Human Fab Fragments Research Articles

Preclinical Evaluation of VB6-845: An Anti-EpCAM Immunotoxin with Reduced Immunogenic Potential

VB6-845 is a recombinant immunotoxin comprised of deBouganin (a de-immunized plant toxin) genetically linked to an epithelial cell adhesion molecule (EpCAM)-targeting humanized Fab fragment (4D5MOCB). EpCAM is highly expressed on a wide range of epithelial tumors but has limited expression on most normal epithelia and therefore represents an excellent target for immunotherapy. A comprehensive preclinical evaluation was performed to determine the safety and suitability of VB6-845 as a systemically administered drug for the treatment of solid tumors. Efficacy studies in mice demonstrated that VB6-845 specifically and potently targeted EpCAM-positive tumors. In a dose-ranging study in Sprague-Dawley rats, single doses of VB6-845 were well-tolerated resulting in a no-observable adverse effect level (NOAEL) of 100 mg/kg whereas repeated doses of VB6-845 resulted in vascular leak-associated symptoms particularly at higher dose levels. However, much higher doses in Cynomolgus monkeys were well-tolerated when given as a 3-hour infusion mimicking the intended route of administration in the clinic. In addition, VB6-845 proved to be minimally immunogenic in monkeys. The toxicological data obtained in Cynomolgus monkeys indicated an excellent safety profile with a NOAEL value of 30 mg/kg (equivalent to a 10 mg/kg dose in humans). These results are supportive of an exploratory Phase I trial.

Inhibiting Alternative Pathway Complement Activation by Targeting the Factor D Exosite

By virtue of its amplifying property, the alternative complement pathway has been implicated in a number of inflammatory diseases and constitutes an attractive therapeutic target. An anti-factor D Fab fragment (AFD) was generated to inhibit the alternative complement pathway in advanced dry age-related macular degeneration. AFD potently prevented factor D (FD)-mediated proteolytic activation of its macromolecular substrate C3bB, but not proteolysis of a small synthetic substrate, indicating that AFD did not block access of the substrate to the catalytic site. The crystal structures of AFD in complex with human and cynomolgus FD (at 2.4 and 2.3 Å, respectively) revealed the molecular details of the inhibitory mechanism. The structures show that the AFD-binding site includes surface loops of FD that form part of the FD exosite. Thus, AFD inhibits FD proteolytic function by interfering with macromolecular substrate access rather than by inhibiting FD catalysis, providing the molecular basis of AFD-mediated inhibition of a rate-limiting step in the alternative complement pathway.

Plasma Half-life Extension of Small Recombinant Antibodies by Fusion to Immunoglobulin-binding Domains

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.

Protein trafficking, ergosterol biosynthesis and membrane physics impact recombinant protein secretion in Pichia pastoris

BackgroundThe increasing availability of 'omics' databases provide important platforms for yeast engineering strategies since they offer a lot of information on the physiology of the cells under diverse growth conditions, including environmental stresses. Notably, only a few of these approaches have considered a performance under recombinant protein production conditions. Recently, we have identified a beneficial effect of low oxygen availability on the expression of a human Fab fragment in Pichia pastoris. Transcriptional analysis and data mining allowed for the selection of potential targets for strain improvement. A first selection of these candidates has been evaluated as recombinant protein secretion enhancers.ResultsBased on previous transcriptomics analyses, we selected 8 genes for co-expression in the P. pastoris strain already secreting a recombinant Fab fragment. Notably, WSC4 (which is involved in trafficking through the ER) has been identified as a novel potential target gene for strain improvement, with up to a 1.2-fold increase of product yield in shake flask cultures. A further transcriptomics-based strategy to modify the yeast secretion system was focused on the ergosterol pathway, an aerobic process strongly affected by oxygen depletion. By specifically partially inhibiting ergosterol synthesis with the antifungal agent fluconazole (inhibiting Erg11p), we tried to mimic the hypoxic conditions, in which the cellular ergosterol content was significantly decreased. This strategy led to an improved Fab yield (2-fold) without impairing cellular growth. Since ergosterol shortage provokes alterations in the plasma membrane composition, an important role of this cellular structure in protein secretion is suggested. This hypothesis was additionally supported by the fact that the addition of non-ionic surfactants also enhanced Fab secretion.ConclusionsThe current study presents a systems biotechnology-based strategy for the engineering of the industrially important yeast P. pastoris combining the use of host specific DNA microarray technologies and physiological studies under well defined environmental conditions. Such studies allowed for the identification of novel targets related with protein trafficking and ergosterol biosynthesis for improved recombinant protein production. Nevertheless, further studies will be required to elucidate the precise mechanisms whereby membrane biogenesis and composition impact on protein secretion in P. pastoris.

