a b s t r a c tDespite large investments in drug development, the overall success rate of drugs during clinical development remains low. One prominent explanation is flawed preclinical research, in which the use and outcome of animal models is pivotal to bridge the translational gap to the clinic. Therefore, the selection of a validated and predictive animal model is essential to address the clinical question. In this review, the current challenges and limitations of animal models are discussed, with a focus on the fit-forpurpose validation. Moreover, guidance is provided on the selection, design and conduct of an animal model, including the recommendation of assessing both efficacy and safety endpoints. In order to improve the clinical translation, the use of humanized mouse models and preclinical applications of clinical features are discussed. On top, the translational value of animal models could be further enhanced when combined with emerging alternative translational approaches. Focal points:Bedside Animal models are essential for translation of drug findings from bench to bedside. Hence, critical evaluation of the face and predictive validity of these models is important. Reversely, clinical bedside findings that were not predicted by animal testing should be back translated and used to refine the animal models. BenchsideProper design, execution and reporting of animal model results help to make preclinical data more reproducible and translatable to the clinic. IndustryDesign of an animal model strategy is part of the translational plan rather than (a) single experiment (s). Data from animal models are essential in predicting the clinical outcome for a specific drug in development. CommunityReview, standardization and refinement of animal models by disease expert groups helps to improve rigor of animal model testing. It is important that the applied animal models are validated fit-forpurpose according to stringent criteria and reproducible. GovernmentsAs during drug development fit-for-purpose animal models are key for success in clinical translation, financial investments and support from the government to develop, optimize, validate and run such translation tools are important. Over time, this will be of benefit for patients and healthcare institutions. Regulatory agenciesPreclinical testing of a drug in an animal model is not a prerequisite for regulatory agencies before entering clinical trials, but does unquestionably provide valuable data on the expected clinical performance of the drug. Hence, testing in animal models is largely recommended from both a business and patient perspective. In addition, inclusion of safety parameters in animal models will help to build the required safety data package of drugs in development.
TNF has remarkable antitumor activities; however, therapeutic applications have not been possible because of the systemic and lethal proinflammatory effects induced by TNF. Both the antitumor and inflammatory effects of TNF are mediated by the TNF receptor p55 (p55TNFR) (encoded by the Tnfrsf1a gene). The antitumor effect stems from an induction of cell death in tumor endothelium, but the cell type that initiates the lethal inflammatory cascade has been unclear. Using conditional Tnfrsf1a knockout or reactivation mice, we found that the expression level of p55TNFR in intestinal epithelial cells (IECs) is a crucial determinant in TNFinduced lethal inflammation. Remarkably, tumor endothelium and IECs exhibited differential sensitivities to TNF when p55TNFR levels were reduced. Tumor-bearing Tnfrsf1a +/-or IEC-specific p55TNFR-deficient mice showed resistance to TNF-induced lethality, while the tumor endothelium remained fully responsive to TNF-induced apoptosis and tumors regressed. We demonstrate proof of principle for clinical application of this approach using neutralizing anti-human p55TNFR antibodies in human TNFRSF1A knockin mice. Our results uncover an important cellular basis of TNF toxicity and reveal that IEC-specific or systemic reduction of p55TNFR mitigates TNF toxicity without loss of antitumor efficacy.
Background:The atypical chemokine receptor CXCR7 is highly expressed in various types of cancer. Results: CXCR7 Nanobodies were generated and show inhibition of -arrestin2 signaling and secretion of angiogenic CXCL1 in vitro. Anti-CXCR7 Nanobodies reduce tumor growth by inhibiting angiogenesis. Conclusion: CXCR7 inhibition by Nanobodies inhibit head and neck tumor formation. Significance: Anti-CXCR7 therapies are potential novel treatments against head and neck cancer.
IntroductionThe pleiotropic cytokine interleukin-6 (IL-6) plays an important role in the pathogenesis of different diseases, including rheumatoid arthritis (RA). ALX-0061 is a bispecific Nanobody® with a high affinity and potency for IL-6 receptor (IL-6R), combined with an extended half-life by targeting human serum albumin. We describe here the relevant aspects of its in vitro and in vivo pharmacology.MethodsALX-0061 is composed of an affinity-matured IL-6R-targeting domain fused to an albumin-binding domain representing a minimized two-domain structure. A panel of different in vitro assays was used to characterize the biological activities of ALX-0061. The pharmacological properties of ALX-0061 were examined in cynomolgus monkeys, using plasma levels of total soluble (s)IL-6R as pharmacodynamic marker. Therapeutic effect was evaluated in a human IL-6-induced acute phase response model in the same species, and in a collagen-induced arthritis (CIA) model in rhesus monkeys, using tocilizumab as positive control.ResultsALX-0061 was designed to confer the desired pharmacological properties. A 200-fold increase of target affinity was obtained through affinity maturation of the parental domain. The high affinity for sIL-6R (0.19 pM) translated to a concentration-dependent and complete neutralization of sIL-6R in vitro. In cynomolgus monkeys, ALX-0061 showed a dose-dependent and complete inhibition of hIL-6-induced inflammatory parameters, including plasma levels of C-reactive protein (CRP), fibrinogen and platelets. An apparent plasma half-life of 6.6 days was observed after a single intravenous administration of 10 mg/kg ALX-0061 in cynomolgus monkeys, similar to the estimated expected half-life of serum albumin. ALX-0061 and tocilizumab demonstrated a marked decrease in serum CRP levels in a non-human primate CIA model. Clinical effect was confirmed in animals with active drug exposure throughout the study duration.ConclusionsALX-0061 represents a minimized bispecific biotherapeutic of 26 kDa, nearly six times smaller than monoclonal antibodies. High in vitro affinity and potency was demonstrated. Albumin binding as a half-life extension technology resulted in describable and expected pharmacokinetics. Strong IL-6R engagement was shown to translate to in vivo effect in non-human primates, demonstrated via biomarker deregulation as well as clinical effect. Presented results on preclinical pharmacological properties of ALX-0061 are supportive of clinical development in RA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0651-0) contains supplementary material, which is available to authorized users.
