Identification of engineered IMGT Fc variants in IMGT/mAb-DB, a database of therapeutic antibodies and fusion proteins

Antibody-dependent cellular cytotoxicity (ADCC) is mediated through the engagement of the Fc segment of antibodies with Fcγ receptors (FcγRs) on immune cells upon binding of tumor or viral antigen. The co-crystal structure of FcγRIII in complex with Fc revealed that Fc binds to FcγRIII asymmetrically with two Fc chains contacting separate regions of the FcγRIII by utilizing different residues. To fully explore this asymmetrical nature of the Fc-FcγR interaction, we screened more than 9,000 individual clones in Fc heterodimer format in which different mutations were introduced at the same position of two Fc chains using a high throughput competition AlphaLISA® assay. To this end, we have identified a panel of novel Fc variants with significant binding improvement to FcγRIIIA (both Phe-158 and Val-158 allotypes), increased ADCC activity in vitro, and strong tumor growth inhibition in mice xenograft human tumor models. Compared with previously identified Fc variants in conventional IgG format, Fc heterodimers with asymmetrical mutations can achieve similar or superior potency in ADCC-mediated tumor cell killing and demonstrate improved stability in the CH2 domain. Fc heterodimers also allow more selectivity toward activating FcγRIIA than inhibitory FcγRIIB. Afucosylation of Fc variants further increases the affinity of Fc to FcγRIIIA, leading to much higher ADCC activity. The discovery of these Fc variants will potentially open up new opportunities of building the next generation of therapeutic antibodies with enhanced ADCC effector function for the treatment of cancers and infectious diseases.

Many therapeutic proteins possessing a small size are rapidly cleared from circulation. Half-life extension strategies have therefore become increasingly important to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Here, we performed a comparative analysis of the half-life extension properties of various bacterial immunoglobulin-binding domains (IgBDs) derived from Staphylococcus protein A (SpA), Streptococcus protein G (SpG), and Finegoldia (formerly Peptostreptococcus) protein L (PpL). These domains, composed of 50-60 amino acid residues, were fused to the C terminus of a single-chain Fv and a bispecific single-chain diabody, respectively. All fusion proteins were produced in mammalian cells and retained their antigen-binding properties. The half-lives of the antibody molecules were prolonged to varying extents for the different IgBDs. The strongest effects in mice were observed for domain C3 of SpG (SpG(C3)) followed by domains B and D of SpA, suggesting that SpG(C3) is particularly useful to extend the plasma half-life of small proteins.

Antibody fragments are emerging as promising biopharmaceuticals because of their relatively small size and other unique properties. However, compared with full-size antibodies, these antibody fragments lack the ability to bind the neonatal Fc receptor (FcRn) and have reduced half-lives. Fc engineered to bind antigens but preserve interactions with FcRn and Fc fused with monomeric proteins currently are being developed as candidate therapeutics with prolonged half-lives; in these and other cases, Fc is a dimer of two CH2-CH3 chains. To further reduce the size of Fc but preserve FcRn binding, we generated three human soluble monomeric IgG1 Fcs (mFcs) by using a combination of structure-based rational protein design combined with multiple screening strategies. These mFcs were highly soluble and retained binding to human FcRn comparable with that of Fc. These results provide direct experimental evidence that efficient binding to human FcRn does not require human Fc dimerization. The newly identified mFcs are promising for the development of mFc fusion proteins and for novel types of mFc-based therapeutic antibodies of small size and long half-lives.

Comparing Phylogeny and the Predicted Pathogenicity of Protein Variations Reveals Equal Purifying Selection across the Global Human mtDNA Diversity

Effect of the ADCC-Modulating Mutations and the Selection of Human IgG Isotypes on Physicochemical Properties of Fc

