Aug 10.
Article in English | MEDLINE | ID: mdl-31544881

ABSTRACT

Bispecific antibodies (bsAbs) are often composed of several polypeptide chains that have to be expressed adequately to enable optimal assembly and yield of the bsAb. κλ bodies are a bispecific format with a native IgG structure, composed of two different light chains that pair with a common heavy chain. Introduction of non-optimal codons into the sequence of a particular polypeptide is an effective strategy for down modulating its expression. Here we applied this strategy but restricted the modification of the codon content to the constant domain of one light chain. This approach facilitates parallel optimization of several bsAbs by using the same modified constant domains. Partial sequence de-optimization reduced expression of the targeted polypeptide. Stable cell pools could be isolated displaying increased bispecific antibody titers as well as changes in the abundance of undesired by-products that require elimination during downstream processing. Thus, modulating the relative expression of polypeptides can have a significant impact on bsAb titer and product related impurities; which are important factors for large scale manufacturing for clinical supply.

7.
MAbs ; 9(2): 231-239, 2017.
Article in English | MEDLINE | ID: mdl-28001485

ABSTRACT

When production of bispecific antibodies requires the co-expression and assembly of three or four polypeptide chains, low expression of one chain can significantly limit assembly and yield. κλ bodies, fully human bispecific antibodies with native IgG structure, are composed of a common heavy chain and two different light chains, one kappa and one lambda. No engineering is applied to force pairing of the chains, thus both monospecific and bispecific antibodies are secreted in the supernatant. In this context, stoichiometric expression of the two light chains allows for maximal assembly of the bispecific antibody. In this study, we selected a κλ body with suboptimal characteristics due to low kappa chain expression. Codon optimization to increase expression of the kappa chain did not improve bispecific yield. Surprisingly, progressive introduction of non-optimal codons into the sequence of the lambda chain resulted in lowering its expression for an optimal tuning of the relative distribution of monospecific and bispecific antibodies. This codon de-optimization led to doubling of the κλ body yield. These results indicate that assembly of different proteins into a recombinant complex is an interconnected process and that reducing the expression of one polypeptide can actually increase the overall yield.


Subject(s)
Antibodies, Bispecific/biosynthesis , Protein Engineering/methods , Animals , Codon , Humans , Immunoglobulin G/biosynthesis , Immunoglobulin kappa-Chains/biosynthesis , Immunoglobulin kappa-Chains/genetics , Immunoglobulin lambda-Chains/biosynthesis , Immunoglobulin lambda-Chains/genetics
8.
J Exp Med ; 213(2): 177-87, 2016 Feb 08.
Article in English | MEDLINE | ID: mdl-26809444

ABSTRACT

Evidence has recently emerged that butyrophilins, which are members of the extended B7 family of co-stimulatory molecules, have diverse functions in the immune system. We found that the human and mouse genes encoding butyrophilin-2A2 (BTN2A2) are regulated by the class II trans-activator and regulatory factor X, two transcription factors dedicated to major histocompatibility complex class II expression, suggesting a role in T cell immunity. To address this, we generated Btn2a2-deficient mice. Btn2a2(-/-) mice exhibited enhanced effector CD4(+) and CD8(+) T cell responses, impaired CD4(+) regulatory T cell induction, potentiated antitumor responses, and exacerbated experimental autoimmune encephalomyelitis. Altered immune responses were attributed to Btn2a2 deficiency in antigen-presenting cells rather than T cells or nonhematopoietic cells. These results provide the first genetic evidence that BTN2A2 is a co-inhibitory molecule that modulates T cell-mediated immunity.


