Electron and X-ray diffraction studies of influenza neuraminidase complexed with monoclonal antibodies

Complexes of influenza virus neuraminidase both with antigen-binding (Fab) fragments and with whole monoclonal antibody molecules have been crystallized. Uniformly thin platelet microcrystals suitable for structure analysis by electron diffraction, yielding reflections to approximately 4.3 A resolution, have been grown from one neuraminidase-Fab complex, that of N9 neuraminidase with 32/3 Fab, and thicker crystals of a second neuraminidase-Fab complex (N9 neuraminidase-NC35 Fab) diffract X-rays to approximately 4.0 A resolution. Electron microscope lattice images of microcrystals both of Fab and of immunoglobulin G complexed with neuraminidase have been interpreted in terms of negatively stained images of the respective individual complex protomers. The sites of binding of the antibodies to the antigen are consistent with the notion that single amino acid changes observed in monoclonal variants of neuraminidase occur in binding epitopes for the antibody used for their selection.     

Valency of CD3 binding and internalization of the CD3 cell-surface complex control T cell responses to second signals: distinction between effects on protein kinase C, cytoplasmic free calcium, and proliferation

We have studied the relationship of valency of CD3 stimulation and modulation of the CD3 receptor complex with biochemical and proliferative responses of T cells. Anti-CD3 Fab, as well as F(ab')2 and whole antibody caused rapid modulation of the CD3 antigen, whereas anti-CD3 conjugated to Sepharose did not. In the absence of monocytes, T cells stimulated with anti-CD3 Fab, F(ab')2, or F(ab')2-Sepharose showed differences in their ability to respond to second signals given by PMA, IL 1, IL 2, or antibodies to Tp67 and Tp44. None of the anti-CD3 signals alone caused resting T cells to produce IL 2, and only the Sepharose-bound anti-CD3 F(ab')2 caused T cells to express high levels of functional IL 2 receptors. Anti-CD3 F(ab')2-Sepharose-stimulated T cells produced IL 2 and proliferated in response to each of the second signals. Because anti-CD3-Sepharose did not cause modulation of the CD3 antigen, the ability of the Sepharose-bound antibody to induce T cells to express IL 2 receptors and to respond to individual second signals may be related to lack of modulation rather than valency of binding. Anti-CD3 Fab-stimulated T cells responded to PMA but required combinations of other second signals. T cells stimulated with unmodified anti-CD3 antibody or F(ab')2 fragments responded to PMA but did not respond to any other second signals alone or in combination. Stimulations that resulted in modulation (i.e., anti-CD3 whole antibody, anti-CD3 F(ab')2, or anti-CD3 Fab fragments) caused an increase in cytoplasmic calcium levels in resting T cells but blocked proliferation of T cells in response to mitogenic lectins or CD2 stimulation. Anti-CD3 F(ab')2 on Sepharose, however, did not block T cell proliferation. Whole bivalent anti-CD3 antibody or F(ab')2 fragments, but not monovalent Fab fragments, caused a rapid translation of protein kinase C activity from cytosol to membrane in the Jurkat T cell line. Because all of these modulate the receptor, these data indicate that the functional difference between monovalent and bivalent binding to CD3 is related to antibody valency and not to antigenic modulation. The use of Fab anti-CD3 stimulation that requires combinations of second signals for proliferation allowed an analysis of the functional relationships between IL 1, anti-Tp67, and anti-Tp44.     

Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains.

The display of proteins on the surface of phage offers a powerful means of selecting for rare genes encoding proteins with binding activities. Recently we found that antibody heavy and light chain variable (V) domains fused as a single polypeptide chain to a minor coat protein of filamentous phage fd, could be enriched by successive rounds of phage growth and panning with antigen. This allows the selection of antigen-binding domains directly from diverse libraries of V-genes. Now we show that heterodimeric Fab fragments can be assembled on the surface of the phage by linking one chain to the phage coat protein, and secreting the other into the bacterial periplasm. Furthermore by introducing an amber mutation between the antibody chain and the coat protein, we can either display the antibody on phage using supE strains of bacteria, or produce soluble Fab fragment using non-suppressor strains. The use of Fab fragments may offer advantages over single chain Fv fragments for construction of combinatorial libraries.     

The influence of antibody fragment format on phage display based affinity maturation of IgG

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.

In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab.     

