Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 13 de 13
Filter
1.
Methods ; 60(1): 99-110, 2013 Mar 15.
Article in English | MEDLINE | ID: mdl-23500657

ABSTRACT

The use of next generation sequencing (NGS) for the analysis of antibody sequences both in phage display libraries and during in vitro selection processes has become increasingly popular in the last few years. Here, our methods developed for DNA preparation, sequencing and data analysis are presented. A key parameter has also been to develop new software designed for high throughput antibody sequence analysis that is used in combination with publicly available tools. As an example of our methods, we provide data from the extensive analysis of five scFv libraries generated using different heavy chain CDR3 diversification strategies. The results not only confirm that the library designs were correct but also reveal differences in quality not easily identified by standard DNA sequencing approaches. The very large number of reads permits extensive sequence coverage after the selection process. Furthermore, as samples can be multiplexed, costs decrease and more information is gained per NGS run. Using examples of results obtained post phage display selections against two antigens, frequency and clustering analysis identified novel antibody fragments that were then shown to be specific for the target antigen. In summary, the methods described here demonstrate how NGS analysis enhances quality control of complex antibody libraries as well as facilitates the antibody discovery process.


Subject(s)
Antibodies/chemistry , Bacteriophages/genetics , Drug Discovery , High-Throughput Nucleotide Sequencing , Peptide Library
2.
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
3.
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
4.
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
5.
Protein Expr Purif ; 72(2): 209-16, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20399863

ABSTRACT

Many research projects in life sciences require purified biologically active recombinant protein. In addition, different formats of a given protein may be needed at different steps of experimental studies. Thus, the number of protein variants to be expressed and purified in short periods of time can expand very quickly. We have therefore developed a rapid and flexible expression system based on described episomal vector replication to generate semi-stable cell pools that secrete recombinant proteins. We cultured these pools in serum-containing medium to avoid time-consuming adaptation of cells to serum-free conditions, maintain cell viability and reuse the cultures for multiple rounds of protein production. As such, an efficient single step affinity process to purify recombinant proteins from serum-containing medium was optimized. Furthermore, a series of multi-cistronic vectors were designed to enable simultaneous expression of proteins and their biotinylation in vivo as well as fast selection of protein-expressing cell pools. Combining these improved procedures and innovative steps, exemplified with seven cytokines and cytokine receptors, we were able to produce biologically active recombinant endotoxin free protein at the milligram scale in 4-6weeks from molecular cloning to protein purification.


Subject(s)
Cloning, Molecular/methods , Plasmids/genetics , Recombinant Proteins/biosynthesis , Animals , Biotin/genetics , Biotin/metabolism , Cells, Cultured , Cytokines/biosynthesis , Cytokines/genetics , Enzyme-Linked Immunosorbent Assay , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Humans , Interleukins/biosynthesis , Interleukins/genetics , Mice , Rats , Receptors, Cytokine/biosynthesis , Receptors, Cytokine/genetics , Recombinant Proteins/genetics
6.
Protein Eng Des Sel ; 30(9): 575-582, 2017 09 01.
Article in English | MEDLINE | ID: mdl-28444391

ABSTRACT

Antibody phage display technology has supported the emergence of numerous therapeutic antibodies. The development of bispecific antibodies, a promising new frontier in antibody therapy, could be facilitated by new phage display approaches that enable pairs of antibodies to be co-selected based on co-engagement of their respective targets. We describe such an approach, making use of two complementary leucine zipper domains that heterodimerize with high affinity. Phagemids encoding a first antibody fragment (scFv) fused to phage coat protein via the first leucine zipper are rescued in bacteria expressing a second scFv fused to the second leucine zipper as a soluble periplasmic protein, so that it is acquired by phage during assembly. Using a soluble scFv specific for a human CD3-derived peptide, we show that its acquisition by phage displaying an irrelevant antibody is sufficiently robust to drive selection of rare phage (1 in 10(5)) over three rounds of panning. We then set up a model selection experiment using a cell line expressing the chemokine receptor CCR5 fused to the CD3 peptide together with a panel of phage clones capable displaying either an anti-CCR5 scFv or an irrelevant antibody, with or without the capacity to acquire the soluble anti-CD3 scFv. In this experiment we showed that rare phage (1 in 10(5)) capable of displaying the two different scFvs can be specifically enriched over four rounds of panning. This approach has the potential to be applied to the identification of pairs of ligands capable of co-engaging two different user-defined targets, which would facilitate the discovery of novel bispecific antibodies.


Subject(s)
Antibodies, Bispecific/biosynthesis , Antibodies, Monoclonal/biosynthesis , Bacteriophages/genetics , Peptide Library , Recombinant Fusion Proteins/genetics , Single-Chain Antibodies/biosynthesis , Amino Acid Sequence , Animals , Antibodies, Bispecific/genetics , Antibodies, Monoclonal/genetics , Antibody Specificity , Bacteriophages/immunology , CD3 Complex/genetics , CD3 Complex/immunology , CHO Cells , Capsid Proteins/genetics , Capsid Proteins/immunology , Cricetulus , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli/virology , Gene Expression , Humans , Leucine Zippers , Peptides/genetics , Peptides/immunology , Receptors, CCR5/genetics , Receptors, CCR5/immunology , Recombinant Fusion Proteins/immunology , Sequence Analysis, DNA , Single-Chain Antibodies/genetics
7.
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
8.
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
9.
MAbs ; 7(2): 294-302, 2015.
Article in English | MEDLINE | ID: mdl-25608219

