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Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti–4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor–mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP–4-1BBL (RG7826) and CD19–4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen–mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP–4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer–bearing rhesus monkey. Combination of FAP–4-1BBL with tumor antigen–targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP– or CD19–4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8(+) T cells. FAP– and CD19–4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.

New challenges and opportunities in nonclinical safety testing of biotherapeutics were presented and discussed at the 5th European BioSafe Annual General Membership meeting in November 2015 in Ludwigshafen. This article summarizes the presentations and discussions from both the main and the breakout sessions. The following topics were covered in six main sessions: The following questions were discussed across 4 breakout sessions (i-iv) and a case-study based general discussion (v).

The translatability of mouse models for the clinical development of cancer immunotherapies remains limited because of substantial differences between the murine and the human immune systems, as well as dissimilarities in the tumor biology. Non-human primates (NHPs) display good homologies towards the human immune system, based on the development of vaccines to several human pathogens. Tumor-bearing monkeys (TBMs) are NHPs that spontaneously developed cancer with progression patterns similar to humans, potentially bridging the experimental gap between early preclinical models and cancer patients treated with immunotherapeutic agents. TBMs are prevalently rhesus macaques (Macaca mulata) and the most common cancer types are of gastrointestinal, urogenital and endocrine origin. Rhesus macaques are genetically similar to humans and share many characteristics of aging. In both, humans and rhesus macaques, cancer incidence increases with age with the greatest incidence in those over 60 years of age and 20 years, respectively. TBMs were recently employed to assess the tumor targeting and the pharmacodynamics of the FAP-expressing tumor stroma-targeted immunocytokine FAP-IL2v (Evers et al, AACR 2014, Abstract 2592) and costimulatory agonist FAP-4-1BBL (Claus et al, Science translational Medicine, 2019). In the latter study, we could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. These data were the basis to investigate tumor targeting of FAP-4-1BBL in an on-going clinical imaging study. In the present work, we validated further TBMs as translational models for cancer immunotherapy, by performing an imaging/biomarker study in animals exposed to a second FAP-targeted TNFRSF agonist. Two breast cancer-bearing rhesus monkeys (one triple-negative and one Luminal A) were first pre-immunized with a diphtheria/pertussis/tetanus (DTP) vaccine, and then exposed to a single injection of the TNFRSF agonist. In both animals, we could measure a strong systemic immune activation, induction of the TNFRSF agonist in T cells and also tumor regression. These observations validated the applied pre-immunization strategy to induce the TNFRSF protein expression, and confirmed the target to be pursued in the clinical setting. In conclusion, we consider TBMs as valuable translational animal models to generate proof-of-mechanism evidence in small “signal-seeking” preclinical studies. Tumor targeting, biodistribution, peripheral and intra-tumoral pharmacodynamic activity, pharmacokinetics, immunogenicity, intra-tumoral metabolic activity and tumor regression can all be assessed in TBMs. Because of the similar tumor stroma biology shared between humans and rhesus macaques, TBMs are particularly well suited to test FAP-targeting agents. We anticipate that testing cancer immunotherapy compounds in TBMs could be of high predictability for clinical behavior. Citation Format: Maurizio Ceppi, Michael Hettich, Volker Teichgräber, Wouter Driessen, Dietrich Tuerck, Estelle Marrer-Berger, Stefan Evers, Flavio Crameri, Bruno Gomes, Jürgen Bachl, Christian Klein, Christina Claus, Maria Amann, Oliver Krieter, Greg Dugan, David Caudell, Jason Grayson, Sai Kumar Solingapuram Kiran, Mark Cline. Tumor-bearing non-human primates: An unrivaled model for translational cancer immunology research [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6135.

Introduction: Carnitine palmitoyltransferases (CPT1 and 2) regulate the rate of fatty acid oxidation through intake of long-chain fatty acids (LCFA) into mitochondria. Inhibition of CPT can reduce ...