Subject(s)
Genes, MHC Class II , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Animals , Antigen-Presenting Cells/immunology , Butyrophilins , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/immunology , Cell Line , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/immunology , Gene Expression Regulation , Humans , Immunity, Cellular , Membrane Glycoproteins/deficiency , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins/genetics , Nuclear Proteins/immunology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Regulatory Factor X Transcription Factors , Trans-Activators/genetics , Trans-Activators/immunology , Transcription Factors/genetics , Transcription Factors/immunology
9.
PLoS Pathog ; 11(11): e1005276, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26587982

ABSTRACT

Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein's globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Arenavirus/immunology , Hemorrhagic Fevers, Viral/immunology , Polysaccharides/immunology , Animals , HIV Antibodies/immunology , HIV-1/immunology , Humans , Mice, Inbred C57BL , Molecular Sequence Data
10.
J Biol Chem ; 290(45): 26943-26953, 2015 Nov 06.
Article in English | MEDLINE | ID: mdl-26363066

ABSTRACT

The IL-6 signaling complex is described as a hexamer, formed by the association of two IL-6·IL-6 receptor (IL-6R)·gp130 trimers, with gp130 being the signal transducer inducing cis- and trans-mediated signaling via a membrane-bound or soluble form of the IL-6R, respectively. 25F10 is an anti-mouse IL-6R mAb that binds to both membrane-bound IL-6R and soluble IL-6R with the unique property of specifically inhibiting trans-mediated signaling events. In this study, epitope mapping revealed that 25F10 interacts at site IIb of IL-6R but allows the binding of IL-6 to the IL-6R and the recruitment of gp130, forming a trimer complex. Binding of 25F10 to IL-6R prevented the formation of the hexameric complex obligate for trans-mediated signaling, suggesting that the cis- and trans-modes of IL-6 signaling adopt different mechanisms for receptor complex assembly. To study this phenomenon also in the human system, we developed NI-1201, a mAb that targets, in the human IL-6R sequence, the epitope recognized by 25F10 for mice. Interestingly, NI-1201, however, did not selectively inhibit human IL-6 trans-signaling, although both mAbs produced beneficial outcomes in conditions of exacerbated IL-6 as compared with a site I-directed mAb. These findings shed light on the complexity of IL-6 signaling. First, triggering cis- versus trans-mediated IL-6 signaling occurs via distinctive mechanisms for receptor complex assembly in mice. Second, the formation of the receptor complex leading to cis- and trans-signaling biology in mice and humans is different, and this should be taken into account when developing strategies to inhibit IL-6 clinically.


Subject(s)
Interleukin-6/chemistry , Interleukin-6/metabolism , Receptors, Interleukin-6/chemistry , Receptors, Interleukin-6/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Cytokine Receptor gp130/chemistry , Cytokine Receptor gp130/metabolism , Female , Genetic Complementation Test , Humans , Interleukin-6/deficiency , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Knockout , Models, Molecular , Molecular Sequence Data , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , NIH 3T3 Cells , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Quaternary , Rats , Receptors, Interleukin-6/genetics , Sequence Homology, Amino Acid , Signal Transduction
11.
J Mol Biol ; 427(16): 2647-62, 2015 Aug 14.
Article in English | MEDLINE | ID: mdl-26013163

ABSTRACT

Hu 15C1 is a potent anti-human Toll-like receptor 4 (TLR4) neutralizing antibody. To better understand the molecular basis of its biological activity, we used a multidisciplinary approach to generate an accurate model of the Hu 15C1-TLR4 complex. By combining site-directed mutagenesis, in vitro antibody evolution, affinity measurements and X-ray crystallography of Fab fragments, we identified key interactions across the Hu 15C1-TLR4 interface. These contact points were used as restraints to predict the structure of the Fab region of Hu 15C1 bound to TLR4 using computational molecular docking. This model was further evaluated and validated by additional site-directed mutagenesis studies. The predicted structure of the Hu 15C1-TLR4 complex indicates that the antibody antagonizes the receptor dimerization necessary for its activation. This study exemplifies how iterative cycles of antibody engineering can facilitate the discovery of components of antibody-target interactions.


Subject(s)
Antibodies, Neutralizing/immunology , Antigen-Antibody Complex/ultrastructure , Binding Sites, Antibody/immunology , Immunoglobulin Fab Fragments/ultrastructure , Toll-Like Receptor 4/immunology , Amino Acid Sequence , Animals , Antigen-Antibody Complex/immunology , CHO Cells , Cell Line , Cell Surface Display Techniques , Computer Simulation , Cricetinae , Cricetulus , Crystallography, X-Ray , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Humans , Immunoglobulin Fab Fragments/immunology , Models, Molecular , Molecular Docking Simulation , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Sequence Alignment , Species Specificity , Surface Plasmon Resonance
12.
Nat Commun ; 6: 6113, 2015 Feb 12.
Article in English | MEDLINE | ID: mdl-25672245

ABSTRACT

Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies.


