Mats Ohlin

Recombining germline-derived CDR sequences for creating diverse single- framework antibody libraries

We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 × 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and functional variation in antibody molecules.

Exploiting sequence space : Shuffling in vivo formed complementarity determining regions into a master framework

A novel approach in molecular design is presented, where in vivo formed complementarity determining regions (CDR) from antibody genes were shuffled into a specific framework region. A synthetic gene library of soluble VH-fragments was created and the complexity of the library was determined by sequencing. The synthetic genes were diverse and contained random combinations of CDR from different germlines. All CDR were randomised in one step and by using in vivo formed CDR, the length, sequence and combination were varied simultaneously.

Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research

Background: Microarrays of plant-derived oligosaccharides are potentially powerful tools for the high throughput discovery and screening of antibodies, enzymes, and carbohydrate-binding proteins. Results: Oligosaccharide microarrays were produced, and their utility was demonstrated in several applications. Conclusion: A new generation of oligosaccharide microarrays will make an important contribution to plant glycomic research. Significance: High throughput screening technology enables the more effective production of carbohydrate active enzymes and molecular probes. Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

Light chain shuffling of a high affinity antibody results in a drift in epitope recognition

Human polyclonal and monoclonal antibodies against pathogens and toxins are potentially useful in the treatment of various diseases. A number of human monoclonal antibodies with protective capacity in vitro have been established by conventional hybridoma technology. However, with the development of phage-display technology, the possibility of specifically tailoring antigen-binding properties has improved substantially. We show here that the reactivity of a high affinity, virus-neutralizing human antibody against the AD-2 epitope of cytomegalovirus gB can be modified by introducing other Vkappa sequences together with the original VH sequence. The fine specificity, as determined by the requirement of particular amino acid residues in the epitope, is shifted in these new antibody fragments. It was also evident that the VH/Vkappa pairing was not promiscuous, since antibody fragments selected by phage display retained light chain sequences very similar to the original hybridoma-derived light chain, proving that a high affinity interaction was very dependent on a co-operativity between both variable domains. These findings show that phage display technology might modify the binding properties of pre-existing, high affinity antibodies.

Real time analysis of antibody-antigen reaction kinetics.

Surface plasmon resonance, i.e. detection of changes in refractive index on a surface, was used in a biosensor to evaluate the dissociation/association rate and affinity constants of human monoclonal IgG and IgM antibodies and Tab fragments. The results showed that an observed difference in affinity constants between intact and fragmented IgG anti‐tetanus antibody was related to approximately 10‐fold differences in dissociation rate constants, since the association rate constants were in the same range, i.e. 2–3×105 (m‐1s‐1). Affinity constants, as determined by conventional solid phase enzyme immunoassays, were substantially higher than the constants produced by the biosensor. Human monoclonal IgM anti‐Tnα antibodies showed, furthermore, one order of magnitude higher association rate constants, as compared with the IgG antibodies, but since the dissociation rate constants were more than ten times higher, the resulting affinity constants of the anti‐carbohydrate IgM antibodies were still somewhat lower than those of the IgG antibodies.

Does endogenous glycosylation prevent the use of mouse monoclonal antibodies as cancer therapeutics

Monoclonal antibodies have many potential therapeutic benefits. However, when applied to humans, mouse monoclonal antibodies have several disadvantages. Here Carl Borrebaeck and colleagues describe a strategy to overcome the anti-Gal activity, thought to be one of the reasons why mouse mAbs have a limited half-life.

Selection of binders from phage displayed antibody libraries using the BIAcore™ biosensor

In this report we show that phage displayed antibodies can be selected based on dissociation rate constants, using a BIAcore biosensor. To demonstrate the principle, two Fab phage stocks displaying antibodies specific for hen egg lysozyme or phenyloxazolone were mixed in a ratio of 1:10 and injected over the biosensor chip containing immobilized lysozyme. Antigen-specific bound phages were eluted and analysed for specificity and phage titer. This procedure enriched for phages carrying specific antibodies. Selection of high affinity binders from phage libraries was then demonstrated with the BIAcore when phages were eluted and collected at different time points. Soluble antibody fragments were subsequently expressed and their kinetic parameters were determined. The time of elution was directly proportional to the affinity, due to decreased dissociation rate constants. This procedure offers a rapid and simple approach for selecting binders from phage libraries differing in antibody dissociation rate constants.

