Treatment of pemphigus vulgaris and foliaceus with efgartigimod, a neonatal Fc receptor inhibitor: a phase II multicentre, open-label feasibility trial.

BACKGROUND: Pemphigus vulgaris and pemphigus foliaceus are potentially life-threatening autoimmune disorders triggered by IgG autoantibodies against mucosal and epidermal desmogleins. There is an unmet need for fast-acting drugs that enable patients to achieve early sustained remission with reduced corticosteroid reliance. OBJECTIVES: To investigate efgartigimod, an engineered Fc fragment that inhibits the activity of the neonatal Fc receptor, thereby reducing serum IgG levels, for treating pemphigus. METHODS: Thirty-four patients with mild-to-moderate pemphigus vulgaris or foliaceus were enrolled in an open-label phase II adaptive trial. In sequential cohorts, efgartigimod was dosed at 10 or 25 mg kg-1 intravenously with various dosing frequencies, as monotherapy or as add-on therapy to low-dose oral prednisone. Safety endpoints comprised the primary outcome. The study is registered at ClinicalTrials.gov (identifier NCT03334058). RESULTS: Adverse events were mostly mild and were reported by 16 of 19 (84%) patients receiving efgartigimod 10 mg kg-1 and 13 of 15 (87%) patients receiving 25 mg kg-1 , with similar adverse event profiles between dose groups. A major decrease in serum total IgG and anti-desmoglein autoantibodies was observed and correlated with improved Pemphigus Disease Area Index scores. Efgartigimod, as monotherapy or combined with prednisone, demonstrated early disease control in 28 of 31 (90%) patients after a median of 17 days. Optimized, prolonged treatment with efgartigimod in combination with a median dose of prednisone 0·26 mg kg-1 per day (range 0·06-0·48) led to complete clinical remission in 14 of 22 (64%) patients within 2-41 weeks. CONCLUSIONS: Efgartigimod was well tolerated and exhibited an early effect on disease activity and outcome parameters, providing support for further evaluation as a therapy for pemphigus.

Orally administered L. lactis secreting an anti-TNF Nanobody demonstrate efficacy in chronic colitis.

Inflammatory bowel disease (IBD) is a chronic inflammatory gastrointestinal disorder. Systemic treatment of IBD patients with anti-tumor necrosis factor (TNF)-alpha antibodies has proven to be a highly promising approach, but several drawbacks remain, including side effects related to systemic administration and high cost of treatment. Lactococcus lactis was engineered to secrete monovalent and bivalent murine (m)TNF-neutralizing Nanobodies as therapeutic proteins. These therapeutic proteins are derived from fragments of heavy-chain camelid antibodies and are more stable than conventional antibodies. L. lactis-secreted anti-mTNF Nanobodies neutralized mTNF in vitro. Daily oral administration of Nanobody-secreting L. lactis resulted in local delivery of anti-mTNF Nanobodies at the colon and significantly reduced inflammation in mice with dextran sulfate sodium (DSS)-induced chronic colitis. In addition, this approach was also successful in improving established enterocolitis in interleukin 10 (IL10)(-/-) mice. Finally, L. lactis-secreted anti-mTNF Nanobodies did not interfere with systemic Salmonella infection in colitic IL10(-/-) mice.In conclusion, this report details a new therapeutic approach for treatment of chronic colitis, involving in situ secretion of anti-mTNF Nanobodies by orally administered L. lactis bacteria. Therapeutic application of these engineered bacteria could eventually lead to more effective and safer management of IBD in humans.

Properties, production, and applications of camelid single-domain antibody fragments.

Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies) have several advantages for biotechnological applications. They are well expressed in microorganisms and have a high stability and solubility. Furthermore, they are well suited for construction of larger molecules and selection systems such as phage, yeast, or ribosome display. This minireview offers an overview of (1) their properties as compared to conventional antibodies, (2) their production in microorganisms, with a focus on yeasts, and (3) their therapeutic applications.

Llama-derived phage display antibodies in the dissection of the human disease oculopharyngeal muscular dystrophy.

Functional analysis of the estimated 30,000 genes of the human genome requires fast and reliable high-throughput methods to study spatio-temporal protein dynamics. To explore the suitability of heavy-chain antibodies (HCAbs) for studying mechanisms underlying human disease, we used oculopharyngeal muscular dystrophy (OPMD) as a paradigm for the expanding group of protein aggregation disorders that is characterized by subcellular dislocalization and aggregation of mutant protein. OPMD is caused by a moderate alanine expansion in the poly-A binding protein nuclear 1 (PABPN1) and is associated with intranuclear PABPN1 deposition exclusively in muscle. An experimental approach was designed in which the primary sequence of the PABPN1 gene was employed for generating a prokaryotic expression construct that permitted its expression in the host Escherichia coli. The purified product was used for immunization of a llama as well as for the selection of an antigen-specific antibody fragment from the derived phage display library. This single-domain antibody was able to recognize the native gene product in mammalian cell lines and in human muscle tissue by immunocytochemical, immunohistochemical and immunoblot analysis. Our results suggest that phage display derived heavy-chain antibodies can be used in proteomics to study the localization and function of hypothetical gene products, relevant to human disease.