Generation of Human Fab Antibody Libraries: PCR Amplification and Assembly of Light- and Heavy-Chain Coding Sequences

The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human Fab (fragment antigen binding) antibody libraries. In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final Fab products that are used for cloning.

The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies

The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.

Certolizumab pegol for the treatment of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease that affects the joints. The cause of RA is unknown. TNF plays a pivotal role in the pathogenesis of RA. Since the late 1990s, the development of biologic agents that target TNF-α has revolutionized the approach to treating RA. Five inhibitors of TNF-α are currently available for clinical use. Certolizumab pegol is a novel TNF-α inhibitor, consisting of a humanized Fab fragment fused to a 40 KDa polyethylene glycol moiety. After a loading dose of 400 mg at weeks 0, 2 and 4, the drug is administered as maintenance therapy at 4-week intervals by subcutaneous injection. It is demonstrated that it improves the signs and symptoms of disease, quality of life and slows the progressive destruction of joints. Certolizumab pegol was generally well tolerated when used as a monotherapy or in combination with methotrexate, with most adverse events being of mild-to-moderate intensity, with a response rate similar to that of other biological therapy available f...

Human kappa light chain targeted Pseudomonas exotoxin A — identifying human antibodies and Fab fragments with favorable characteristics for antibody–drug conjugate development

Antibody–drug conjugates (ADC) represent promising agents for targeted cancer therapy. To allow rational selection of human antibodies with favorable characteristics for ADC development a screening tool was designed obviating the need of preparing individual covalently linked conjugates. Therefore, α-kappa-ETA′ was designed as a fusion protein consisting of a human kappa light chain binding antibody fragment and a truncated version of Pseudomonas exotoxin A. α-kappa-ETA′ specifically bound to human kappa light chains of human or human–mouse chimeric antibodies and Fab fragments. Antibody-redirected α-kappa-ETA′ specifically inhibited proliferation of antigen-expressing cell lines at low toxin and antibody concentrations. Selected antibodies that efficiently delivered α-kappa-ETA′ in the novel assay system were used to generate scFv-based covalently linked immunotoxins. These molecules efficiently triggered apoptosis of target cells, indicating that antibodies identified in our assay system can be converted to functional immunoconjugates. Finally, a panel of human epidermal growth factor receptor (EGFR) antibodies was screened — demonstrating favorable characteristics with antibody 2F8. These data suggest that antibodies with potential for Pseudomonas exotoxin A-based ADC development can be identified using the novel α-kappa-ETA′ conjugate.

