Hilde Revets

Asthma is an inflammatory lung disease that is initiated and directed by Th2 and inhibited by Th1 cytokines. Microbial infections have been shown to prevent allergic responses by inducing the secretion of the Th1 cytokines IL-12 and IFN-γ. In this study, we examined whether administration of lipoprotein I (OprI) from Pseudomonas aeruginosa could prevent the inflammatory and physiological manifestations of asthma in a murine model of OVA-induced allergic asthma. OprI triggered dendritic cells to make IL-12 and TNF-α, with subsequent IFN-γ production from T cells. OprI stimulation of dendritic cells involved both TLR2 and TLR4. Intranasal coadministration of OprI with OVA allergen resulted in a significant decrease in airway eosinophilia and Th2 (IL-4 and IL-13) cytokines and this effect was sustained after repeated allergen challenge. The immediate suppressive effect of OprI (within 2 days of administration) was accompanied by an increase in Th1 cytokine IFN-γ production and a significant, but transient infiltration of neutrophils. OprI did not redirect the immune system toward a Th1 response since no increased activation of locally recruited Th1 cells could be observed upon repeated challenge with allergen. Our data show for the first time that a bacterial lipoprotein can modulate allergen-specific Th2 effector cells in an allergic response in vivo for a prolonged period via stimulation of the TLR2/4 signaling pathway.

Nanobodies are the smallest fragments of naturally occurring heavy-chain antibodies that have evolved to be fully functional in the absence of a light chain. As such, the cloning and selection of antigen-specific nanobodies obviate the need for construction and screening of large libraries, and for lengthy and unpredictable invitro affinity maturation steps. The unique and well-characterised properties enable nanobodies to excel conventional therapeutic antibodies in terms of recognising uncommon or hidden epitopes, binding into cavities or active sites of protein targets, tailoring of half-life, drug format flexibility, low immunogenic potential and ease of manufacture. Moreover, the favourable biophysical and pharmacological properties of nanobodies, together with the ease of formatting them into multifunctional protein therapeutics, leaves them ideally placed as a new generation of antibody-based therapeutics. This review describes the state of the art on nanobodies and illustrates their potential as c...

Abstract: Secretory leukocyte protease inhibitor (SLPI), an epithelial‐specific serine protease inhibitor of the whey acidic protein (WAP) family, exerts broad tissue‐protective functions: on the one hand, it counteracts inflammatory responses and invading pathogens, and on the other hand, it repairs the damaged tissue. Here, we report that subcutaneous in vivo passage of low‐malignant 3LL‐S cells increases the malignancy of these cancer cells. Applying the subtraction suppressive hybridization method to this cancer model, we identify SLPI as one of the genes whose level of expression directly correlates with the malignancy of the cancer cells. Using transfection experiments, we demonstrate that overexpression of mouse or human SLPI in 3LL‐S cells is sufficient to enhance their malignant behavior, and that this activity of SLPI is related to its ability to inhibit serine proteases. Furthermore, using cDNA dot‐blot hybridization, we show that in human gynecological cancer tissue SLPI expression is frequently increased as compared with normal tissue. This study underscores the promalignant role of SLPI in the development of cancer and its potential usefulness as a biomarker for gynecological cancer.

Nanobodies are the smallest fragments of naturally occurring single-domain antibodies that have evolved to be fully functional in the absence of a light chain. Nanobodies are strictly monomeric, very stable, and highly soluble entities. We identified a nanobody with subnanomolar affinity for the human tumor-associated carcinoembryonic antigen. This nanobody was conjugated to Enterobacter cloacae β-lactamase, and its site-selective anticancer prodrug activation capacity was evaluated. The conjugate was readily purified in high yields without aggregation or loss of functionality of the constituents. In vitro experiments showed that the nanobody–enzyme conjugate effectively activated the release of phenylenediamine mustard from the cephalosporin nitrogen mustard prodrug 7-(4-carboxybutanamido) cephalosporin mustard at the surface of carcinoembryonic antigen-expressing LS174T cancer cells. In vivo studies demonstrated that the conjugate had an excellent biodistribution profile and induced regressions and cures of established tumor xenografts. The easy generation and manufacturing yield of nanobody-based conjugates together with their potent antitumor activity make nanobodies promising vehicles for new generation cancer therapeutics.

The variable domain of functional heavy chain antibodies (VHH) devoid of light chains, found in camels, constitute the smallest intact antigen‐binding domain fragment. Two camel single‐domain fragments, cAb‐Lys2 and cAb‐Lys3, recognizing an overlapping epitope of lysozyme with a dissociation constant of 2 nM and 65 nM, respectively, and a bivalent cAb‐Lys3 were investigated for their ability to target transgenic tumors expressing lysozyme on their membrane. Biodistribution studies revealed that these non‐immunogenic monomeric and bivalent camel single‐domain antigen binders specifically target lysozyme‐expressing tumors and metastatic lesions. The excess of antibody is rapidly eliminated from the blood circulation and no cAb retention was observed in normal organs. The tumor to organ cAb‐ratios at 2 and 8 hr were in the (2.1–10.8):1 and (6.2–23.7):1 range, respectively. The degree and specificity of tumor retention is independent of the affinity of the recombinant camel single‐domain fragments for their antigen and from their univalent monomeric (15 kDa) or bivalent format (33 kDa). This study demonstrates the successful and specific in vivo targeting of tumors by camel single‐domain fragments. It may open perspectives for their future use as tumor‐targeting vehicle, due to their small size, soluble behaviour and because they are non‐immunogenic and interact with epitopes that are less antigenic for conventional antibodies.

Immunity againstLeishmania majorrequires rapid induction of a type 1 immune response in which tumor necrosis factor alpha (TNF-α) plays an essential role. Hence, vaccination strategies that simulate the protective immune response found in hosts that have recovered from natural infection provide a rational approach to combat leishmaniasis. One method for optimizing the qualitative and quantitative immune responses after vaccination is to use an adjuvant. In this study we demonstrate that the OprI lipoprotein (L-OprI) fromPseudomonas aeruginosainduces a long-term cellular (gamma interferon [IFN-γ]) and humoral (immunoglobulin G2a) type 1 immune response against a truncated 32-kDa version (COOHgp63) of the 63-kDa major cell surface glycoprotein gp63. By contrast, immunization with COOHgp63 either fused to OprI nonlipoprotein or with no adjuvant did not result in the induction of type 1 immune responses. The adjuvanticity of L-OprI is strongly dependent on its capacity to induce TNF-α, since generation of type 1 immune responses is clearly delayed and impaired in TNF-α^−/−mice. Vaccination with L-OprICOOHgp63 fusion protein protected BALB/c mice againstL. majorinfection for at least 19 weeks. Vaccinated mice were largely free of lesions or clearly controlled lesion size on termination of the experiment. The control of disease progression in mice vaccinated with L-OprICOOHgp63 was associated with enhancement of antigen-specific IFN-γ production. These data indicate that bacterial lipoproteins constitute appropriate adjuvants to include in vaccines against diseases in which type 1 immune responses are important for protection.