Gertrudis Rojas

Phage antibody fragments library combining a single human light chain variable region with immune mouse heavy chain variable regions

We describe the construction of a phage antibody fragments library which combines, in a single cloning step, a synthetic human light chain variable region (V(L)) with a diverse set of heavy chain variable regions, from a mouse immunized with the prostate specific antigen (PSA). Despite V(L) restriction, selection from this library rendered two different single chain Fv antibody fragments, specifically recognizing PSA. The human V(L), used as a general partner for mouse heavy chains, was constructed by linking the germline A27 gene and the J(K)1 minigene segment, both of which are prominently involved in human antibody responses. Our approach offers a fast and simple way to produce half-human molecules, while keeping the advantage of immunizing animals for high affinity antibodies.

Structure and molecular interactions of a unique antitumor antibody specific for N-glycolyl GM3

N-Glycolyl GM3 ganglioside is an attractive target antigen for cancer immunotherapy, because this epitope is a molecular marker of certain tumor cells and not expressed in normal human tissues. The murine monoclonal antibody 14F7 specifically recognizes N-glycolyl GM3 and shows no cross-reactivity with the abundant N-acetyl GM3 ganglioside, a close structural homologue of N-glycolyl GM3. Here, we report the crystal structure of the 14F7 Fab fragment at 2.5 Å resolution and its molecular model with the saccharide moiety of N-glycolyl GM3, NeuGcα3Galβ4Glcβ. Fab 14F7 contains a very long CDR H3 loop, which divides the antigen-binding site of this antibody into two subsites. In the docking model, the saccharide ligand is bound to one of these subsites, formed solely by heavy chain residues. The discriminative feature of N-glycolyl GM3 versus N-acetyl GM3, its hydroxymethyl group, is positioned in a hydrophilic cavity, forming hydrogen bonds with the carboxyl group of Asp H52, the indole NH of Trp H33 and the hydroxyl group of Tyr H50. For the hydrophobic methyl group of N-acetyl GM3, this environment would not be favorable, explaining why the antibody specifically recognizes N-glycolyl GM3, but not N-acetyl GM3. Mutation of Asp H52 to hydrophobic residues of similar size completely abolished binding. Our model of the antibodycarbohydrate complex is consistent with binding data for several tested glycolipids as well as for a variety of 14F7 mutants with replaced VL domains.

Fine epitope specificity of antibodies against interleukin-2 explains their paradoxical immunomodulatory effects

The functional dichotomy of antibodies against interleukin-2 (IL-2) is thought to depend upon recognition of different cytokine epitopes. Beyond functional studies, the only molecular evidence obtained so far located the epitopes recognized by the immunoenhancing antibodies S4B6 and JES6–5H4 within the predicted interface of IL-2 with the α receptor subunit, explaining the preferential stimulation of effector cells displaying only β and γ receptor chains. A consistent functional map of the epitope bound by the immunoregulatory antibody JES6-1A12 has now been delineated by screening the interactions of phage-displayed antigen variants (with single and multiple mutations) and antigen mimotopes. The target determinant resides in a region between the predicted interfaces with α and β/γ receptor subunits, supporting the dual inhibitory role of the antibody on both interactions. Binding by JES6-1A12 would thus convert complexed IL-2 into a very weak agonist, reinforcing the advantage of T regulatory cells (displaying the high affinity αβγ heterotrimeric receptor) to capture the cytokine by competition and expand over effector cells, ultimately resulting in the observed strong tolerogenic effect of this antibody. Detailed knowledge of the epitopes recognized by anti-IL-2 antibodies with either immunoenhancing or immunoregulatory properties completes the molecular scenario underlying their use to boost or inhibit immune responses in multiple experimental systems. The expanded functional mapping platform now available could be exploited to study other interactions involving related molecular pairs with the final goal of optimizing cytokine and anti-cytokine therapies.

