Geert Raes

131I-labeled Anti-HER2 Camelid sdAb as a Theranostic Tool in Cancer Treatment

Purpose: Camelid single-domain antibody-fragments (sdAb) have beneficial pharmacokinetic properties, and those targeted to HER2 can be used for imaging of HER2-overexpressing cancer. Labeled with a therapeutic radionuclide, they may be used for HER2-targeted therapy. Here, we describe the generation of a 131I-labeled sdAb as a theranostic drug to treat HER2-overexpressing cancer. Experimental Design: Anti-HER2 sdAb 2Rs15d was labeled with 131I using [131I]SGMIB and evaluated in vitro. Biodistribution was evaluated in two HER2+ murine xenograft models by micro-SPECT/CT imaging and at necropsy, and under challenge with trastuzumab and pertuzumab. The therapeutic potential of [131I]SGMIB-2Rs15d was investigated in two HER2+ tumor mouse models. A single-dose toxicity study was performed in mice using unlabeled [127I]SGMIB-sdAb at 1.4 mg/kg. The structure of the 2Rs15d–HER2 complex was determined by X-ray crystallography. Results: [131I]SGMIB-2Rs15d bound specifically to HER2+ cells (Kd = 4.74 ± 0.39 nmol/L). High and specific tumor uptake was observed in both BT474/M1 and SKOV-3 tumor xenografted mice and surpassed kidney levels by 3 hours. Extremely low uptake values were observed in other normal tissues at all time points. The crystal structure revealed that 2Rs15d recognizes HER2 Domain 1, consistent with the lack of competition with trastuzumab and pertuzumab observed in vivo. [131I]SGMIB-2Rs15d alone, or in combination with trastuzumab, extended median survival significantly. No toxicity was observed after injecting [127I]SGMIB-2Rs15d. Conclusions: These findings demonstrate the theranostic potential of [131I]SGMIB-2Rs15d. An initial scan using low radioactive [*I]SGMIB-2Rs15d allows patient selection and dosimetry calculations for subsequent therapeutic [131I]SGMIB-2Rs15d and could thereby impact therapy outcome on HER2+ breast cancer patients. Clin Cancer Res; 23(21); 6616–28. ©2017 AACR.

Reprint of: The non-mammalian MIF superfamily

Macrophage migration inhibitory factor (MIF) was first described as a cytokine 50 years ago, and emerged in mammals as a pleiotropic protein with pro-inflammatory, chemotactic, and growth-promoting activities. In addition, MIF has gained substantial attention as a pivotal upstream mediator of innate and adaptive immune responses and with pathologic roles in several diseases. Of less importance in mammals is an intrinsic but non-physiologic enzymatic activity that points to MIFs evolution from an ancient defense molecule. Therefore, it is not surprising that mif-like genes also have been found across a range of different organisms including bacteria, plants, protozoa, helminths, molluscs, arthropods, fish, amphibians and birds. While Genebank analysis identifying mif-like genes across species is extensive, contained herein is an overview of the non-mammalian MIF-like proteins that have been most well studied experimentally. For many of these organisms, MIF contributes to an innate defense system or plays a role in development. For parasitic organisms however, MIF appears to function as a virulence factor aiding in the establishment or persistence of infection by modulating the host immune response. Consequently, a combined targeting of both parasitic and host MIF could lead to more effective treatment strategies for parasitic diseases of socioeconomic importance.