Construction and characterization of phage display library: recognition of mouse serologically detected male (SDM) antigen

Improvement of animal embryo sexing depends upon high-titer serologically detected male (SDM) antibody fragments. SDM sera collected from isogenic C57BL/7 female mice after inoculation with male spleen cells were characterized and used for construction of a recombinant Fab antibody library against SDM antigen, and used for analysis of the binding capacity and specificity to SDM antigen. The heavy-chain Fd and full-length light-chain kappa were amplified by RT-PCR from a mouse (#6) that'ed high-titer antiserum. The amplified product was inserted into the pComb3 vector followed by co-infections with the help phage VCSM 13 for construction of the phage library, which gave 1.5x10(7) colonies with the titer of 3.2x10(11) pfu/ml by a recombination rate of 80%. Sequence analysis of the PCR products of plasmid DNA of E5 clones showed that V(H) and V(kappa) had common characteristics shared by other known variable region of antibodies. The Fab antibody libraries against SDM antigen were enriched by three cycles of affinity enrichment with male spleen cells, and two cycles of non-specific absorption with female spleen cells. The ELISA results showed that 9 of 15 clones had binding capacity to the SDM antigen. This is the first report on a phage display library of SDM antigen. The mouse Fab antibody library could be used for identifying SDM antigen, and for the development of sex determination of early embryos in mammals.     

Single chain Fab (scFab) fragment

Background  

The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display.     

Nonlabeled secondary antibodies augment/maintain the binding of primary, specific antibodies to cell membrane antigens.

BACKGROUND: In studies on surface membrane antigen expression using immunofluorescence techniques, it is commonly observed that direct staining gives weaker signals than the signals following indirect staining with fluorochrome-conjugated secondary antibodies. This is most marked when cells have also been permeabilized in order to stain intracellular protein. The commonly accepted explanation for this observation is that fluorochrome-conjugated secondary antibodies bind to a higher number of binding sites on the primary antibody, as compared to the binding of conjugated primary antibodies to the membrane antigens. Another hypothesis might be that the antibody/antibody complexes formed on the membranes when using the indirect technique may have an augmented ability to bind the membrane epitopes. The present study was performed in order to check this hypothesis. MATERIALS AND METHODS: Peripheral blood mononuclear cells were stained with fluorochrome-conjugated anti-CD antibodies directly without or with a second-step application of nonconjugated goat anti-mouse IgG antibodies, followed by different fixation and permeabilization methods. The cells were analyzed by flow cytometry. RESULTS: A second-step application of nonconjugated goat anti-mouse IgG antibodies following direct staining with fluorochrome-conjugated anti-CD antibodies gave a significant increase in membrane antigen expression on permeabilized cells as compared to direct staining alone. The secondary antibody must be bivalent, since whole IgG or F(ab')(2) fragments of the goat anti-mouse antibodies showed effects, while Fab fragments did not. CONCLUSIONS: Nonlabeled secondary antibodies are able to influence the binding of primary, specific antibodies to cell membrane antigens on cells treated with permeabilizing agents necessary for staining intracellular proteins. The improved membrane antigen expression seems to be due to the formation of a network of primary and secondary antibodies on the cell surface, with increased ability for maintaining binding to CD antigens.     

Antibody engineering: the use of Escherichia coli as an expression host.

The hypervariable loops of an antibody molecule are supported on the relatively conserved beta-sheeted frameworks of the heavy- and light-chain variable domains (designated VH and VL domains, respectively). Residues within and flanking these loops interact with antigen and confer the specificity and affinity of antigen binding on the immunoglobulin molecule. Thus, the isolation and expression of VH and VL domain genes are of particular interest both for analysis of the determinants of antibody specificity and for generation of fragments with binding affinities for use in therapy and diagnosis. The PCR can now be used to isolate diverse repertoires of antibody VH and VL domain genes from antibody-producing cells from different species, including humans and mice. The genes can be expressed as either secreted or surface-bound Fv or Fab fragments, using Escherichia coli expression systems, and the desired antigen-binding specificity screened for or, preferably, selected. The use of E. coli as an expression host allows the required antigen-binding specificity to be isolated in clonal form in a matter of days. The VH and VL domain genes can also be hypermutated and higher-affinity variants isolated by screening or selection. Thus, the use of this technology should allow the isolation of novel binding specificities or specificities that are difficult to generate by hybridoma technology. It will also facilitate the isolation of human-derived Fv/Fab fragments that may be less immunogenic in therapy. This approach therefore has almost unlimited potential in the generation of therapeutics with binding specificities to order. The fragments can be used either alone or linked to effector functions in the form of antibody-constant domains or toxins. The new technology could prove to be a method of choice for the rapid and convenient production of designer antibodies.     

Biomedicals from a soil bug: expanding scFv production host range

Recombinant antibody fragments have a wide range of applications from research to diagnostics and therapy. Of special interest are small fragments like fragment antigen binding (Fab) or single chain fragment variables (scFv) fragments as they can be produced inexpensively in bacterial expression systems. However, recombinant production efficiencies from established production hosts vary significantly leading to inadequate yields. Gene sequences that have been synthetically adapted to match the codon preferences and respective genomic tRNA pool of the host have been used to improve yields but cannot resolve the principal problem. The development of inducible broad host range scFv expression plasmid constructs leads the way to an easy and efficient screening method for the identification of the optimal bacterial expression host.     