A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies.

We report the design, construction, and use of the first very large non-immunized phage antibody library in Fab format, which allows the rapid isolation and affinity analysis of antigen-specific human antibody fragments. Individually cloned heavy and light chain variable region libraries were combined in an efficient two-step cloning procedure, permitting the cloning of a total of 3.7 x 10(10) independent Fab clones. The performance of the library was determined by the successful selection of on average 14 different Fabs against 6 antigens tested. These include tetanus toxoid, the hapten phenyl-oxazolone, the breast cancer-associated MUC1 antigen, and three highly related glycoprotein hormones: human chorionic gonadotropin, human luteinizing hormone, and human follicle-stimulating hormone. In the latter category, a panel of either homone-specific or cross-reactive antibodies were identified. The design of the library permits the monitoring of selections with polyclonal phage preparations and to carry out large scale screening of antibody off-rates with unpurified Fab fragments on BIAcore. Antibodies with off-rates in the order of 10(-2) to 10(-4) s-1 and affinities up to 2.7 nM were recovered. The kinetics of these phage antibodies are of the same order of magnitude as antibodies associated with a secondary immune response. This new phage antibody library is set to become a valuable source of antibodies to many different targets, and to play a vital role in target discovery and validation in the area of functional genomics.

Vernier zone residue 4 of mouse subgroup II kappa light chains is a critical determinant for antigen recognition.

BACKGROUND: During the conversion of murine monoclonal antibodies directed against the human chorionic gonadotropin (hCG) into bacterially expressed single chain fragments (scFv), we found a major reduction of binding activity upon introduction of a primer encoded mutation. OBJECTIVES: In this study we tried to determine which mutation was responsible and on what manner this mutation affected antigen binding (structural effect versus direct involvement of the residue in binding). RESULTS: No binding could be detected, when the wild type residue methionine at position 4 within the Framework region 1 of the Vkappa light chain was substituted by serine in two antibodies with a subgroup II kappa light chain. However, a similar replacement within an anti-hCG antibody with a subgroup IV kappa light chain and thereby having leucine as wild type residue, did not affect the binding characteristics. The mutant scFv's derived from both AB-s sensitive for substitution by serine never reacted with antigen in ELISA. Analysis with surface plasmon resonance revealed a residual binding only on a sensorchip with a high density coating of antigen; however, an increased dissociation, relative to that of the wild type scFv and the absence of reactivity in ELISA suggest a drastically altered affinity. CONCLUSION: A structural explanation for the changed binding characteristics can be the influence of the position 4 residue, as being a constituent of the Vernier zone, on the position of the CDR1 loop of Vkappa, which might harbour residues that directly bind to antigen, or indirectly positions other variable loops of the binding pocket. An increased sensitivity for trypsin digestion supported the hypothesis of a local conformational change in the serine mutant of the subgroup II kappa containing antibody.

Selection of recombinant, library-derived antibody fragments against p24 for human immunodeficiency virus type 1 diagnostics.

By application of combinatorial library technology, we generated the first recombinant antibody fragments directed against the major capsid protein p24 of human immunodeficiency virus type 1 (HIV-1). A library of single-chain Fv fragments (scFvs) was constructed by using the antibody variable-region (V) genes of B cells derived from the spleen of a viral lysate-immunized mouse. Antibodies were selected by panning or by enrichment with biotinylated antigen, yielding four different families of antibody fragments. The different types of scFvs were characterized by affinity measurements, by antigen recognition on Western blots, and by pepscan analysis. The epitope of one of the scFvs is located near the residues involved in CypA binding, thereby making it an attractive candidate for therapeutic applications. Comparison of the V gene sequence of this scFV with that of a previously described monoclonal antibody reactive against this immunodominant epitope revealed the usage of the identical combination of VH and Vkappa regions. Thus, this is one of the rare examples in which the original combination in a library-derived antibody fragment was retrieved. After appropriate affinity and format improvements, the best of our recombinant scFvs may form the basis for a sensitive p24 assay as a measure of viral load. In addition, anti-p24 scFvs could be expressed as intracellular antibodies (intrabodies) to aid in the treatment of HIV infections.

Absolute conservation of residue 6 of immunoglobulin heavy chain variable regions of class IIA is required for correct folding.

