A broad range of Fab stabilities within a host of therapeutic IgGs

Although the functional properties of IgGs are well known, little has been published concerning the stability of whole IgG molecules. Stability is, however, a requirement for the development of antibodies for therapeutic or diagnostic applications. The hypervariable antigen-binding region (Fv) is responsible for stability variations between IgGs of identical subclass. To determine the range of stabilities that may be expected for human(ized) antibodies, differential scanning calorimetry was performed on 17 human(ized) antibodies from various in-house programs. The antigen-binding fragments (Fabs) of these antibodies exhibited thermal unfolding transitions with midpoints (T(M)s) varying from 57 to 82 degrees C. Antibodies with very low Fab stabilities were found to aggregate and express poorly. Fab instability was often associated with high levels of uncommonly observed amino acids or CDR loop lengths particularly within the variable heavy chain domain. Overall, the study provides a thermostability range for IgGs and suggests possible stability guidelines for developing antibody diagnostics or therapeutics.     

The murine pan T cell marker CD96 is an adhesion receptor for CD155 and nectin-1

The CD155 ligand CD96 is an immunoglobulin-like protein tentatively allocated to the repertoire of human NK receptors. We report here that the CD96/CD155-interaction is preserved between man and mouse although both receptors are only moderately conserved in amino acid sequence. Moreover, murine CD96 (mCD96) binds to nectin-1, a receptor related to CD155. Applying newly generated monoclonal antibodies specifically recognizing mCD96, an expression profile is revealed resembling closely that of human CD96 (hCD96) on cells of hematopoietic origin. A panel of anti-mCD96 but also recently established anti-mCD155 antibodies effectively prevents formation of CD96/CD155-complexes. This was exploited to demonstrate that the only available receptor for mCD96 present on thymocytes is mCD155. Moreover, T cell adhesion to insect cells expressing mCD155 is blocked by these antibodies depending on the T cell subtype. These results suggest a function of the CD96/CD155-adhesion system in T cell biology.     

Comparison of naturally acquired antibody responses against the C-terminal processing products of Plasmodium vivax Merozoite Surface Protein-1 under low transmission and unstable malaria conditions in Sri Lanka

We report here, for the first time, a comparison of naturally acquired antibody responses to the 42 and 19 kDa C-terminal processing products of Plasmodium vivax Merozoite Surface Protein-1 assayed by ELISA using p42 and p19 baculovirus-derived recombinant proteins, respectively. Test populations comprised patients with microscopy confirmed acute P. vivax infections from two regions endemic for vivax malaria where low transmission and unstable malaria conditions prevail, and a non-endemic urban area, in Sri Lanka. The antibody prevalence to the two proteins, both at the individual and population levels, tend to respond more to p42 than to p19 in all test areas, where >14% of individuals preferentially recognized p42, compared with <2% for p19. In patients with no previous exposure to malaria, 21% preferentially recognized p42, whereas none exclusively recognized p19. A significantly lower prevalence of anti-p19 IgM, but not anti-p42 IgM, was observed among residents from endemic areas compared with their non-endemic counterparts. Individuals from both endemic areas produced significantly less anti-p19 IgM compared with anti-p42 IgM. IgG1 was the predominant IgG isotype for both antigens in all individuals. With increasing exposure to malaria in both endemic areas, anti-p19 antibody responses were dominated by the functionally important IgG1 and IgG3 isotypes, with a concurrent reduction in IgM that was lacking in the non-endemic residents. This antibody switch was also reflected for PvAMA-1 as we previously reported with the identical battery of sera. In contrast, the antibody switch for p42 was restricted to endemic residents with more extensive exposure. These results suggest that an IgM-dominated antibody response against the p42 polymorphic region in endemic residents may interfere with the development of an IgG-dominated "protective" isotype shift to p19, that may complicate vaccine development.     