Selection of phage displayed antibodies based on kinetic constants

The display of antibody fragments on the surface of filamentous bacteriophages and the selection of binders from antibody libraries have provided powerful tools to generate human antibodies. We reported recently a new concept (SAP system) for the selection of specific phages by linking antigenic recognition and phage replication, using a soluble fusion protein containing the antigen and a fragment of the M13 coat protein 3. In this investigation, a model library has been composed using six different antibody fragments which were characterized individually regarding their kass, kdiss and Ka. All Fab fragments were specific for a 15 amino acid region of the V3 loop of gp120 (HIV-1). We demonstrated that the SAP system could discriminate between the kinetic parameters of each clone, using different selection strategies. Phages expressing high affinity clones were selected preferentially using low doses of antigen but clones of lower affinity also could be selected by increasing the antigen concentration or using a preselection procedure. Phages expressing antibody fragment with high association or low dissociation rate constants were retrieved by utilizing short contact times between antigen and antibody or antigen-chase conditions.

Structural domains involved in human cytomegalovirus glycoprotein B-mediated cell-cell fusion

A novel fusion assay was established to determine fusion activity with cocultivated human foreskin fibroblasts of stable transfectants derived from human astrocytoma cells (U373) expressing authentic or mutagenized human cytomegalovirus glycoprotein B (HCMV gB; gpUL55). Compared to transfectants expressing authentic HCMV gB, those expressing gB forms with a deletion of hydrophobic domain 1 (hd1; aa 714-747) or with deletions of specific segments in the cytoplasmic tail (aa 811-825 and 871-906) exhibited significantly reduced heterologous fusogenicity. HCMV gB-specific monoclonal antibodies (MAbs) as well as MAb against cellular annexin II prevented fusion of the transfectant expressing authentic gB. Comparable surface exposure of HCMV gB or its derivatives was demonstrated in all transfectants by FACS analysis. Our observations are compatible with the notion that indigenous fusion activity of HCMV gB depends on the extracellular hd1 domain and on the conformation of the cytoplasmic tail.

Antigenic domain 1 of human cytomegalovirus glycoprotein B induces a multitude of different antibodies which, when combined, results in incomplete virus neutralization.

Glycoprotein B (gB, gpUL55) is the major antigen for the induction of neutralizing antibodies against human cytomegalovirus (HCMV), making it an attractive molecule for active and passive immunoprophylaxis. The region between aa 552 and 635 of HCMV gB (termed AD-1) has been identified as the immunodominant target for the humoral immune response following natural infection. AD-1 represents a complex domain which requires a minimal continuous sequence of more than 70 aa for antibody binding. Neutralizing as well as non-neutralizing antibodies can bind to AD-1 in a competitive fashion. The fine specificity of AD-1-binding monoclonal antibodies (MAbs) and affinity-purified human polyclonal antibodies was analysed by using recombinant proteins containing single amino acid substitutions spanning the entire AD-1 domain. Our results revealed that all MAbs had individual patterns of binding to the mutant proteins indicating the presence of a considerable number of distinct antibody-binding sites on AD-1. The neutralization capacity of antibodies could not be predicted from their binding pattern to AD-1 mutant proteins. Polyclonal human antibodies purified from different convalescent sera showed identical binding patterns to the mutant proteins suggesting that the combined antibody specificities present in human sera are comparable between individuals. Neutralization capacities of polyclonal human AD-1 antibodies did not exceed 50% indicating that, during natural infection, a considerable proportion of non-neutralizing antibodies are induced and thus might provide an effective mechanism to evade complete virus neutralization.