Subject(s)
Antibodies, Bispecific/isolation & purification , Immunoglobulin G/isolation & purification , Immunoglobulin Heavy Chains/isolation & purification , Protein Engineering/methods , Antibodies, Monoclonal/metabolism , Chromatography, High Pressure Liquid , Humans , Immunoglobulin Light Chains/metabolism , Immunoglobulin kappa-Chains/metabolism , Neutralization Tests , Peptide Library , T-Lymphocytes/immunology
13.
Biochim Biophys Acta ; 1843(12): 2913-25, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25229427

ABSTRACT

Deoxyribose-phosphate aldolase (EC 4.1.2.4), which converts 2-deoxy-d-ribose-5-phosphate into glyceraldehyde-3-phosphate and acetaldehyde, belongs to the core metabolism of living organisms. It was previously shown that human cells harbor deoxyribose phosphate aldolase activity but the protein responsible of this activity has never been formally identified. This study provides the first experimental evidence that DERA, which is mainly expressed in lung, liver and colon, is the human deoxyribose phosphate aldolase. Among human cell lines, the highest DERA mRNA level and deoxyribose phosphate aldolase activity were observed in liver-derived Huh-7 cells. DERA was shown to interact with the known stress granule component YBX1 and to be recruited to stress granules after oxidative or mitochondrial stress. In addition, cells in which DERA expression was down-regulated using shRNA formed fewer stress granules and were more prone to apoptosis after clotrimazole stress, suggesting the importance of DERA for stress granule formation. Furthermore, the expression of DERA was shown to permit cells in which mitochondrial ATP production was abolished to make use of extracellular deoxyinosine to maintain ATP levels. This study unraveled a previously undescribed pathway which may allow cells with high deoxyribose-phosphate aldolase activity, such as liver cells, to minimize or delay stress-induced damage by producing energy through deoxynucleoside degradation.

14.
Nat Biotechnol ; 32(5): 485-9, 2014 May.
Article in English | MEDLINE | ID: mdl-24752077

ABSTRACT

Heterogeneity in the N-glycans on therapeutic proteins causes difficulties for protein purification and process reproducibility and can lead to variable therapeutic efficacy. This heterogeneity arises from the multistep process of mammalian complex-type N-glycan synthesis. Here we report a glycoengineering strategy--which we call GlycoDelete--that shortens the Golgi N-glycosylation pathway in mammalian cells. This shortening results in the expression of proteins with small, sialylated trisaccharide N-glycans and reduced complexity compared to native mammalian cell glycoproteins. GlycoDelete engineering does not interfere with the functioning of N-glycans in protein folding, and the physiology of cells modified by GlycoDelete is similar to that of wild-type cells. A therapeutic human IgG expressed in GlycoDelete cells had properties, such as reduced initial clearance, that might be beneficial when the therapeutic goal is antigen neutralization. This strategy for reducing N-glycan heterogeneity on mammalian proteins could lead to more consistent performance of therapeutic proteins and modulation of biopharmaceutical functions.


Subject(s)
Polysaccharides/genetics , Protein Engineering/methods , Recombinant Proteins/genetics , Animals , Glycosylation , Humans , Mice , Polysaccharides/chemistry , Polysaccharides/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
15.
J Biol Chem ; 289(22): 15309-18, 2014 May 30.
Article in English | MEDLINE | ID: mdl-24737331