Novel half-life extended anti-MIF nanobodies protect against endotoxic shock

Sepsis—leading to septic shock—is the leading cause of death in intensive care units. The systemic inflammatory response to infection, which is initiated by activated myeloid cells, plays a key role in the lethal outcome. Macrophage migration inhibitory factor (MIF) is an upstream immunoregulatory mediator, released by myeloid cells, that underlies a common genetic susceptibility to different infections and septic shock. Accordingly, strategies that are aimed at inhibiting the action of MIF have therapeutic potential. Here, we report the isolation and characterization of tailorable, small, affinity‐matured nanobodies (Nbs; single‐domain antigen‐binding fragments derived from camelid heavy‐chain Abs) directed against MIF. Of importance, these bioengineered Nbs bind both human and mouse MIFs with nanomolar affinity. NbE5 and NbE10 inhibit key MIF functions that can exacerbate septic shock, such as the tautomerase activity of MIF (by blocking catalytic pocket residues that are critical for MIFs conformation and receptor binding), the TNF‐inducing potential, and the ability of MIF to antagonize glucocorticoid action. A lead NbE10, tailored to be a multivalent, half‐life extended construct (NbE10‐NbAlb8‐NbE10), attenuated lethality in murine endotoxemia when administered via single injection, either prophylactically or therapeutically. Hence, Nbs, with their structural and pharmacologic advantages over currently available inhibitors, may be an effective, novel approach to interfere with the action of MIF in septic shock and other conditions of inflammatory endorgan damage.—Sparkes, A., De Baetselier, P., Brys, L., Cabrito, I., Sterckx, Y. G.‐J., Schoonooghe, S., Muyldermans, S., Raes, G., Bucala, R., Vanlandschoot, P., Van Ginderachter, J. A., Stijlemans, B. Novel half‐life extended anti‐MIF nanobodies protect against endotoxic shock. FASEB J. 32, 3411–3422 (2018). www.fasebj.org

SAT0075 The use of macrophage mannose receptor-targeting nanobodies and spect imaging to study joint inflammation in mice with collagen-induced arthritis

Background Rheumatoid arthritis (RA) is a chronic autoimmune disease that occurs in 0.5-1.0% of the population worldwide. The primary affected organ is the small diarthrodial joint, where the synovial membrane, cartilage and bone tissue will be damaged, ultimately leading to joint deformity and disability of the patient. In the pathogenesis of RA, the synovial membrane becomes hyperplastic and will be infiltrated with T cells, B cells, neutrophils and macrophages. A hallmark of RA is the progressive destruction of bone tissue caused by an elevated bone resorption by osteoclasts, multinuclear cells derived from the monocyte/macrophage lineage. Objectives Our goal was to provide a method to visualize and quantify joint inflammation by the use of an animal model of RA, namely collagen-induced arthritis (CIA). We focused on the macrophage mannose receptor (MMR), since this protein is a well described marker for macrophages, which are numerously present in inflamed tissues. Methods CIA was induced in DBA/1 mice by the injection of collagen type II in Complete Freund’s adjuvant. Flow cytometry and qPCR were used to study the expression of MMR in vitro in macrophages and osteoclasts and in vivo in CIA. SPECT/CT imaging with 99mTc-labeled nanobodies generated against MMR was performed to visualize and quantify MMR expression in the joints of mice. Results MMR expression was shown to be highly upregulated in cultures of bone marrow-derived macrophages and osteoclasts by qPCR and by flow cytometry using MMR-targeting nanobodies. Ex vivo, we identified MMR in lymph nodes, spleen and bone marrow of naïve and arthritic mice. Interestingly, we detected expression of MMR in the synovial fluid, and to a lesser extent in synovium, of mice with CIA. More specifically, MMR was present on CD11b+F4/80+ macrophages isolated from the synovial fluid of the inflamed joints. SPECT/CT imaging was used to detect MMR in vivo in mice with CIA. Therefore, nanobodies against MMR were radioactively labeled with 99mTc, while nanobodies targeting a bacterial enzyme were used as controls. We observed high signals of MMR in lymph nodes, spleen and liver in naïve conditions as well as after immunization. Importantly, the joints of arthritic mice displayed high retention of MMR nanobody. The signal from SPECT imaging was significantly higher in mice with arthritic symptoms compared to naïve animals or immunized mice without clinical symptoms. Conclusions The use of MMR nanobodies in SPECT/CT imaging generates the possibility to track and quantify inflammatory macrophages in vivo in arthritic joints. In vivo quantification of joint inflammation by non-invasive techniques would be a great help in diagnosis and monitoring of disease processes as well as testing the efficiency of (new) drugs. Disclosure of Interest None Declared