Key Points Blockade of VWF-A1 by ALX-0081 induces reperfusion of a thrombus-occluded middle cerebral artery without provoking cerebral bleeding. The interaction between GPIb and VWF is not only essential for platelet adhesion but also for initial thrombus stabilization.
The human epidermal growth factor receptor 3 (HER3) is an interesting target for antitumor therapy. For optimal HER3 signaling inhibition, a biparatopic Nanobody construct (MSB0010853) was developed that binds 2 different HER3 epitopes. In addition, MSB0010853 contains a third HER3 epitope that binds albumin to extend its circulation time. MSB0010853 is cross-reactive with HER3 and albumin of mouse origin. We aimed to gain insight into MSB0010853 biodistribution and tumor uptake by radiolabeling the Nanobody construct with 89 Zr. Methods: MSB0010853 was radiolabeled with 89 Zr. Dose-and time-dependent tumor uptake was studied in nude BALB/c mice bearing a subcutaneous HER3 overexpressing H441 non-small cell lung cancer xenograft. Dose-dependent biodistribution of 89 Zr-MSB0010853 was assessed ex vivo at 24 h after intravenous injection. Protein doses of 5, 10, 25, 100, and 1,000 mg were used. Time-dependent biodistribution of MSB0010853 was analyzed ex vivo at 3, 6, 24, and 96 h after intravenous administration of 25 mg of 89 Zr-MSB0010853. PET imaging and biodistribution were performed 24 h after administration of 25 mg of 89 Zr-MSB0010853 to mice bearing human H441, FaDu (high HER3 expression), or Calu-1 (no HER3 expression) tumor xenografts. Results: Radiolabeling of MSB0010853 with 89 Zr was performed with a radiochemical purity of greater than 95%. Ex vivo biodistribution showed protein dose-and time-dependent distribution of 89 Zr-MSB0010853 in all organs. Uptake of 89 Zr-MSB0010853 in H441 tumors was only time-dependent. Tumor could be visualized up to at least 96 h after injection. The highest mean SUV of 0.6 6 0.2 was observed at 24 h after injection of 25 mg of 89 Zr-MSB0010853. 89 Zr-MSB0010853 tumor uptake correlated with HER3 expression and was highest in H441 (6.2 6 1.1 percentage injected dose per gram [%ID/g]) and lowest in Calu-1 (2.3 6 0.3 %ID/g) xenografts. Conclusion: 89 Zr-MSB0010853 organ distribution and tumor uptake in mice are time-dependent, and tumor uptake correlates with HER3 expression. In contrast to tumor uptake except for kidney uptake, organ distribution of 89 Zr-MSB0010853 is protein dose-dependent for the tested doses. 89 Zr-MSB0010853 PET imaging gives insight into the in vivo behavior of MSB0010853.
The heterodimeric cytokine interleukin (IL) 23 comprises the IL12-shared p40 subunit and an IL23-specific subunit, p19. Together with IL12 and IL27, IL23 sits at the apex of the regulatory mechanisms shaping adaptive immune responses. IL23, together with IL17, plays an important role in the development of chronic inflammation and autoimmune inflammatory diseases. In this context, we generated monovalent antihuman IL23 variable heavy chain domain of llama heavy chain antibody (VHH) domains (Nanobodies®) with low nanomolar affinity for human interleukin (hIL) 23. The crystal structure of a quaternary complex assembling hIL23 and several nanobodies against p19 and p40 subunits allowed identification of distinct epitopes and enabled rational design of a multivalent IL23-specific blocking nanobody. Taking advantage of the ease of nanobody formatting, multivalent IL23 nanobodies were assembled with properly designed linkers flanking an antihuman serum albumin nanobody, with improved hIL23 neutralization capacity in vitro and in vivo, as compared to the monovalent nanobodies. These constructs with long exposure time are excellent candidates for further developments targeting Crohn’s disease, rheumatoid arthritis, and psoriasis.
Tumor necrosis factor (TNF)-induced inflammation prevents its broad application as an antitumor agent. We here report that addition of ZnSO 4 to the drinking water of mice induces expression of heat shock protein 70 (HSP70) in several organs, notably the gastrointestinal track. Zinc conferred doseresponsive protection against TNF-induced hypothermia, systemic induction of interleukin-6 and NO x , as well as against TNF-induced bowel cell death and death of the organism. The protective effect of zinc was completely absent in mice deficient in the major HSP70-inducible gene, hsp70.1, whereas transgenic mice constitutively expressing the human HSP70.A gene, under control of a B-actin promoter, was also protected against TNF, indicating that an increase in HSP70 is necessary and sufficient to confer protection. The therapeutic potential of the protection induced by ZnSO 4 was clearly shown in a TNF/IFN;-based antitumor therapy using three different tumor models. In hsp70.1 wild-type mice, but not in hsp70.1-deficient mice, zinc very significantly protected against lethality but left the antitumor effect intact. We conclude that zinc protects against TNF in a HSP70-dependent way and that protection by zinc could be helpful in developing a safer anticancer therapy with TNF/IFN;. [Cancer Res 2007;67(15):7301-7]
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