Generation of a Neutralizing Human Monoclonal Antibody Fab Fragment to Surface Antigen 1 ofToxoplasma gondiiTachyzoites

A combinatorial immunoglobulin gene library was constructed from lymphocytes in peripheral blood of a patient with toxoplasmosis and screened for production of human monoclonal antibody Fab fragments to recombinant surface antigen 1 (SAG1) of Toxoplasma gondii. Two Fab clones, Tox203 and Tox1403, which consisted of a common heavy chain and different light chains, showed positive staining on the entire surface of tachyzoites in confocal microscopy. Sequence analysis of the heavy-chain gene revealed that the closest germ line V segments were VH3-23. The germ line D segment was D1-7, and the closest germ line J segment was JH4. In the light-chain genes, the closest germ line V segment was Vκ1-17 with the Jκ1 or Jκ4 segments. The dissociation constants of these Fab fragments with recombinant SAG1 were 3.09 × 10(-9) M for Tox203 and 2.01 × 10(-8) M for Tox1403, indicating that the affinity of Tox203 was 7 times higher than that of Tox1403. Preincubation of T. gondii tachyzoites with Tox203 significantly inhibited their attachment to cultured MDBK cells. Passive immunization of mice with Tox203 also significantly reduced mortality after challenge with T. gondii tachyzoites. This is the first report of bacterial expression of human monoclonal antibody Fab fragments to SAG1 of T. gondii. These results also demonstrate that human Fab fragments to SAG1 might be applicable for immunoprophylaxis of toxoplasmosis.

Production of a humanized Fab fragment of a neutralizing antibody against rabies virus

This paper describes the production of a functional humanized Fab fragment of a neutralizing antibody against the rabies virus. It is a prototype of a therapeutic agent that could be an alternative to equine anti-rabies immunoglobulins and anti-rabies serum immunoglobulins obtained from the blood of vaccinated human donors. Variable fragments of the high-affinity light and heavy chains neutralizing an antibody against the rabies virus were cloned and sequenced. Mouse constant regions were replaced by human constant regions followed by expression of the humanized Fab fragments in a yeast expression system. The immunochemical properties of the Fab fragments were evaluated using ELISA, polyacrylamide gel electrophoresis, and the Western blot assay. The binding capability of the produced humanized Fab fragments surpasses that of the parental antibody. A high degree of humanization was confirmed using sera against human immunoglobulins. The yield of the humanized Fab fragments was 21 mg per liter of culture medium. The purified Fab fragment preparation did not contain traces of the isolated light and heavy chains of the humanized Fab fragments.

Monoclonal antibodies isolated from human B cells neutralize a broad range of H1 subtype influenza A viruses including swine-origin Influenza virus (S-OIV)

The new H1N1 swine-origin influenza virus (S-OIV) strain is a global health problem. The elucidation of the virus-host relationship is crucial for the control of the new infection. Two human monoclonal antibody Fab fragments (HMab) neutralizing the novel H1N1 influenza strain at very low concentrations were cloned before the emergence of S-OIV from a patient who had a broad-range H1N1 serum neutralizing activity. The two HMabs neutralized all tested H1N1 strains, including S-OIV and a swine strain with IC(50) ranging from 2 to 7 microg/ml. Data demonstrate that infection with previously circulating H1N1 strains can elicit antibodies neutralizing S-OIV. Finally, the human genes coding for the neutralizing HMabs could be used for generating full human monoclonal IgGs that can be safely administered being potentially useful in the prophylaxis and the treatment of this human infection.

Tumor Necrosis Factor-Alpha Antagonists: Differential Clinical Effects by Different Biotechnological Molecules

Inhibitors of tumor necrosis factor-alpha have deeply changed the therapy of several inflammatory human diseases. For instance, clinical management of rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis have profoundly benefited after the introduction of new therapeutic tools, such as antagonist of TNF-alpha molecule. These drugs include etanercept, a soluble TNF-alpha receptor antagonist, three anti-TNF-alpha antibodies, adalimumab, infliximab, golimumab and certolizumab a humanized Fab fragment combined with polyethylene glycol. These compounds efficiently inhibit several TNF-alpha biological-mediated effects, however, they have also shown differential clinical efficacy in several trials from different autoimmune diseases. It is of clinical relevance that non-responders to one of these drugs often positively responded to another. Different mechanisms of action and diversity in pharmacokinetics of these three compounds may partially explain different clinical effects. However, partially diverse pathogenetic mechanisms in different diseases also contribute to differential therapeutic responses. Therefore, these apparently homogeneous agents can not be considered equivalent in their clinically efficacy. Differential therapeutic actions of these drugs may be advantageously used in clinical practice and further improve the great potential of individual TNF-alpha inhibitors.