Myasthenia Gravis (MG) is a rare autoimmune disease presenting with auto-antibodies that affect the neuromuscular junction. In addition to symptomatic treatment options, novel therapeutics include monoclonal antibodies (mAbs). IMGT®, the international ImMunoGeneTics information system®, extends the characterization of therapeutic antibodies with a systematic description of their mechanisms of action (MOA) and makes them available through its database for mAbs and fusion proteins, IMGT/mAb-DB. Using available literature data combined with amino acid sequence analyses from mAbs managed in IMGT/2Dstructure-DB, the IMGT® protein database, biocuration allowed us to define in a standardized way descriptions of MOAs of mAbs that target molecules towards MG treatment. New therapeutic targets include FcRn and molecules such as CD38, CD40, CD19, MS4A1, and interleukin-6 receptor. A standardized graphical representation of the MOAs of selected mAbs was created and integrated within IMGT/mAb-DB. The main mechanisms involved in these mAbs are either blocking or neutralizing. Therapies directed to B cell depletion and plasma cells have a blocking MOA with an immunosuppressant effect along with Fc-effector function (MS4A1, CD38) or FcγRIIb engager effect (CD19). Monoclonal antibodies targeting the complement also have a blocking MOA with a complement inhibitor effect, and treatments targeting T cells have a blocking MOA with an immunosuppressant effect (CD40) and Fc-effector function (IL6R). On the other hand, FcRn antagonists present a neutralizing MOA with an FcRn inhibitor effect. The MOA of each new mAb needs to be considered in association with the immunopathogenesis of each of the subtypes of MG in order to integrate the new mAbs as a viable and safe option in the therapy decision process. In IMGT/mAb-DB, mAbs for MG are characterized by their sequence, domains, and chains, and their MOA is described.

Blockade Of CD47 Using SIRPαFc: Role Of The Fc Region In Anti-Leukemic Activity and Tolerability

IMGT®, the international ImMunoGeneTics information system®1, (CNRS and Université Montpellier 2) is the global reference in immunogenetics and immunoinformatics. By its creation in 1989, IMGT® marked the advent of immunoinformatics, which emerged at the interface between immunogenetics and bioinformatics. IMGT® is specialized in the immunoglobulins (IG) or antibodies, T cell receptors (TR), major histocompatibility (MH), and proteins of the IgSF and MhSF superfamilies. IMGT® has been built on the IMGT-ONTOLOGY axioms and concepts, which bridged the gap between genes, sequences, and three-dimensional (3D) structures. The concepts include the IMGT® standardized keywords (concepts of identification), IMGT® standardized labels (concepts of description), IMGT® standardized nomenclature (concepts of classification), IMGT unique numbering, and IMGT Colliers de Perles (concepts of numerotation). IMGT® comprises seven databases, 15,000 pages of web resources, and 17 tools, and provides a high-quality and integrated system for the analysis of the genomic and expressed IG and TR repertoire of the adaptive immune responses. Tools and databases are used in basic, veterinary, and medical research, in clinical applications (mutation analysis in leukemia and lymphoma) and in antibody engineering and humanization. They include, for example IMGT/V-QUEST and IMGT/JunctionAnalysis for nucleotide sequence analysis and their high-throughput version IMGT/HighV-QUEST for next-generation sequencing (500,000 sequences per batch), IMGT/DomainGapAlign for amino acid sequence analysis of IG and TR variable and constant domains and of MH groove domains, IMGT/3Dstructure-DB for 3D structures, contact analysis and paratope/epitope interactions of IG/antigen and TR/peptide-MH complexes and IMGT/mAb-DB interface for therapeutic antibodies and fusion proteins for immune applications (FPIA).

IMGT/DomainGapAlign is the online tool of IMGT(®), the international ImMunoGeneTics information system(®), for the analysis of amino acid sequences and two-dimensional (2D) structures of domains. IMGT/DomainGapAlign allows the analysis of the closest variable (V) and constant (C) domains of immunoglobulins (IG) or antibodies, T cell receptors (TR), and immunoglobulin superfamily (IgSF) proteins, and of the groove (G) domains of major histocompatibility (MH; in humans, HLA for human leukocyte antigen) and MH superfamily proteins. IMGT/DomainGapAlign aligns the user own sequences against the IMGT domain reference directory, displays amino acid changes, creates IMGT gaps, and delimits the domain strands and loops (and helix for G domain) according to the IMGT unique numbering. IMGT/DomainGapAlign is coupled to the IMGT/Collier-de-Perles tool that draws standardized IMGT Colliers de Perles. The analysis is based on the IMGT-ONTOLOGY concepts of identification, classification, description, and numerotation generated from the axioms of the Formal IMGT-ONTOLOGY or IMGT-Kaleidoscope. IMGT/DomainGapAlign provides an invaluable help for antibody engineering and antibody humanization as it precisely defines the standardized framework regions (FR-IMGT) and complementarity determining regions (CDR-IMGT) to be grafted. IMGT/DomainGapAlign is freely available at http://www.imgt.org.

