Lena Danielsson

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.

Human monoclonal antibodies produced from L-leucine methyl ester-treated and in vitro immunized peripheral blood lymphocytes.

Primary in vitro immunization of peripheral blood lymphocytes has been studied for the production of human monoclonal antibodies. Depletion of a lysosome-rich cell population, containing large granular lymphocytes, monocytes, and a subset of T suppressor cells, was shown to be crucial before the cells could be immunized in vitro. The in vitro immunized lymphocytes were used to produce human x human and human x mouse hybridomas secreting monoclonal antibodies specific for digoxin, hemocyanin, or a recombinant fragment of gp120 of human immunodeficiency virus.

Sequence determination of variable region genes of two human monoclonal antibodies against Neisseria meningitidis

Nucleotide sequences for the variable regions of both the heavy and light chains for two human monoclonal antibodies have been determined. Both antibodies are directed against an outer membrane protein of Neisseria meningitidis, and their genes show a low resemblance to germline sequences.

The Use of In Vitro Immunization, Cloning of Variable Regions, and SCID Mice for the Production of Human Monoclonal Antibodies

Antibody-dependent immunotherapy dates back several decades, to when polyclonal rabbit serum was initially tested in hematopoetic malignancies. The advent of monoclonal antibodies significantly increased the therapeutic expectations in passive immunization, and the possibility to treat and cure cancer was especially exciting for most scientists. Despite a significant effort and the use of sophisticated drug conjugates the therapeutic efficacy against tumors has only been proven in a minority of the clinical studies. The reasons for this are legion. The absolute majority of monoclonal antibodies has been of mouse origin, which results in the elicitation of a human immune response against these molecules, the production of human antimouse antibody (HAMA), and a short serum half-life of the administered antibody. Production of HAMA, with the subsequently reduced efficacy of the monoclonal antibody, is presently considered the most serious problem related to human immunotherapy based on xenogeneically derived monoclonal antibodies.

A combinatory antibody-antigen microarray assay for high-content screening of single-chain fragment variable clones from recombinant libraries

We have developed a combinatory antibody–antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a “spot-on-spot” print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcα-serine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation.

Charges drive selection of specific antibodies by phage display

Phage display technology has emerged as a leading approach to select proteins with improved properties for many different types of applications. The selection typically selects not only for improved binding properties but also for other factors such as efficiency of protein production and folding in Escherichia coli, the host in which the proteins and the phage are produced. Furthermore, the selection methodology is likely to influence the character of retrieved variants. We have now defined the extent whereby the charge of the displayed proteins influence the selection process, resulting in an increased average positive charge among selected proteins in comparison to the proteins that are harbored in the library before selection. Implications of and possible routes to minimize this effect are discussed.

Epitope mapping of a new anti-Tn antibody detecting gastric cancer cells

Here, we introduce a novel scFv antibody, G2-D11, specific for two adjacent Tn-antigens (GalNAc-Ser/Thr) binding equally to three dimeric forms of the epitope, Ser-Thr, Thr-Thr and Thr-Ser. Compared to other anti-Tn reagents, the binding of G2-D11 is minimally influenced by the peptide structure, which indicates a high degree of carbohydrate epitope dominance and a low influence from the protein backbone. With a high affinity (KDapp = 1.3 × 10-8 M) and no cross-reactivity to either sialyl-Tn epitope or blood group A antigens, scFv G2-D11 is an excellent candidate for a well-defined anti-Tn-antigen reagent. Detailed immunohistochemical evaluation of tissue sections from a cohort of 80 patients with gastric carcinoma showed in all cases positive tumor cells. The observed staining was localized to the cytoplasm and in some cases to the membrane, whereas the surrounding tissue was completely negative demonstrating the usefulness of the novel Tn-antigen binding antibody.

A novel monoclonal antibody targeting carboxymethyllysine, an advanced glycation end product in atherosclerosis and pancreatic cancer

Advanced glycation end products are formed by non-enzymatic reactions between proteins and carbohydrates, causing irreversible lysine and arginine alterations that severely affect protein structure and function. The resulting modifications induce inflammation by binding to scavenger receptors. An increase in advanced glycation end products is observed in a number of diseases e.g. atherosclerosis and cancer. Since advanced glycation end products also are present in healthy individuals, their detection and quantification are of great importance for usage as potential biomarkers. Current methods for advanced glycation end product detection are though limited and solely measure total glycation. This study describes a new epitope-mapped single chain variable fragment, D1-B2, against carboxymethyllysine, produced from a phage library that was constructed from mouse immunizations. The phage library was selected against advanced glycation end product targets using a phage display platform. Characterization of its binding pattern was performed using large synthetic glycated peptide and protein libraries displayed on microarray slides. D1-B2 showed a preference for an aspartic acid, three positions N-terminally from a carboxymethyllysine residue and also bound to a broad collection of glycated proteins. Positive immunohistochemical staining of mouse atherosclerotic plaques and of a tissue microarray of human pancreatic tumors confirmed the usability of the new scFv for advanced glycation end product detection in tissues. This study demonstrates a promising methodology for high-throughput generation of epitope-mapped monoclonal antibodies against AGE.

Intra-tumour IgA1 is common in cancer and is correlated with poor prognosis in bladder cancer.

A high frequency of IgA1-positive tumour cells was found in tissue micro-arrays of oesophagus, colon, testis, lung, breast, bladder and ovarian cancer. IgA1 was observed in the cytoplasm and the plasma membrane. A correlation was found between intra-tumour IgA1 and poor overall survival in a large cohort of bladder cancer patients (n = 99, p = 0.011, log-rank test). The number of IgA1-positive tumour cells was also found to be higher in female than male bladder cancer patients. The presence of IgA1 was confirmed in formalin-fixed paraffin-embedded ovarian carcinoma samples using LC-MS/MS analysis. Uptake of IgA1 was also observed in breast cancer and melanoma cell lines when cultivated in the presence of serum from healthy individuals, indicating a possible origin of the IgA1 antibodies in cancer cells.