Ana Cauerhff

Bio: Ana Cauerhff is an academic researcher from Facultad de Ciencias Exactas y Naturales. The author has contributed to research in topics: Lumazine synthase & Epitope. The author has an hindex of 18, co-authored 28 publications receiving 1519 citations. Previous affiliations of Ana Cauerhff include National Institute of Standards and Technology & National University of Rosario.

Single domain antibodies: promising experimental and therapeutic tools in infection and immunity.

Abstract: Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.

Single domain antibodies from llama effectively and specifically block T cell ecto-ADP-ribosyltransferase ART2.2 in vivo

Abstract: The purpose of our study was to develop a tool for blocking the function of a specific leukocyte ecto-enzyme in vivo. ART2.2 is a toxin-related ecto-enzyme that transfers the ADP-ribose moiety from NAD onto other cell surface proteins. ART2.2 induces T cell death by activating the cytolytic P2×7 purinoceptor via ADP-ribosylation. Here, we report the generation of ART2.2-blocking single domain antibodies from an immunized llama. The variable domain of heavy-chain antibodies (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses. Their long CDR3 endows VHH domains with the extraordinary capacity to extend into and block molecular clefts. Following intravenous injection, the ART2.2-specific VHH domains effectively shut off the enzymatic and cytotoxic activities of ART2.2 in lymphatic organs. This blockade was highly specific (blocking ART2.2 but not the related enzymes ART1 or ART2.1), rapid (within 15 min after injection), and reversible (24 h after injection)...

Structural mechanism for affinity maturation of an anti-lysozyme antibody

Abstract: In the immune response against a typical T cell-dependent protein antigen, the affinity maturation process is fast and is associated with the early class switch from IgM to IgG. As such, a comprehension of the molecular basis of affinity maturation could be of great importance in biomedical and biotechnological applications. Affinity maturation of anti-protein antibodies has been reported to be the result of small structural changes, mostly confined to the periphery of the antigen-combining site. However, little is understood about how these small structural changes account for the increase in the affinity toward the antigen. Herein, we present the three-dimensional structure of the Fab fragment from BALB/c mouse mAb F10.6.6 in complex with the antigen lysozyme. This antibody was obtained from a long-term exposure to the antigen. mAb F10.6.6, and the previously described antibody D44.1, are the result of identical or nearly identical somatic recombination events. However, different mutations in the framework and variable regions result in an ≈103 higher affinity for the F10.6.6 antibody. The comparison of the three-dimensional structures of these Fab-lysozyme complexes reveals that the affinity maturation produces a fine tuning of the complementarity of the antigen-combining site toward the epitope, explaining at the molecular level how the immune system is able to increase the affinity of an anti-protein antibody to subnanomolar levels.

Enzyme immobilisation in biocatalysis : Why, what and how

Abstract: In this tutorial review, an overview of the why, what and how of enzyme immobilisation for use in biocatalysis is presented. The importance of biocatalysis in the context of green and sustainable chemicals manufacture is discussed and the necessity for immobilisation of enzymes as a key enabling technology for practical and commercial viability is emphasised. The underlying reasons for immobilisation are the need to improve the stability and recyclability of the biocatalyst compared to the free enzyme. The lower risk of product contamination with enzyme residues and low or no allergenicity are further advantages of immobilised enzymes. Methods for immobilisation are divided into three categories: adsorption on a carrier (support), encapsulation in a carrier, and cross-linking (carrier-free). General considerations regarding immobilisation, regardless of the method used, are immobilisation yield, immobilisation efficiency, activity recovery, enzyme loading (wt% in the biocatalyst) and the physical properties, e.g. particle size and density, hydrophobicity and mechanical robustness of the immobilisate, i.e. the immobilised enzyme as a whole (enzyme + support). The choice of immobilisate is also strongly dependent on the reactor configuration used, e.g. stirred tank, fixed bed, fluidised bed, and the mode of downstream processing. Emphasis is placed on relatively recent developments, such as the use of novel supports such as mesoporous silicas, hydrogels, and smart polymers, and cross-linked enzyme aggregates (CLEAs).

Nanobodies: Natural Single-Domain Antibodies

Abstract: Sera of camelids contain both conventional heterotetrameric antibodies and unique functional heavy (H)-chain antibodies (HCAbs). The H chain of these homodimeric antibodies consists of one antigen-binding domain, the VHH, and two constant domains. HCAbs fail to incorporate light (L) chains owing to the deletion of the first constant domain and a reshaped surface at the VHH side, which normally associates with L chains in conventional antibodies. The genetic elements composing HCAbs have been identified, but the in vivo generation of these antibodies from their dedicated genes into antigen-specific and affinity-matured bona fide antibodies remains largely underinvestigated. However, the facile identification of antigen-specific VHHs and their beneficial biochemical and economic properties (size, affinity, specificity, stability, production cost) supported by multiple crystal structures have encouraged antibody engineering of these single-domain antibodies for use as a research tool and in biotechnology and medicine.

Role of Biocatalysis in Sustainable Chemistry

Abstract: Based on the principles and metrics of green chemistry and sustainable development, biocatalysis is both a green and sustainable technology. This is largely a result of the spectacular advances in molecular biology and biotechnology achieved in the past two decades. Protein engineering has enabled the optimization of existing enzymes and the invention of entirely new biocatalytic reactions that were previously unknown in Nature. It is now eminently feasible to develop enzymatic transformations to fit predefined parameters, resulting in processes that are truly sustainable by design. This approach has successfully been applied, for example, in the industrial synthesis of active pharmaceutical ingredients. In addition to the use of protein engineering, other aspects of biocatalysis engineering, such as substrate, medium, and reactor engineering, can be utilized to improve the efficiency and cost-effectiveness and, hence, the sustainability of biocatalytic reactions. Furthermore, immobilization of an enzyme ...

Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9

Abstract: Variable regions 1 and 2 (V1/V2) of human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein are critical for viral evasion of antibody neutralization, and are themselves protected by extraordinary sequence diversity and N-linked glycosylation. Human antibodies such as PG9 nonetheless engage V1/V2 and neutralize 80% of HIV-1 isolates. Here we report the structure of V1/V2 in complex with PG9. V1/V2 forms a four-stranded β-sheet domain, in which sequence diversity and glycosylation are largely segregated to strand-connecting loops. PG9 recognition involves electrostatic, sequence-independent and glycan interactions: the latter account for over half the interactive surface but are of sufficiently weak affinity to avoid autoreactivity. The structures of V1/V2-directed antibodies CH04 and PGT145 indicate that they share a common mode of glycan penetration by extended anionic loops. In addition to structurally defining V1/V2, the results thus identify a paradigm of antibody recognition for highly glycosylated antigens, which-with PG9-involves a site of vulnerability comprising just two glycans and a strand.