Deciphering the molecular bases of the biological effects of antibodies against Interleukin-2: A versatile platform for fine epitope mapping

Elucidating the network of interactions established by Interleukin-2 is a key step to understanding its role as a master regulator of the immune system. Binding of this cytokine by specific antibodies gives rise to different classes of immune complexes that boost or inhibit immune responses. The molecular bases of such functional dichotomy are likely related to the nature of the recognized epitopes, making it necessary to perform fine epitope mapping studies. The current work was aimed at developing a versatile platform to do so. This was accomplished by display of human and mouse Interleukin-2 on filamentous phages, together with extensive mutagenesis of both antigens and high throughput screening of binding properties of more than 200 variants. Detailed molecular pictures of the epitopes were thus delineated for four antibodies against either human or mouse Interleukin-2, which refined and, in some cases, modified the conclusions derived from previous mapping studies with peptide libraries. Overlapping surface patches on mouse Interleukin-2 that also coincide with the predicted interface between the cytokine and its receptor alpha chain were shown to be recognized by two monoclonal antibodies that promote enhancement of immune responses, shedding new light on the structural bases of their biological activity. Our strategy was powerful enough to reveal multiple binding details and could be used to map the epitopes recognized by other antibodies and to explore additional interactions involving Interleukin-2 and related cytokines, thus contributing to our understanding of the complex structure-function relationships within the immune system.

Biologically active vascular endothelial growth factor as a bacterial recombinant glutathione S‐transferase fusion protein

Human VEGF121 (vascular endothelial growth factor isoform 121) was produced as a recombinant fusion protein with GST (glutathione S‐transferase) in Escherichia coli. After affinity purification with glutathione, the GST–VEGF121 fusion protein preparation was used to obtain antibodies in mice against commercial hrVEGF (human recombinant VEGF) through immunization. It was also employed successfully to select specific antihuman VEGF antibody fragments of human origin employing phage‐display technology. The fusion protein preparation was separated in monomeric, dimeric and oligomeric forms using size‐exclusion chromatography. The dimers were recognized by a soluble VEGF receptor 2–Fc chimaera, and stimulated the growth of human umbilical‐vein endothelial cells in vitro in a similar fashion to a commercial hrVEGF. The presence of GST in the fusion protein apparently did not affect the correct assembly of dimers and display of residues critical for receptor recognition. The two‐step purification method reported in the present paper involves no laborious renaturalization methods, yields 10 mg/l of the mixture of different aggregation states after affinity chromatography, and 5 mg/l of the biologically active dimer after gel filtration, thus providing a source of material for the development of new anti‐angiogenic therapeutic molecules.

A combinatorial mutagenesis approach for functional epitope mapping on phage-displayed target antigen: application to antibodies against epidermal growth factor.

Although multiple different procedures to characterize the epitopes recognized by antibodies have been developed, site-directed mutagenesis remains the method of choice to define the energetic contribution of antigen residues to binding. These studies are useful to identify critical residues and to delineate functional maps of the epitopes. However, they tend to underestimate the roles of residues that are not critical for binding on their own, but contribute to the formation of the target epitope in an additive, or even cooperative, way. Mapping antigenic determinants with a diffuse energetic landscape, which establish multiple individually weak interactions with the antibody paratope, resulting in high affinity and specificity recognition of the epitope as a whole, is thus technically challenging. The current work was aimed at developing a combinatorial strategy to overcome the limitations of site-directed mutagenesis, relying on comprehensive randomization of discrete antigenic regions within phage-displayed antigen libraries. Two model antibodies recognizing epidermal growth factor were used to validate the mapping platform. Abrogation of antibody recognition due to the introduction of simultaneous replacements was able to show the involvement of particular amino acid clusters in epitope formation. The abundance of some of the original residues (or functionally equivalent amino acids sharing their physicochemical properties) among the set of mutated antigen variants selected on a given antibody highlighted their contributions and allowed delineation of a detailed functional map of the corresponding epitope. The use of the combinatorial approach could be expanded to map the interactions between other antigens/antibodies.

Isolation of a novel neutralizing antibody fragment against human vascular endothelial growth factor from a phage-displayed human antibody repertoire using an epitope disturbing strategy.