ABSTRACT

Inflammation is mediated mainly by leukocytes that express both Toll-like receptor 4 (TLR4) and Fc γ receptors (FcγR). Dysregulated activation of leukocytes via exogenous and endogenous ligands of TLR4 results in a large number of inflammatory disorders that underlie a variety of human diseases. Thus, differentially blocking inflammatory cells while sparing structural cells, which are FcγR-negative, represents an elegant strategy when targeting the underlying causes of human diseases. Here, we report a novel tethering mechanism of the Fv and Fc portions of anti-TLR4 blocking antibodies that achieves increased potency on inflammatory cells. In the presence of ligand (e.g. lipopolysaccharide (LPS)), TLR4 traffics into glycolipoprotein microdomains, forming concentrated protein platforms that include FcγRs. This clustering produces a microenvironment allowing anti-TLR4 antibodies to co-engage TLR4 and FcγRs, increasing their avidity and thus substantially increasing their inhibitory potency. Tethering of antibodies to both TLR4 and FcγRs proves valuable in ameliorating inflammation in vivo. This novel mechanism of action therefore has the potential to enable selective intervention of relevant cell types in TLR4-driven diseases.


Subject(s)
Inflammation/immunology , Macrophages/immunology , Receptors, IgG/immunology , Toll-Like Receptor 4/immunology , Animals , Antibodies, Monoclonal/immunology , Binding Sites , CHO Cells , Cell Line , Cricetulus , Dimerization , Female , Humans , Inflammation/metabolism , Macrophages/cytology , Membrane Microdomains/immunology , Membrane Microdomains/metabolism , Mice , Mice, Inbred C57BL , Receptors, IgG/metabolism , Toll-Like Receptor 4/chemistry , Toll-Like Receptor 4/metabolism , U937 Cells
16.
Methods Mol Biol ; 1131: 3-20, 2014.
Article in English | MEDLINE | ID: mdl-24515456

ABSTRACT

The quality of the target antigen is very important in order to generate a good antibody, in particular when binding to a conformational epitope is desired. The use of mammalian cells for recombinant protein expression provides an efficient machinery for the correct folding and posttranslational modification of proteins. In this chapter, we describe a process to rapidly generate semi-stable human cell lines secreting a recombinant protein of interest into the culture medium. Simple disposable bioreactors that can be used in any standard cell culture laboratory enable the production of recombinant protein in the multi-milligram range. The protein can be readily purified from the culture supernatant by immobilized metal affinity chromatography. In addition, by inserting a tag recognized by a co-expressed biotin ligase, the protein can be biotinylated during the secretion process. This greatly facilitates the immobilization of the protein for assay development or for antibody isolation using in vitro selection technologies.


Subject(s)
Antibodies, Monoclonal/immunology , Antigens/immunology , Animals , Antigens/genetics , Antigens/metabolism , Bioreactors , Chromatography, Affinity , Humans , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism
17.
Methods Mol Biol ; 1131: 253-61, 2014.
Article in English | MEDLINE | ID: mdl-24515471

ABSTRACT

Magnetic microspheres represent an interesting alternative to conventional chromatography resins in automated high-throughput protocols replacing centrifugation and filtration by magnetic separation. Some magnetic microspheres have unique features like high magnetite content and non-porous surface which allows them to migrate very fast in magnetic fields while binding target molecules with a low unspecific adsorption. Here, we describe the use of protein A or protein G-coated magnetic microspheres to purify quickly monoclonal antibodies from crude serum-free supernatants without the need of preliminary clarification or purification step. Using this method, multiple samples can be processed in parallel, a high level of purity can be obtained, and the purified IgG maintain their biological activity.


Subject(s)
Antibodies, Monoclonal/isolation & purification , Bacterial Proteins/chemistry , Microspheres , Staphylococcal Protein A/chemistry
18.
J Immunol ; 192(4): 1641-50, 2014 Feb 15.
Article in English | MEDLINE | ID: mdl-24442438

ABSTRACT

B cells play a major role in the pathogenesis of many autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and type I diabetes mellitus, as indicated by the efficacy of B cell-targeted therapies in these diseases. Therapeutic effects of the most commonly used B cell-targeted therapy, anti-CD20 mAb, are contingent upon long-term depletion of peripheral B cells. In this article, we describe an alternative approach involving the targeting of CD79, the transducer subunit of the B cell AgR. Unlike anti-CD20 mAbs, the protective effects of CD79-targeted mAbs do not require cell depletion; rather, they act by inducing an anergic-like state. Thus, we describe a novel B cell-targeted approach predicated on the induction of B cell anergy.