INTRODUCTIONThe “IMGT unique numbering” (or “IMGT_unique_numbering”) concept is a major concept of numerotation (generated from the NUMEROTATION axiom) of IMGT-ONTOLOGY, the global reference in immunogenetics and immunoinformatics, built by IMGT, the international ImMunoGeneTics information system. The “IMGT unique numbering” concept, described here, allows one to number domain types that are characteristic of protein superfamilies, whatever the species, the molecule type or the chain type. Three leafconcepts (a leafconcept is a concept that corresponds to the finest level of granularity) have been defined, respectively, for the variable (V) domain and constant (C) domain of the immunoglobulin superfamily (IgSF) and for the groove (G) domain of the major histocompatibility (MH) superfamily (MhSF). The IMGT unique numbering concept has been a great breakthrough in immunogenetics and systems biology in allowing, for the first time, the bridging of the gap between amino acid (and nucleotide) sequences of any V, C, or G domain and their two-dimensional (2D) and three-dimensional (3D) structures. The IMGT unique numbering concept has been fundamental in the creation of the IMGT Collier de Perles and in the standardization of the description of mutations, amino acid changes, polymorphisms, and contact analysis in the IMGT databases, tools, and web resources.

IMGT®, the international ImMunoGeneTics information system®, http://www.imgt.org/, is at the forefront of the immunogenetics and immunoinformatics fields with more than 30 years of experience. IMGT® makes available databases and tools to the scientific community pertaining to the adaptive immune response, based on the IMGT-ONTOLOGY. We focus on the recent features of the IMGT® databases, tools, reference directories and web resources, within the three main axes of IMGT® research and development. Axis I consists in understanding the adaptive immune response, by deciphering the identification and characterization of the immunoglobulin (IG) and T cell receptor (TR) genes in jawed vertebrates. It is the starting point of the two other axes, namely the analysis and exploration of the expressed IG and TR repertoires based on comparison with IMGT reference directories in normal and pathological situations (Axis II) and the analysis of amino acid changes and functions of 2D and 3D structures of antibody and TR engineering (Axis III).

IMGT®, the international ImMunoGeneTics information system®(http://www.imgt.org) is the global reference in immunogenetics and immunoinformatics. By its creation in 1989 by Marie-Paule Lefranc (Université de Montpellier and CNRS), IMGT® marked the advent of immunoinformatics, which emerged at the interface between immunogenetics and bioinformatics. IMGT® is specialized in the immunoglobulins (IG) or antibodies, T cell receptors (TR), major histocompatibility (MH) and proteins of the IgSF and MhSF superfamilies. IMGT® is built on the IMGT-ONTOLOGY axioms and concepts, which bridged the gap between genes, sequences and 3D structures. The concepts include the IMGT® standardized keywords (identification), IMGT® standardized labels (description), IMGT® standardized nomenclature (classification), IMGT unique numbering and IMGT Colliers de Perles (numerotation). IMGT® comprises 7 databases, 17 online tools and 15 000 pages of web resources, and provides a high-quality and integrated system for analysis of the genomic and expressed IG and TR repertoire of the adaptive immune responses, including NGS high-throughput data. Tools and databases are used in basic, veterinary and medical research, in clinical applications (mutation analysis in leukemia and lymphoma) and in antibody engineering and humanization. The IMGT/mAb-DB interface was developed for therapeutic antibodies and fusion proteins for immunological applications (FPIA). IMGT® is freely available at http://www.imgt.org.