Following the clinical success of Bevacizumab, a humanized monoclonal antibody that blocks the interaction between vascular endothelial growth factor (VEGF) and its receptors, the search for new neutralizing antibodies targeting this molecule has continued until now. We used a human VEGF variant containing three mutations in the region recognized by Bevacizumab to direct antibody selection towards recognition of other epitopes. A total of seven phage-displayed antibody fragments with diverse binding properties in terms of inter-species cross-reactivity and sensitivity to chemical modifications of the antigen were obtained from a human phage display library. All of them were able to recognize not only the selector mutated antigen, but also native VEGF. One of these phage-displayed antibody fragments, denominated 2H1, was shown to compete with the VEGF receptor 2 for VEGF binding. Purified soluble 2H1 inhibited in a dose dependent manner the ligand-receptor interaction and abolished VEGF-dependent proliferation of human umbilical vein endothelial cells. Our epitope disturbing strategy based on a triple mutant target antigen was successful to focus selection on epitopes different from a known one. Similar approaches could be used to direct phage isolation towards the desired specificity in other antigenic systems.

Engineering the binding site of an antibody against N-glycolyl GM3: from functional mapping to novel anti-ganglioside specificities.

The structurally related gangliosides N-glycolyl GM3 and N-acetyl GM3 are potential targets for tumor immunotherapy. 14F7 is a monoclonal antibody able to discriminate the tumor-specific antigen N-glycolyl GM3 from the closely related N-acetyl GM3 on the basis of the presence of a single additional hydroxyl group in the former. A combinatorial phage display strategy, based on the screening of a large library followed by refined mutagenesis, allowed a thorough exploration of the binding chemistry of this unique antibody. Three essential features of the heavy chain variable region were identified: two aromatic rings (in positions 33 and 100D) contributing to the binding site architecture and an arginine residue (position 98) critical for recognition. Directed evolution of 14F7 resulted in novel variants that cross-react with the tumor-associated antigen N-acetyl GM3 and display recurrent replacements: the substitution W33Q and the appearance of additional arginine residues at several positions of CDR H1. Successful conversion of such engineered variable regions into whole cross-reactive anti-GM3 immunoglobulins validated our phage-based approach to study and modify the lead antibody 14F7. The resulting family of closely related antibodies offers new tools to study the mechanisms of cell death induced by antibodies targeting gangliosides. In vitro directed evolution was useful to overcome the technical limitations to obtain anti-ganglioside antibodies. The case of 14F7 illustrates the power of combining library screening with focused site-directed randomization for a comprehensive scanning of protein interactions.

High throughput functional epitope mapping: Revisiting phage display platform to scan target antigen surface

Antibody engineering must be accompanied by mapping strategies focused on identifying the epitope recognized by each antibody to define its unique functional identity. High throughput fine specificity determination remains technically challenging. We review recent experiences aimed at revisiting the oldest and most extended display technology to develop a robust epitope mapping platform, based on the ability to manipulate target-derived molecules (ranging from the whole native antigen to antigen domains and smaller fragments) on filamentous phages. Single, multiple and combinatorial mutagenesis allowed comprehensive scanning of phage-displayed antigen surface that resulted in the identification of clusters of residues contributing to epitope formation. Functional pictures of the epitope(s) were thus delineated in the natural context. Successful mapping of antibodies against interleukin-2, epidermal growth factor and its receptor, and vascular endothelial growth factor showed the versatility of these procedures, which combine the accuracy of site-directed mutagenesis with the high throughput potential of phage display.

Fine epitope mapping based on phage display and extensive mutagenesis of the target antigen.

The residues contributing to the formation of the epitope recognized by a monoclonal antibody can be defined in several ways. Mutagenesis on the target antigen, followed by screening of the reactivity of the new variants with the antibody, is particularly powerful to reveal the functional contribution of each amino acid in the context of the native antigen. The current protocol provides a relatively simple procedure to study the surface of the target antigen in the search for residues involved in recognition. If the antigen is successfully displayed on the surface of filamentous bacteriophages, it can be quickly scanned by simultaneous mutagenesis of multiple solvent-exposed residues and high-throughput screening of the new variants with the antibody to be mapped. Once a few amino acids critically involved in recognition are defined, they can be used as starting points for a comprehensive exploration of the antigenic region by randomization of their whole neighborhood. The analysis of binding and sequence data allows delineating a detailed picture of the epitope recognized by the antibody under investigation.