Recoverin as a cancer-retina antigen

In photoreceptor cells the Ca²⁺-binding protein recoverin controls phosphorylation of the visual receptor rhodopsin by inhibiting rhodopsin kinase (GRK-1). It can also serve as a paraneoplastic antigen in the development of retinal degeneration in some patients with cancer. The aberrant expression of recoverin in cancer cells and the presence of autoantibodies against recoverin are essential for the occurrence of cancer-associated retinopathy, which finally results in the apoptosis of photoreceptor cells. Noteworthy in cancer patients, the aberrant recoverin expression and the appearance of autoantibodies against recoverin are more frequent than paraneoplastic syndromes. We suggest the term “cancer-retina antigens” for this kind of proteins like recoverin that are solely expressed in retina and tumor tissues and evoke antibodies and/or T cells in patients with cancer. The rare development of a paraneoplastic syndrome is possibly caused by this immune response and probably depends on further events allowing to overcome the blood–retina barrier and the immune privileged status of the retina. It is still unknown whether aberrantly expressed recoverin could have a specific function in cancer cells, though it is suggested that it can be functionally associated with G-protein-coupled receptor kinases. This paper reviews the present knowledge on paraneoplastic syndromes associated with the aberrant expression of recoverin. A possible application of recoverin as a potential target for immunotherapy of cancer is discussed.This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2005 (PIVAC 5)”, held in Athens, Greece, on 20–21 September 2005.     

Failure of layer-by-layer multilayers composed of neutravidin-biotin-labeled antibody for sandwich fluoroimmunosensing

The high-density attachment of active antibodies or other recognition molecules to the capture surface is one of the fundamental processes in route to developing effective biosensors. One method applied frequently to enzymatic sensor systems has been the layer-by-layer assembly of the bioactive surface. Cui et al. [Cui, X., Pei, R., Wang, Z., Yang, F., Ma, Y., Dong, S., Yang, X., 2003. Biosens. Bioelectron. 18, 59–67] extended this concept to immunosensors, where they formed multilayers composed of avidin and biotinylated antibody and reported this construct to be a potent way to form an effective surface for surface plasmon resonance biodetection. We reexamined this concept in an effort to establish a simple method to improve the activity of polystyrene capture surfaces used in sandwich fluoroimmunoassays for the detection of the target, staphylococcal enterotoxin B (SEB). Using multilayers prepared by alternating between NeutrAvidin and either biotinylated mono or polyclonal anti-SEB antibody, we found that virtually all the SEB-binding activity was derived from the final layer added; and that additional layers provided no observable enhancement in fluoroimmunoassay signal strength.     

β-glucosidase from <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>: a new and efficient purification procedure and use as a suitable marker in immuno-enzymatic assay

The β-glucosidase enzyme β-glu2 isolated from Sclerotinia sclerotiorum was purified and used as tracer in enzyme linked immunosorbent assay. A novel purification procedure of the protein was developed that consists of ammonium sulphate precipitation, gel filtration on Sephacryl S-200-HR column, ion exchange chromatography on DEAE-Toyopearl and polybuffer exchanger PBE 94 TM chromatofocusing. The pI value was 4.45. K _m and V _max values for the enzyme towards p-nitrophenyl-β-D-glucopyranoside were respectively 0.45 mM and 0.2 U/mL. Thermal stability showed that β-glu2 has a half-life of 85 min at 55 °C and of 25 min at 65 °C. β-glu2 was conjugated to goat anti-rabbit antibodies with glutaraldehyde as cross linking agent according to the one-step method. Conjugates were purified by HPLC gel filtration on TSK 2000. Enzymatic and immunological activities of the β-glucosidase conjugate component were tested by the ELISA method.     