Interaction of platelet factor 4 with human platelets

Human washed resting platelets bound 125I-labeled platelet factor 4 in a reaction which was saturable and approached equilibrium within 15-30 min. Scatchard plot analysis of the binding isotherms suggested a single class of specific binding sites. Excess of unlabeled protein and low- and high-affinity heparin competed for platelet factor 4 binding sites on the platelet surface and caused a partial displacement of this molecule. Anti-platelet factor 4 Fab fragments caused inhibition of binding of 125I-platelet factor 4 to platelets. Most of the labeled platelet factor 4 which was bound to intact platelets was recovered in the Triton X-100-insoluble cytoskeletal fraction prepared from the same platelets after their stimulation by thrombin. The association with the cytoskeleton was inhibited by anti-platelet factor 4 Fab fragments and by low-affinity heparin. Anti-platelet factor 4 125I-labeled Fab fragments bound to resting platelets, and this binding was greatly increased following platelet stimulation with thrombin. This suggested that endogenously secreted platelet factor 4 also binds to the platelet surface. No significant binding to platelets of 125I-labeled beta-thromboglobulin and 125I-labeled anti-beta-thromboglobulin Fab fragments was observed. Fab fragments of monospecific anti-human platelet factor 4 antibody raised in rabbits inhibited platelet aggregation and secretion induced by low concentrations of thrombin. Fab fragments of anti-beta-thromboglobulin antibody had no inhibitory effect. We suggest that the binding of alpha-granule-derived platelet factor 4 to the specific sites on the surface of platelets may modulate platelet aggregation and secretion induced by low levels of platelet agonists.     

Production of humanized Fab fragment against human high affinity IgE receptor in Pichia pastoris

Binding of allergen-IgE complexes to the high affinity IgE receptor (Fc epsilonRI) on mast cells and basophils leads to the release of various mediaters such as histamine. Fab fragments prepared by the papain digestion of humanized antibody against human Fc epsilonRI inhibited the release of histamine from human basophils. Here we established an expression system to directly produce Fab fragments of the humanized anti-human Fc epsilonRI antibody in methylotropic yeast, P. pastoris. Fab fragments were efficiently secreted into the medium at a concentration of 10-40 mg/L using a signal sequence from the P. pastoris phosphatase gene. They were consisted of disulfide-linked light and heavy chains correctly starting from the first amino acid residues by proper cleavage of the signal peptides. The obtained Fab fragments inhibited the binding between IgE and Fc epsilonRI as efficiently as the counterpart prepared by papain digestion of the whole antibody.     

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody–antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1–20 mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS–PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.     

Rationalization of gene regulation by a eukaryotic transcription factor: calculation of regulatory region occupancy from predicted binding affinities

The antigen-binding fragments (Fab) of antibodies are powerful tools in clinical therapy, molecular diagnostics and basic research. However, their principal applications require pure recombinant molecules in large amounts, which are challenging to obtain. Severe limitations in yield, folding and functionality are commonly encountered in bacterial production of Fab fragments. Secretion into the oxidizing periplasm generally results in low yield, whereas expression in the reducing cytoplasmic environment produces unfolded or non-functional protein. We hypothesized that an impaired reducing environment of the cytoplasm would permit correctly folded, functional cytoplasmic expression of Fabs with high yield. We used the Escherichia coli strain FA113, which has no activity of both thioredoxin and glutathione reductase, and thus has an oxidizing cytoplasmic environment. With the newly constructed vector pFAB1 we tested the cytoplasmic expression of two Fab fragments, which recognize the integral membrane protein NhaA, a bacterial Na(+)/H(+) antiporter. These antibodies differ in terms of DNA sequence and stability. Both antibody fragments were produced to very high yields (10-30 mg/l from bacterial cultures at an A(600 nm)=1.2-1.3). This is a factor 50-250 times higher than any other reported over-expression strategy for Fab fragments and currently represents the highest production rate ever been reported for antibody Fab fragments in bacteria grown to similar cell densities. The fragments are fully functional and can be efficiently purified by His-tag chromatography. Expression of active Fab fragments in the bacterial cytoplasm unlocks the possibility of using antibody specific targeting in an intracellular environment. Such a capacity opens new perspectives for investigating metabolic and regulatory pathways in vivo and also provides a powerful selection system for functional genomics.     