Affinity ligands for immunoglobulins based on the multicomponent Ugi reaction

This report describes a novel use of the four-component Ugi reaction to generate a solid-phase library suitable for the purification of immunoglobulins and their fragments by affinity chromatography. An aldehyde-functionalised Sepharose™ solid-support constituted one component in the four-component reaction, whereas the other three components (a carboxylic acid, a primary or secondary amine and an isonitrile) were varied in a combinatorial fashion to generate a tri-substituted peptoidal scaffold structure which provides a degree of rigidity and functionality suitable for rational investigation of immunoglobulin binding. The Ugi ligand library was initially screened chromatographically against whole human IgG and its fragments (Fc and Fab) to yield a Fab-specific lead ligand based on its ability to bind Fab differentially over Fc. Preparative chromatography of IgG from human serum showed 100% of IgG was adsorbed from the 20mg/ml crude stock and subsequently eluted with a purity of 81.0% as determined by SDS-PAGE analysis under non-optimised conditions. High purity Fab and IgG isolation was achieved from both yeast and E. coli host cell proteins according to silver-stained SDS-PAGE lane densitometry. The ligand density and spacer-arm chemistry of the immobilised ligand was optimised to define an affinity adsorbent which binds 73.06mgIgG/ml moist gel (dynamic binding capacity at 10% breakthrough) and a static binding capacity of 16.1±0.25mg Fab/ml moist resin displaying an affinity constant Kd=(2.6±0.3)×10−6M. The lead candidate was modelled in silico and docked into a human Fab fragment (PDB: 1AQK) to suggest a putative binding interface to the constant CH1-CL Fab terminal through six defined hydrogen bond interactions together with putative hydrophobic interactions.

An Engineered Human Antibody Fab Fragment Specific for Pseudomonas aeruginosa PcrV Antigen Has Potent Antibacterial Activity

Pseudomonas aeruginosa is an opportunistic pathogen that can cause acute lung injury and mortality through the delivery of exotoxins by the type III secretion system (TTSS). PcrV is an important structural protein of the TTSS. An engineered human antibody Fab fragment that binds to the P. aeruginosa PcrV protein with high affinity has been identified and has potent in vitro neutralization activity against the TTSS. The instillation of a single dose of Fab into the lungs of mice provided protection against lethal pulmonary challenge of P. aeruginosa and led to a substantial reduction of viable bacterial counts in the lungs. These results demonstrate that blocking of the TTSS by a Fab lacking antibody Fc-mediated effector functions can be sufficient for the effective clearance of pulmonary P. aeruginosa infection.

Antiaggregatory and proangiogenic effects of a novel recombinant human dual specificity anti-integrin antibody.