An unexpected dose related increase in oral squamous cell carcinomas was observed in a standard 2-year carcinogenicity study with a novel calcium channel blocker, in which Wistar rats received daily doses of 0, 1.5, 7, 20, or 40 mg/kg of the compound mixed with a standard diet containing fibers from barley. This finding was associated with an increased incidence of severe (destructive) periodontitis and the formation of oro-nasal fistulae at the 2 highest doses. Five assays of the compound for genotoxicity were negative indicating that a genotoxic effect was highly improbable. To investigate the underlying pathogenic mechanisms a second 2-year study in the same strain of rats was initiated and the influence of the diet and/or a possible local irritancy by the drug was assessed. In this second study the compound was administered by oral gavage at daily doses of 0, 7, or 40 mg/kg (later reduced to 20 mg/kg due to systemic intolerance) to rats maintained either on the standard diet or on a low fiber diet assumed to be less aggressive in terms of inducing periodontal lesions. Dose dependent gingival overgrowth (a class-related effect) was observed in the incisor and molar teeth area of all treated groups but was independent of the diet used. No oral tumors were found in the standard diet or low fiber diet controls and all treatment groups fed the low fiber diet, whereas in the high-dose group fed the standard diet a total of 8 oral squamous cell carcinomas were detected in association with an increased incidence of severe periodontitis. These results indicate that the increased incidence of squamous cell carcinomas observed upon chronic administration of the compound is not due to a direct tumorigenic effect of the drug. Tumor formation is attributable to severe periodontal disease favored by the diet and class related gingival overgrowth.

The gene and protein expression changes after exposure to a toxic compound might help elucidate its mechanism of action. In this paper we investigated the effect of carbon tetrachloride (CCl4) on the gene and protein expression in rat livers. Adult Wistar rats were administered CCl4 and livers were harvested 6 or 24 h thereafter. The analysis of mitochondrial proteins on 2D gels showed the upregulation of two proteins involved in stress (catalase and uricase). Among the downregulated proteins, enzymes related to the metabolism of lipids and aminoacids were affected. Additionally, α-2-macroglobulin and senescence marker protein, two proteins whose decrease in expression has been connected to hepatocyte damage, were decreased. Several of the upregulated genes are involved in stress response, DNA and protein damage, and repair. Genes coding for several enzymes involved in different metabolic pathways, including some P450, were downregulated in the treated animals. In conclusion, a single dose of CCl4 caused gene and protein expression changes that can be related to its mechanism of toxicity. Results from both technologies support previous publications and provide possible new toxicity markers. However, the correlation between gene and protein expression at a given time point is less apparent, partly as a result of different regulatory mechanisms between gene and protein expression.

Overdose of acetaminophen (APAP) causes acute hepatotoxicity in rodents and man. The mechanism underlying APAP-induced liver injury remains unclear, but experimental evidence strongly suggests that activation of APAP and subsequent formation of protein adducts are involved in hepatotoxicity. Using proteomics technologies, we constructed a two-dimensional protein database for mouse liver, comprising 256 different gene products and investigated the proteins affected after APAP-induced hepatotoxicity. Adult male mice received a single dose of APAP (100 or 300 mg/kg) or its nontoxic regioisomer 3-acetamidophenol (AMAP, 300 mg/kg). The extent of liver damage was assessed 8 h after administration by increased liver enzyme release and histopathology. Changes in the protein level were studied by comparison of the intensities of the corresponding spots on two-dimensional (2-D) gels. The expression level of about 35 of the identified proteins was modified due to treatment with APAP or AMAP. The observed changes were usually in the order of 10-50% of the control value and were more marked in the high- than in the low-dose of APAP-treated animals. Most of the changes caused by AMAP occurred in a subset of the proteins modified by APAP. Many of the proteins showing changed expression levels are either known targets for covalent modification by N-acetyl-p-benzoquinoneimine (NAPQI) or involved in the regulation of mechanisms that are believed to drive APAP-induced hepatotoxicity.