Production of Humanized Fab Fragment against Human High Affinity IgE Receptor in Pichia pastoris

Binding of allergen-IgE complexes to the high affinity IgE receptor (FcεRI) on mast cells and basophils leads to the release of various mediaters such as histamine. Fab fragments prepared by the papain digestion of humanized antibody against human FcεRI inhibited the release of histamine from human basophils. Here we established an expression system to directly produce Fab fragments of the humanized anti-human FcεRI antibody in methylotropic yeast, P. pastoris. Fab fragments were efficiently secreted into the medium at a concentration of 10-40 mg/L using a signal sequence from the P. pastoris phosphatase gene. They were consisted of disulfide-linked light and heavy chains correctly starting from the first amino acid residues by proper cleavage of the signal peptides. The obtained Fab fragments inhibited the binding between IgE and FcεRI as efficiently as the counterpart prepared by papain digestion of the whole antibody.     

The effect of antigen concentration,antibody valency and size,and tumor architecture on antibody binding in multicell spheroids

Intact IgG₁ and F(ab′)₂ anti-carcinoembryonic antigen antibodies penetrate human colon adenocarcinoma multicell spheroids much more slowly than Fab fragments and the molecular weight and the binding site valency appear to be the most important factor in determining the rate of penetration. The rate is also influenced considerably by the number of antigen binding sites per cell, with a high antigen concentration slowing penetration appreciably. The tumor cell architecture appears to have a minor effect on antibody penetration when compared to antibody size or antigen concentration.     

Comparison of two forms of catalytic antibody displayed on yeast-cell surface

Two forms of the Fab fragment of the catalytic antibody 6D9 were individually displayed on yeast-cell surface in fusion to the C-terminal half of -agglutinin: one was 6D9 Fab1, in which the light chain of the Fab (Lc) fragment is displayed on cell surface and the heavy chain of the Fab (Fd) fragment is secreted and linked to the Lc fragment with a disulfide bond; the other was 6D9 Fab2, in which the Fd fragment is displayed on cell surface and the Lc fragment is secreted and linked to the Fd fragment with a disulfide bond. Analysis by flow cytometry indicated that some 6D9 Fab2 fragments were unable to construct an appropriate conformation, and that most of the 6D9 Fab1 fragments displayed on yeast-cell surface exhibited higher binding affinity, stability, and catalytic activity. Conformation of the surface-displayed hetero-dimeric Fab fragment mainly depended on the intermolecular disulfide bond between the Lc and Fd fragments. The conformation of 6D9 Fab1 was more stable than that of Fab2. In the reducing environment of solution containing 25 nM DTT, the function of 6D9 Fab2 was almost completely lost. The successful display of 6D9 Fab1 on yeast-cell surface provides a novel approach to the engineering of catalytic antibodies.     

Crystallisation of membrane proteins mediated by antibody fragments

X-ray structures of three different membrane proteins in complex with antibody fragments have been published. The binding of Fv or Fab fragments enlarges the hydrophilic part of integral membrane proteins, thereby providing additional surface for crystal contacts and space for the detergent micelle. In all reported cases, antibody binding was either essential for the crystallisation of the membrane protein or it substantially improved the diffraction quality of the crystals. Antibody-fragment-mediated crystallisation appears to be a valuable tool in particular for membrane proteins with very small hydrophilic or flexible domains.     

The influence of antibody fragment format on phage display based affinity maturation of IgG

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.   In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab.     

The disulfide bonds in antibody variable domains: effects on stability, folding in vitro, and functional expression in Escherichia coli.

The formation of the disulfide bonds in the variable domains VH and VL of the antibody McPC603 was found to be essential for the stability of all antigen binding fragments investigated. Exposure of the Fv fragment to reducing conditions in vitro resulted in irreversible denaturation of both VH and VL. In vitro refolding of the reduced Fv fragment was only possible when the disulfide bonds were allowed to form under oxidizing conditions. The analysis of a series of mutants of the Fv fragment, the Fab fragment and the single-chain Fv fragment, all secreted into the periplasm of Escherichia coli, in which each of the cysteine residues of the variable domains was replaced by a series of other amino acids, showed that functional antigen binding fragments required the presence of both the disulfide bond in VH and the one in VL. These results were also used to devise an alternative expression system based on the production of insoluble fusion proteins consisting of truncated beta-galactosidase and antibody domains, enzymatic cleavage, and refolding and assembly in vitro. This strategy should be useful for providing access to unstable antibody domains and fragments.     

DNA-binding properties of the antibody specific for the Dewar photoproduct of thymidylyl-(3-5')-thymidine

A monoclonal antibody (DEM-1) specific for the Dewar photoproduct is used for detection and quantification of photolesions in DNA. To help understand the molecular recognition of damaged DNA by the antibody protein, we have cloned and sequenced the variable region genes of DEM-1. We have also prepared Fab fragments of DEM-1 (DEM1Fab), and synthesized two kinds of 3'-biotinylated oligonucleotides of different lengths containing a central Dewar photoproduct of TpT to analyze the effects of the antigen size on the binding rates by means of surface plasmon resonance (SPR). Results obtained from SPR analyses suggest that DEM1Fab may recognize tetranucleotide unit as the epitope.