beta(3)-Integrins are involved in platelet aggregation via alpha(IIb)beta(3) [glycoprotein (GP)IIb-GPIIIa], and in angiogenesis via endothelial alpha(V)beta(3). Cross-reactive ligands with antiaggregatory and proangiogenic effects, both desirable in peripheral vasculopathies, have not yet been described. In vitro and in vivo characterization of antiaggregatory and proangiogenic effects of two recombinant human Fab fragments, with emphasis on beta(3)-integrins. Recombinant Fab fragments were obtained by phage display technology. Specificity, affinity and IC(50) were determined by immunodot assays, enzyme-linked immunosorbent assay (ELISA), and Scatchard plot analysis, and by means of human umbilical vein endothelial cells (HUVECs). Functional analyses included ELISA for interaction with fibrinogen binding to GPIIb-GPIIIa, flow cytometry for measurement of activation parameters and competitive inhibition experiments, human platelet aggregometry, and proliferation, tube formation and the chorioallantoic membrane (CAM) assay for measurement of angiogenic effects. We observed specific and high-affinity binding to an intact GPIIb-GPIIIa receptor complex of two human Fab autoantibody fragments, with no platelet activation. Dose-dependent fibrinogen binding to GPIIb-GPIIIa and platelet aggregation were completely inhibited. One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody (mAb) 7E3, whereas the other Fab fragment bound to cultured HUVECs, suggesting cross-reactivity with alpha(V)beta(3), and also demonstrated proangiogenic effects in tube formation and CAM assays. These Fab fragments are the first entirely human anti-GPIIb-GPIIIa Fab fragments with full antiaggregatory properties; furthermore, they do not activate platelets. The unique dual-specificity anti-beta(3)-integrin Fab fragment may represent a new tool for the study and management of peripheral arterial vasculopathies.

Characteristics of Fc-independent human antimannan antibody-mediated alternative pathway initiation of C3 deposition to Candida albicans

The complement system has an important role in host resistance to systemic candidiasis but regulation of complement activation by Candida albicans remains poorly defined. Previous studies have identified a requirement for naturally occurring antimannan IgG antibody in initiation of C3 opsonization of C. albicans through either the classical or alternative pathway. This study characterized antibody-dependent initiation of the alternative pathway using the recombinant human monoclonal antimannan Fab fragment M1 and its full-length IgG1 antibody M1g1. Kinetic analysis of C3b deposition onto C. albicans with flow cytometry demonstrated the ability of M1g1 to restore the activity of either the classical or alternative pathway to the yeast-absorbed normal human serum, but the Fc-free M1 Fab restored only the activity of the alternative pathway. This Fc-independent, antimannan Fab-mediated C3 deposition through the alternative pathway was also observed in a serum-free assay containing the six alternative pathway proteins and in C1q- or C2-depleted serum but not in factor B-depleted serum. M1- or M1g1-dependent alternative pathway initiation of C3b deposition occurred in an asynchronous manner at discrete sites that expanded to cover the entire cell surface over time as revealed with immunofluorescence microscopy, in contrast to a uniform appearance of initial C3 deposition through the classical pathway. Furthermore, antimannan Fab M1 promoted the assembly of the alternative pathway convertase on the cell surface seen as colocalization of C3 and factor B with immunofluorescence microscopy. Thus, human antimannan antibody has a distinct Fc-independent effector function in regulation of C3 deposition to C. albicans.

Introduction: Uveal melanoma

Introduction: Uveal melanoma

Functional humanization of an anti-CD16 Fab fragment: obstacles of switching from murine to human or light chains

An alphaCD30xalphaCD16 bispecific monoclonal antibody (MAb) was previously shown to induce remission of Hodgkin's disease refractory to chemo- and radiotherapy through specific activation of natural killer (NK) cells, but the appearance of a human anti-mouse antibody (HAMA) response prevented its use for prolonged therapy. Here, we describe an effort to humanize the Fab arm directed against FcgammaRIII (CD16), which-in context with the previously humanized CD30 Fab fragment-provides the necessary component for the design of a clinically useful bispecific antibody. Thus, the CDRs of the anti-CD16 mouse IgG1/lambda MAb A9 were grafted onto human Ig sequences. In a first attempt, the murine V(lambda) domain was converted to a humanized lambda chain, which led, however, to complete loss of antigen-binding activity and extremely poor folding efficiency upon periplasmic expression in Escherichia coli. Hence, its CDRs were transplanted onto a human kappa light chain in a second attempt, which resulted in a functional recombinant Fab fragment, yet with 100-fold decreased antigen affinity. In the next step, an in vitro affinity maturation was performed, wherein random mutations were introduced into the humanized V(H) and V(kappa) domains through error-prone PCR, followed by a filter sandwich colony screening assay for increased binding activity towards the bacterially produced extracellular CD16 fragment. Finally, an optimized Fab fragment was obtained, which carries nine additional amino acid exchanges and exhibits an affinity that is within a factor of 2 identical to that of the original murine A9 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to binding of the recombinant CD16 antigen in enzyme-linked immunosorbent assay and in cytofluorimetry with CD16-positive granulocytes, thus providing a promising starting point for the preparation of a fully human bispecific antibody that permits the therapeutic recruitment of NK cells.