While studying the expression of single-chain antibodies (scFv) derived from several murine monoclonal antibodies, we found that residue 6 in Framework region 1 of the heavy chain variable domain plays a crucial role in antibody folding. Binding activity of three murine antibodies with a heavy chain variable region (VH) subgroup IIA was completely lost when at this position the wild-type residue glutamine (Q) was substituted by glutamate (E). Increased sensitivity towards trypsin digestion of soluble scFv suggested that the lack of binding activity was caused by incorrect folding of Q6E mutants. Grafting of the three additional class IA derived FR1 residues, based upon the comparison between both classes of VH sequences, on to the 'defect' subgroup IIA sequence, partially restored the antigen binding activity of the Q6E-containing scFv. Our results suggest that residue 6 of the heavy chain may be part of a folding nucleus, involving the first two beta-strands of Framework region 1. The evolutionary conservation of either glutamine or glutamate at position 6 in different antibody families may well indicate that within immunoglobulin VH domains, different family specific folding nuclei have evolved.

Creating and engineering human antibodies for immunotherapy.

Targeting in immunotherapy has traditionally been achieved by using monoclonal rodent antibodies. Despite gene-engineering, there are many problems and limitations associated with the non-human origin, the targeting specificity and the binding strength of these molecules. Now these issues may be addressed in a more rational way, by designing and then shaping, in vitro, the desired human antibodies. This review addresses how this may be achieved by the selection of monoclonal human antibodies from phage display libraries and the engineering of affinity and specificity thereafter. Phage display of antibody fragments has allowed access to large collections of different phage antibodies, created by cloning antibody V-genes from B-cells. Antibodies against any type of antigen may be derived from such repertoires, by rounds of enrichment on antigen and re-amplification. This review presents the state of the art in rational antibody design and creation. It will highlight the strengths of this increasingly important field, which will aid in the generation of tailor-made targeting entities for immunotherapy.

Determination of active single chain antibody concentrations in crude periplasmic fractions.

We have measured active single chain antibody (scFv) concentrations under mass transfer limitation conditions using surface plasmon resonance on the BIAcore. For the creation of a standard curve scFv 4Dwt, derived from monoclonal antibody (mAb) 4D, directed against human chorionic gonadotropin (hCG), was purified by positive affinity chromatography. Determination of the active antibody fraction after purification was performed using anti-FLAG, reactive against a tag sequence C-terminally fused to the scFv. Two independent experiments showed that the activity remaining represented over 75% of the total amount of purified protein. Calibration curves on high density antigen surfaces showed a linear relationship between antibody concentration and binding rates. Periplasmic fractions of six mutant scFvs, also derived from mAb 4D, revealed a clear difference in the amount of soluble active scFv expressed in the periplasm of E. coli compared with the total amount of antibody present, indicating the necessity of measuring active antibody concentrations. This rapid concentration determination method will be particularly useful for accurately comparing affinity constants, using antibody concentrations determined with the BIAcore, of the many different scFv fragments routinely isolated from phage display libraries.

Gene mapping and expression of two immunodominant Epstein-Barr virus capsid proteins.

The genomic localization of two immunodominant genes encoding two proteins of the Epstein-Barr virus capsid antigen (VCA) complex, VCA-p18 and VCA-p40, has been identified. For that purpose, lambda gt11-based cDNA libraries were constructed from HH514.c16 cells induced for virus production. The libraries were screened with a monoclonal antibody, EBV.OT41A, directed against VCA-p40 or with affinity-purified human antibodies against VCA-p18. Sequencing of the inserts of positive plaques showed that VCA-p18 and VCA-p40 are encoded within open reading frames (ORFs) BFRF3 and BdRF1, respectively. Peptide scanning analysis of the predicted protein of ORF BdRF1 resulted in defining the epitope of monoclonal antibody EBV.OT41A at the C-terminal region. The dominant VCA-p18 reactivity of human sera can be completely inhibited by preadsorption with Escherichia coli-expressed BFRF3-beta-galactosidase. Serum of a rabbit immunized with BFRF3-beta galactosidase reacts with a VCA-specific protein of 18 kDa. In addition, BFRF3-beta-galactosidase affinity-purified antibodies react with VCA-p18 of virus-producing cells (HH514.c16). Complete inhibition of viral DNA polymerase activity by phosphonoacetic acid is associated with the absence of RNAs and protein products of both ORFs, indicating that VCA-p18 and VCA-p40 are true late antigens.

Cloning and expression of the Lactococcus lactis subsp. cremoris SK11 gene encoding an extracellular serine proteinase.