不同佐剂肾综合征出血热纯化疫苗体液免疫效果的研究

在肾综合征出血热(HFRS)纯化疫苗原液中分别加入Al(OH)3、IL-2、GM-CSF、CFA等佐剂的一种或两种,以不加佐剂的纯化疫苗原液作为对照,分别免疫BALB/c小鼠,定期进行眼眶采血,用ELISA法检测血清中抗HFRS病毒的抗体水平。用不同佐剂的HFRS纯化疫苗免疫BALB/c小鼠后,其抗体产生的时间和滴度均不同。佐剂组与对照组相比抗体产生早且(或)滴度高,IL-2佐剂组在免后第3天就可以检测到抗体;联合使用两种佐剂组与单独使用一种佐剂组相比,抗体产生早、滴度高。结果表明,上述各种佐剂对HFRS纯化疫苗诱导BALB/c小鼠产生的免疫应答均有一定的增强作用;IL-2对早期免疫应答、早期抗体的产生有显著意义;联合应用两种佐剂疫苗的免疫效果优于只加其中一种佐剂的疫苗。     

Understanding antibody–antigen associations by molecular dynamics simulations: Detection of important intra- and inter-molecular salt bridges

1 NSec molecular dynamics (MD) simulation of anti-hen egg white antibody, HyHEL63 (HH63), complexed with HEL reveals important molecular interactions, not revealed in its X-ray crystal structure. These molecular interactions were predicted to be critical for the complex formation, based on structure–function studies of this complex and 3-other anti-HEL antibodies, HH8, HH10 and HH26, HEL complexes. All four antibodies belong to the same structural family, referred to here as HH10 family. Ala scanning results show that they recognize ‘coincident epitopes’. 1 NSec explicit, with periodic boundary condition, MD simulation of HH63-HEL reveals the presence of functionally important salt-bridges. Around 200 ps in vacuo and an additional 20 ps explicit simulation agree with the observations from 1 Nsec simulation. Intra-molecular salt-bridges predicted to play significant roles in the complex formation, were revealed during MD simulation. A very stabilizing salt-bridge network, and another intra-molecular salt-bridge, at the binding site of HEL, revealed during the MD simulation, is proposed to predipose binding site geometry for specific binding. All the revealed salt-bridges are present in one or more of the other three complexes and/or involve “hot-spot” epitope and paratope residues. Most of these charged epitope residues make large contribution to the binding free energy. The “hot spot” epitope residue Lys97_Y, which significantly contributes to the free energy of binding in all the complexes, forms an intermolecular salt-bridge in several MD conformers. Our earlier computations have shown that this inter-molecular salt-bridge plays a significant role in determining specificity and flexibility of binding in the HH8-HEL and HH26-HEL complexes. Using a robust criterion of salt-bridge detection, this inter-molecular salt-bridge was detected in the native structures of the HH8-HEL and HH26-HEL complexes, but was not revealed in the crystal structure of HH63-HEL complex. The electrostatic strength of this revealed salt-bridge was very strong. During 1 Nsec MD simulation this salt-bridge networks with another inter-molecular salt-bridge to form an inter-molecular salt-bridge triad. Participation of Lys97_Y in the formation of inter-molecular triad further validates the functional importance of Lys97_Y in HH63-HEL associations. These results demonstrate that many important structural details of biomolecular interactions can be better understood when studied in a dynamic environment, and that MD simulations can complement and expand information obtained from static X-ray structure. This study also highlights “hot-spot” molecular interactions in HyHEL63-HEL complex. The publisher or recipient acknowledges right of the U.S. Government to retain a non exclusive, royalty-free license in and to any copyright covering the article.     

Isolation and affinity maturation of hapten-specific antibodies

More and more recombinant antibodies specific for haptens such as drugs of abuse, dyes and pesticides are being isolated from antibody libraries. Thereby isolated antibodies tend to possess lower affinity than their parental, full-size counterparts, and therefore the isolation techniques must be optimized or the antibody genes must be affinity-matured in order to reach high affinities and specificities required for practical applications. Several strategies have been explored to obtain high-affinity recombinant antibodies from antibody libraries: At the selection level, biopanning optimization can be performed through elution with free hapten, analogue pre-incubation and subtractive panning. At the mutagenesis level, techniques such as random mutagenesis, bacterial mutator strains passaging, site-directed mutagenesis, mutational hotspots targeting, parsimonious mutagenesis, antibody shuffling (chain, DNA and staggered extension process) have been used with various degrees of success to affinity mature or modify hapten-specific antibodies. These techniques are reviewed, illustrated and compared.