Conversion of murine antibodies to human antibodies and their optimization for ovarian cancer therapy targeted to the folate receptor.

We previously developed murine and chimeric antibodies against a specific epithelial ovarian carcinoma (EOC) marker, named folate receptor (FR), and promising results were obtained in phase II trials. More recently, we successfully generated a completely human Fab fragment, C4, by conversion of one of the murine anti-FR antibodies to human antibody using phage display and guided selection. However, subsequent efforts to obtain C4 in a dimer format, which seems especially desirable for EOC locoregional treatment, resulted in a highly heterogeneous product upon natural dimerization and in a very poor production yield upon chemical dimerization by a non-hydrolyzable linker to a di-Fab-maleimide (DFM). We therefore designed, constructed and characterized a large Fab dual combinatorial human antibody phage display library obtained from EOC patients and potentially biased toward an anti-tumor response in an effort to obtain new anti-FR human antibodies suitable for therapy. Using this library and guiding the selection on FR-expressing cells with murine/human antibody chains, we generated four new human anti-FR antibody (AFRA) Fab fragments, one of which was genetically and chemically manipulated to obtain a chemical dimer, designated AFRA-DFM5.3, with high yield production and the capability for purification scaled-up to clinical grade. Overall affinity of AFRA-DFM5.3 was in the 2-digit nanomolar range, and immunohistochemistry indicated that the reagent recognized the FR expressed on EOC samples. (131)I-AFRA-DFM5.3 showed high immunoreactivity, in vitro stability and integrity, and specifically accumulated only in FR-expressing tumors in subcutaneous preclinical in vivo models. Overall, our studies demonstrate the successful conversion of murine to completely human anti-FR antibodies through the combined use of antibody phage display libraries biased toward an anti-tumor response, guided selection and chain shuffling, and point to the suitability of AFRA5.3 for future clinical application in ovarian cancer.

Prevalence of anti-Fab antibodies in patients with autoimmune and infectious diseases.

Sensitive ELISA were devised to examine the specificity of circulating IgM and IgA autoantibodies for whole human IgG, Fc and Fab fragments of human IgG. Sera from patients with autoimmune and infectious conditions such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), tuberculosis (TB), infectious mononucleosis (IM) and cystic fibrosis (CF) were studied. Results of the ELISA assays using whole human IgG as antigen revealed that a proportion of patients in each of the groups studied had circulating IgM and IgA rheumatoid factors (RF). Fifteen normal individuals studied were negative. In the latex positive RA group, IgM RF and IgA RF had primarily anti-Fc reactivity (100% and 93% respectively), although 3/15 patients also showed IgM anti-Fab reactivity and one patient had high IgA anti-Fab activity. Patients with SLE and TB who had detectable RF levels also revealed predominantly anti-Fc specificity. In contrast, examination of 25 patients with IM showed positivity for IgM RF activity in 8% of patients using whole IgG as antigen, 24% positivity using purified Fc fragments as antigen and 45% positivity when plates were coated with Fab fragments. Similarly, a large number of CF patients (54%) also showed predominantly IgM anti-Fab activity. Of interest, 69% of the CF patients who were all studied at the time of bacterial infection had detectable IgA RF levels, with 46% of these patients showing both IgA anti-Fc and anti-Fab activity. These findings suggest that autoantibody specificities in autoimmune and infectious diseases are different.