Subject(s)
Autoimmune Diseases/prevention & control , B-Lymphocytes/immunology , CD79 Antigens/immunology , Clonal Anergy/immunology , Animals , Antibodies, Monoclonal/immunology , Autoimmunity/immunology , Female , Lymphocyte Activation/immunology , Lymphocyte Count , Lymphocyte Depletion , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Knockout
19.
J Immunol Methods ; 375(1-2): 20-9, 2012 Jan 31.
Article in English | MEDLINE | ID: mdl-21939661

ABSTRACT

The MHC class-I related receptor or neonatal Fc receptor (FcRn) protects IgG and albumin from degradation by rescuing them in endothelial cells in a pH dependent fashion and consequently increases their respective half-lives. Monoclonal antibody-based therapies are of increasing interest and characterizing the interaction with FcRn is important for the development of an antibody candidate. In order to facilitate the production of soluble FcRn suitable for interaction studies, we generated semi-stable pools co-expressing FcRn α-chain, ß2-microglobulin, biotin ligase and EGFP using a dual promoter, multi-cistronic vector. Human and mouse FcRn were purified in the mg/L range of culture medium and a single purification step was sufficient to reach a high level of purity. The receptors were characterized by ELISA, flow cytometry and surface plasmon resonance and shown to be functional. The single site biotinylation facilitated the directional immobilization of FcRn on the sensor chip and significantly increased the response level of the surface compared to amine coupling used in previous studies. Using this system, the affinity constants of seven IgGs, from various species and isotypes, were determined for human and mouse FcRn, including two hamster isotypes. These results confirm the higher selectivity of the human receptor and the promiscuous binding of mFcRn to IgGs from different species.


Subject(s)
Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Receptors, Fc/genetics , Receptors, Fc/immunology , Animals , Antibodies, Monoclonal/immunology , Biotinylation/methods , Cells, Cultured , Cricetinae , DNA, Complementary/genetics , Genetic Vectors/genetics , Histocompatibility Antigens Class I/biosynthesis , Humans , Immunoglobulin G/immunology , Kinetics , Mice , Protein Binding , Receptors, Fc/biosynthesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Surface Plasmon Resonance/methods
20.
J Biol Chem ; 287(2): 1458-67, 2012 Jan 06.
Article in English | MEDLINE | ID: mdl-22041899

ABSTRACT

Dual-specific antibodies are characterized by an antigen-combining site mediating specific interactions with two different antigens. We have generated five dual-specific single chain variable fragments (scFv) that neutralize the activity of the two chemokines, CXCL9 and CXCL10, to bind to their receptor CXCR3. To better understand how these dual-specific scFvs bind these two chemokines that only share a 37% sequence identity, we mapped their epitopes on human CXCL9 and CXCL10 and identified serine 13 (Ser(13)) as a critical residue. It is conserved between the two chemokines but not in the third ligand for CXCR3, CXCL11. Furthermore, Ser(13) is exposed in the tetrameric structure of CXCL10, which is consistent with our finding that the scFvs are able to bind to CXCL9 and CXCL10 immobilized on glycosaminoglycans. Overall, the data indicate that these dual-specific scFvs bind to a conserved surface involved in CXCR3 receptor interaction for CXCL10 and CXCL9. Thus, structural mimicry between the two targets is likely to be responsible for the observed dual specificity of these antibody fragments.


Subject(s)
Antibody Specificity , Chemokine CXCL10/chemistry , Chemokine CXCL9/chemistry , Molecular Mimicry , Single-Chain Antibodies/chemistry , Animals , Chemokine CXCL10/genetics , Chemokine CXCL10/immunology , Chemokine CXCL11/chemistry , Chemokine CXCL11/genetics , Chemokine CXCL11/immunology , Chemokine CXCL9/genetics , Chemokine CXCL9/immunology , Humans , Macaca fascicularis , Macaca mulatta , Mice , Rabbits , Receptors, CXCR3/chemistry , Receptors, CXCR3/genetics , Receptors, CXCR3/immunology , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology
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