ABSTRACT

pH-dependent antibodies are engineered to release their target at a slightly acidic pH, a property making them suitable for clinical as well as biotechnological applications. Such antibodies were previously obtained by histidine scanning of pre-existing antibodies, a labor-intensive strategy resulting in antibodies that displayed residual binding to their target at pH 6.0. We report here the de novo isolation of pH-dependent antibodies selected by phage display from libraries enriched in histidines. Strongly pH-dependent clones with various affinity profiles against CXCL10 were isolated by this method. Our best candidate has nanomolar affinity for CXCL10 at pH 7.2, but no residual binding was detected at pH 6.0. We therefore propose that this new process is an efficient strategy to generate pH-dependent antibodies.


Subject(s)
Chemokine CXCL10/chemistry , Protein Engineering , Single-Chain Antibodies/chemistry , Chemokine CXCL10/genetics , Humans , Hydrogen-Ion Concentration , Single-Chain Antibodies/genetics
10.
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
11.
PLoS One ; 7(8): e43471, 2012.
Article in English | MEDLINE | ID: mdl-22937053

ABSTRACT

Antibody repertoires are characterized by diversity as they vary not only amongst individuals and post antigen exposure but also differ significantly between vertebrate species. Such plasticity can be exploited to generate human antibody libraries featuring hallmarks of these diverse repertoires. In this study, the focus was to capture CDRH3 sequences, as this region generally accounts for most of the interaction energy with antigen. Sequences from human as well as non-human sources were successfully integrated into human antibody libraries. Next generation sequencing of these libraries proved that the CDRH3 lengths and amino acid composition corresponded to the species of origin. Specific CDRH3 sequences, biased towards the recognition of a model antigen either by immunizing mice or by selecting with phage display, were then integrated into another set of libraries. From these antigen biased libraries, highly potent antibodies were more frequently isolated, indicating that the characteristics of an immune repertoire is transferrable via CDRH3 sequences into a human antibody library. Taken together, these data demonstrate that the properties of naturally or experimentally biased repertoires can be effectively harnessed for the generation of targeted human antibody libraries, substantially increasing the probability of isolating antibodies suitable for therapeutic and diagnostic applications.


Subject(s)
Antibodies/genetics , Animals , Antibodies/immunology , Enzyme-Linked Immunosorbent Assay , Humans , Mice , Peptide Library
12.
MAbs ; 1(3): 288-96, 2009.
Article in English | MEDLINE | ID: mdl-20069756

ABSTRACT

Chemokines are important mediators of the immune response that are responsible for the trafficking of immune cells between lymphoid organs and migration towards sites of inflammation.Using phage display selection and a functional screening approach, we have isolated a panel of single-chain fragment variable (scFv) capable of neutralizing the activity of the human chemokine CXCL10 (hCXCL10). One of the isolated scFv was weakly cross-reactive against another human chemokine CXCL9,but was unable to block its biological activity. We diversified the complementarity determining region 3 (CDR3) of the light chain variable domain (VL) of this scFv and combined phage display with high throughput antibody array screening to identify variants capable of neutralizing both chemokines. Using this approach it is therefore possible to engineer pan-specific antibodies that could prove very useful to antagonize redundant signaling pathways such as the chemokine signaling network.


Subject(s)
Antibodies, Blocking/metabolism , Chemokine CXCL10/immunology , Chemokine CXCL9/immunology , Epitopes/metabolism , Single-Chain Antibodies/metabolism , Antibodies, Blocking/chemistry , Antibodies, Blocking/immunology , Antibody Affinity , Combinatorial Chemistry Techniques , Complementarity Determining Regions/chemistry , Cross Reactions , Epitopes/chemistry , Epitopes/immunology , High-Throughput Screening Assays , Humans , Immunoglobulin Light Chains/chemistry , Peptide Library , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/immunology
13.
Biochem Biophys Res Commun ; 334(2): 370-5, 2005 Aug 26.
Article in English | MEDLINE | ID: mdl-16004969

ABSTRACT

Chemokines are a class of low molecular weight proteins that are involved in leukocytes trafficking. Due to their involvement in recruiting immune cells to sites of inflammation, chemokines, and chemokine receptors have become an attractive class of therapeutic targets. However, when expressed in Escherichia coli chemokines are poorly soluble and accumulate in inclusion bodies. Several purification methods have been described but involve time-consuming refolding, buffer exchange, and purification steps that complicate expression of these proteins. Here, we describe a simple and reliable method to express chemokines as fusions to the protein NusA. The fusion proteins were largely found in the soluble fraction and could be readily purified in a single step. Proteolytic cleavage was used to obtain soluble recombinant chemokines that were found to be very active in a novel in vitro chemotaxis assays. This method could be applied to several alpha and beta human chemokines, suggesting that it is generally applicable to this class of proteins.


Subject(s)
Chemokines/biosynthesis , Chemokines/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Lymphoma/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Animals , Cell Line, Tumor , Chemokines/analysis , Chemokines/genetics , Cloning, Molecular/methods , Lymphoma/genetics , Mice , Protein Engineering , Recombinant Fusion Proteins/analysis , Solubility
SELECTION OF CITATIONS
SEARCH DETAIL