The Lactococcus lactis subsp. cremoris SK11 plasmid-located prtP gene, encoding a cell-envelope-located proteinase (PrtP) that degrades alpha s1-, beta- and kappa-casein, was identified in a lambda EMBL3 gene library in Escherichia coli using immunological methods. The complete prtP gene could not be cloned in E. coli and L. lactis on high-copy-number plasmid vectors. However, using a low-copy-number vector, the complete prtP gene could be cloned in strains MG1363 and SK1128, proteinase-deficient derivatives of L. lactis subsp. lactis 712 and L. lactis subsp. cremoris SK11, respectively. The proteinase deficiency of these hosts was complemented to wild-type (wt) levels by the cloned SK11 prtP gene. The caseinolytic specificity of the proteinase specified by the cloned prtP gene was identical to that encoded by the wt proteinase plasmid, pSK111. The expression of recombinant plasmids containing 3' and 5' deletions of prtP was analyzed with specific attention directed towards the location of the gene products. In this way the expression signals of prtP were localized and overproduction was obtained in L. lactis subsp. lactis. Furthermore, a region at the C terminus of PrtP was identified which is involved in cell-envelope attachment in lactococci. A deletion derivative of prtP was constructed which specifies a C-terminally truncated proteinase that is well expressed and fully secreted into the medium, and still shows the same capacity to degrade alpha s1-, beta- and kappa-casein.

Fibrinogen fragments X, Y, D and E increase levels of plasma fibrinogen and liver mRNAs coding for fibrinogen polypeptides in rats.

Previously, we demonstrated that in vivo regulation of liver fibrinogen synthesis occurs via the fibrinogen mRNA level. However, the molecular regulatory mechanism of fibrinogen synthesis is still not well understood. Fibrinogen or fibrin degradation products might play an important role in regulating fibrinogen synthesis. In our present study, we have injected rats intraperitoneally with purified homologous fragments and measured the liver content of mRNA specific coding for fibrinogen. Increased levels of fibrinogen mRNA and elevated plasma fibrinogen concentrations were observed in rats after administration of fibrinogen degradation products X, Y, DEGTA, Dcate or E. Fragment E or E' has a less stimulatory effect than X, Y or Dcate, whereas cross-linked fibrin degradation product D dimer does not increase fibrinogen synthesis. This article reports for the first time a stimulatory effect of the high molecular weight fibrinogen degradation products on fibrinogen synthesis.

The influence of glucocorticoid on albumin synthesis and its messenger RNA in rat in vivo and in hepatocyte suspension culture.

Corticosteroids are known to stimulate the synthesis of a number of liver-specific proteins. The reports regarding the effect of glucocorticoid on albumin synthesis in vivo and in vitro are controversial. In an attempt to determine the mechanism by which glucocorticoid exerts its influence on hepatic albumin synthesis and to find an explanation for the conflicting data, we have studied the effect of dexamethasone disodium phosphate on albumin synthesis and albumin messenger RNA as determined by the molecular hybridization technique in hepatocytes in rat in vivo and in suspension culture. In hepatocyte suspension culture, addition of 0.48 microM dexamethasone in medium at zero time led to a significant increase (20%) in incorporation of labeled precursor into albumin as compared to control experiments; this was accompanied by a maintainance of the initial level of full-length albumin mRNA for a 9 h period. In hepatocytes cultured without dexamethasone in the medium there was a progressive loss of albumin mRNA content. Despite this finding, dexamethasone was not able to increase the albumin mRNA content in hepatocyte to a level higher than the initial value. Moreover, administration of this hormone either intraperitoneally or intravenously into rats did not lead to enhanced cell-free albumin synthesis or to an increased level of albumin mRNA. These findings suggest that glucocorticoid does not play an essential role in the regulation of albumin synthesis in vivo. In vitro, however, glucocorticoid leads to a preservation of the initial level of albumin mRNA and thus plays a role in the control of spontaneous dedifferentiation of liver cells in culture.

The influence of glucocorticoid on the fibrinogen messenger RNA content of rat liver in vivo and in hepatocyte suspension culture.

The plasma concentration of fibrinogen, one of the major acute-phase proteins produced by the liver, increases during the acute-phase response as a result of enhanced synthesis in liver. Since adrenal-cortical hormones have been thought to have a key role in the regulation of the fibrinogen synthesis, fibrinogen-polypeptide mRNA sequences were determined in the present study, by using a specific complementary-DNA probe, in RNA fractions obtained from rat hepatocytes exposed to glucocorticoids in vitro (hepatocyte suspension cultures) and in vivo. Maximal induction of the fibrinogen-polypeptide mRNA (to 400% of the control value) was found in vitro at 0.1 microM-dexamethasone after 9 h of incubation. The same magnitude of induction was obtained with 20 microM-cortisol or 60 microM-corticosterone. In contrast with the findings in vitro, no induction of the fibrinogen-polypeptide mRNA was observed in the liver at various times after injection of different doses of glucocorticoids into rats. These results suggest that more complex regulatory mechanisms are involved and that glucocorticoids are not the sole regulatory factors in vivo in the enhanced synthesis of fibrinogen during the acute-phase response.