Measles virus (MeV), an enveloped RNA virus belonging to the genus Morbillivirus of the family Paramyxoviridae, is the causative agent of measles, an acute febrile illness with skin rash. MeV has two types of envelope glycoproteins: the hemagglutinin (H) and fusion (F) protein. The H protein initially binds to its receptors, signaling lymphocytic activation molecule family member 1 (SLAMF1) and nectin-4, triggering conformational changes in the F protein that result in virus-to-cell or cell-to-cell membrane fusion. MeV may persist in the brain, which does not express SLAMF1 and nectin-4, leading to subacute sclerosing panencephalitis (SSPE) several years after acute infection. Recently, we have reported that MeV isolates from SSPE patients have hyperfusogenic amino acid changes (e.g., T461I) in the F protein, which enable MeV to spread in the brain by using cell adhesion molecule 1 (CADM1) and CADM2 as cis-acting fusion-triggering molecules. However, F proteins of SSPE isolates, such as the Patient B and OSA-3/Bs/B strains, carry additional changes besides T461I. In this study, we show that specific combinations of amino acid changes in the F proteins from SSPE isolates enable the induction of membrane fusion without CADM1/2 expression. We further demonstrate that these cumulative changes in the F protein allow the virus to exploit other fusion-triggering molecules than CADM1/2. These changes also promote efficient neuronal cell fusion. Our findings suggest that cumulative changes in the F protein may broaden the range of host factors capable of triggering cell fusion, facilitating MeV spread in the brain of SSPE patients.IMPORTANCESubacute sclerosing panencephalitis (SSPE) is a fatal disease caused by persistent infection of measles virus (MeV) in the brain. There is no effective therapy for the disease. MeV isolates from SSPE patients accumulate multiple amino acid changes in the F protein, including hyperfusogenic changes such as the T461I substitution, which allow MeV to spread in the brain by utilizing cell adhesion molecule 1 (CADM1) and CADM2 as cis-acting fusion-triggering molecules. In this study, we show that F proteins of SSPE isolates harboring additional changes besides T461I can induce membrane fusion independently of CADM1 and CADM2. The data also indicate that cumulative changes in the F protein may enable MeV to use other fusion-triggering host molecules than CADM1 and CADM2, facilitating its spread in the brain of SSPE patients. The findings deepen our understanding of the molecular mechanism underlying MeV neuropathogenicity in SSPE.

We describe a novel Drosophila gene, dtl (Drosophila Tat-like), which encodes a 60-kDa protein with RNA binding activity and a methyltransferase (MTase) domain. Dtl has an essential role in Drosophila development. The homologs of DTL recently described include PIMT (peroxisome proliferator-activated receptor-interacting protein with a methyltransferase domain), an RNA-binding protein that interacts with and enhances the nuclear receptor coactivator function, and TGS1, the methyltransferase involved in the formation of the 2,2,7-trimethylguanosine (m3G) cap of non-coding small RNAs. DTL is expressed throughout all of the developmental stages of Drosophila. The dtl mRNA has two ORFs (uORF and dORF). The product of dORF is the 60-kDa PIMT/TGS1 homolog protein that is translated from an internal AUG located 538 bp downstream from the 5' end of the message. This product of dtl is responsible for the formation of the m3G cap of small RNAs of Drosophila. Trimethylguanosine synthase activity is essential in Drosophila. The deletion in the dORF or point mutation in the putative MTase active site results in a reduced pool of m3G cap-containing RNAs and lethality in the early pupa stage. The 5' region of the dtl message also has the coding capacity (uORF) for a 178 amino acid protein. For complete rescue of the lethal phenotype of dtl mutants, the presence of the entire dtl transcription unit is required. Transgenes that carry mutations within the uORF restore the MTase activity but result in only partial rescue of the lethal phenotype. Interestingly, two transgenes bearing a mutation in uORF or dORF in trans can result in complete rescue.

Leukocyte recruitment and effector functions like phagocytosis and respiratory burst are key elements of immunity to infection. Pathogen survival is dependent upon the ability to overwhelm, evade or inhibit the immune system. Pathogenic group A and group B streptococci are well known to produce virulence factors that block the binding of IgA to the leukocyte IgA receptor, Fc alphaRI, thereby inhibiting IgA-mediated immunity. Recently we found Staphylococcus aureus also interferes with IgA-mediated effector functions as the putative virulence factor SSL7 also binds IgA and blocks binding to Fc alphaRI. Herein we report that SSL7 and Fc alphaRI bind many of the same key residues in the Fc region of human IgA. Residues Leu-257 and Leu-258 in domain C alpha2 and residues 440-443 PLAF in C alpha3 of IgA lie at the C alpha2/C alpha3 interface and make major contributions to the binding of both the leukocyte receptor Fc alphaRI and SSL7. It is remarkable this S. aureus IgA binding factor and unrelated factors from streptococci are functionally convergent, all targeting a number of the same residues in the IgA Fc, which comprise the binding site for the leukocyte IgA receptor, Fc alphaRI.