Running title: Non-toxic broad anti-tumor activity of an EGFR×4-1BB bispecific trimerbod

32 p.-4 fig.Purpose: The induction of 4-1BB signaling by agonistic antibodies can drive the activation and proliferation of effector T cells and thereby enhance a T-cell–mediated antitumor response. Systemic administration of anti-4-1BB–agonistic IgGs, although effective preclinically, has not advanced in clinical development due to their severe hepatotoxicity.Experimental Design: Here, we generated a humanized EGFR-specific 4-1BB-agonistic trimerbody, which replaces the IgG Fc region with a human collagen homotrimerization domain. It was characterized by structural analysis and in vitro functional studies. We also assessed pharmacokinetics, antitumor efficacy, and toxicity in vivo.Results: In the presence of a T-cell receptor signal, the trimerbody provided potent T-cell costimulation that was strictly dependent on 4-1BB hyperclustering at the point of contact with a tumor antigen-displaying cell surface. It exhibits significant antitumor activity in vivo, without hepatotoxicity, in a wide range of human tumors including colorectal and breast cancer cell-derived xenografts, and non–small cell lung cancer patient-derived xenografts associated with increased tumor-infiltrating CD8+ T cells. The combination of the trimerbody with a PD-L1 blocker led to increased IFNγ secretion in vitro and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer.Conclusions: These results demonstrate the nontoxic broad antitumor activity of humanized Fc-free tumor-specific 4-1BB-agonistic trimerbodies and their synergy with checkpoint blockers, which may provide a way to elicit responses in most patients with cancer while avoiding Fc-mediated adverse reactions.This work was supported by grants from the European Union [IACT Project (602262), H2020-iNEXT (1676)]; the Spanish Ministry of Science, Innovation and Universities and the Spanish Ministry of Economy and Competitiveness (SAF2017-89437-P, CTQ2017-83810-R, RTC-2016-5118-1, RTC-2017-5944-1), partially supported by the European Regional Development Fund; the Carlos III Health Institute (PI16/00357), co-founded by the Plan Nacional de Investigación and the European Union; the CRIS Cancer Foundation (FCRIS-IFI-2018); and the Spanish Association Against Cancer (AECC, 19084). C. Domínguez-Alonso was supported by a predoctoral fellowship from the Spanish Ministry of Science, Innovation and Universities (PRE2018-083445). M. Zonca was supported by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness, co-founded by the European Social Fund (PTQ-16-08340).Peer reviewe

Maraviroc, a CCR5 antagonist, prevents development of hepatocellular carcinoma in a mouse model.

Chronic liver disease may result in a sequential progression through fibrosis, cirrhosis and lead, eventually, to hepatocellular carcinoma (HCC). Hepatic stellate cells (HSC) seem to be responsible for the fibrogenic response through the activation of an autocrine loop involving the chemokine receptor, CCR5. However, the role of CCR5 in HCC remains poorly understood. Since this receptor is also one of the main ports of entry for the human immunodeficiency virus (HIV), several CCR5 inhibitors are being used in the clinic to reduce viral load. We used one of these inhibitors, maraviroc (MVC), in a mouse model of diet-induced HCC to investigate whether this intervention would reduce disease progression. Animals treated with MVC on top of a normal control diet did not present any evidence of toxicity or any morphological change when compared with non-treated mice. Animals treated with MVC presented higher survival, less liver fibrosis, lower levels of liver injury markers and chemokines, less apoptosis, lower proliferation index, and lower tumor burden than their counterparts receiving only the hepatotoxic diet. In addition, MVC inhibits HSC activation markers such as phosphorylation of p38 and ERK, and increases hepatocyte survival. This study suggests that MVC, a well tolerated and clinically characterized drug, may be used as a preventative treatment for HCC. Clinical studies are needed to demonstrate the efficacy of this drug, or other CCR5 inhibitors, in patients with high risk of developing HCC

Maraviroc, a CCR5 antagonist, ameliorates the development of hepatic steatosis in a mouse model of non-alcoholic fatty liver disease (NAFLD).

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the general population. The NAFLD spectrum ranges from simple steatosis to cirrhosis. The chemokine CCL5/RANTES plays an important role in the progression of hepatic inflammation and fibrosis. The objective of this study was to examine the effects of maraviroc, a CCR5 antagonist, on liver pathology in a NAFLD mouse model. METHODS: A total of 32 male C57BL/6 mice were randomly assigned to one of four groups: (i) control group (chow diet plus tap water); (ii) maraviroc group (chow diet plus maraviroc in drinking water); (iii) high-fat diet (HFD) group (HFD plus tap water); and (iv) maraviroc/HFD group (HFD plus maraviroc). All mice were sacrificed 16 weeks after the beginning of the experiment. Biochemical analyses and liver examinations were performed. RESULTS: Mice in the HFD group showed a tendency towards increased body mass gain and liver damage compared with the maraviroc/HFD group. Moreover, liver weight in the HFD group was significantly higher than in the maraviroc/HFD group. Hepatic triglyceride concentration in the maraviroc/HFD group was significantly lower than in the HFD group. Interestingly, the maraviroc/HFD group exhibited a lower degree of steatosis. Furthermore, hepatic CCL5/RANTES expression was significantly lower in the maraviroc/HFD group than in the HFD group. Overall, no differences were observed between the control group and the maraviroc group. CONCLUSIONS: Maraviroc ameliorates hepatic steatosis in an experimental model of NAFLD.status: publishe

Schematic cartoon of the postulated mechanism by which MVC interferes with HCC progression.

<p>The CDE diet damages resident cells of the liver parenchyma, mainly hepatocytes, inducing oval cell proliferation. As a response, these cells and some collaborating neighbors (such as Kupffer cells) secrete a number of cytokines and chemokines including TGF-β1, CCL3, CCL4, and CCL5. The chemokine cocktail promotes the activation of HSC from a quiescent state into a more aggressive phenotype, whereupon a number of chemokine receptors (CCR1, CCR3, CCR5) are expressed at the HSC membrane. Concomitantly, activated HSC secrete a large number of chemokines and cytokines, some of which perpetuate a positive feedback loop that maintain the activated phenotype of the HSC, whereas other molecules induce fibrosis and tumor progression. Maraviroc blocks the autocrine loop by interfering with CCR5, thus stopping HSC activation, fibrosis, and tumor progression.</p

Relative weight of the liver and the spleen.

<p>A significant increase in the relative weight (weight of the organ divided by the body weight) of both organs was recorded in the animals receiving the CDE diet, compared with the control diet. Among the mice that received the CDE diet, those treated with MVC had a significantly smaller liver (<b>A</b>) and spleen (<b>B</b>) than those who were not treated. ***p<0.001 with respect to control; &p<0.05 with respect to CDE.</p

Representative photographs and microphotographs of the liver.

<p>A clear change in color and general texture was easily appreciate when comparing the liver of animals treated with control diet (<b>A,B</b>) or with the CDE one (<b>C,D</b>). The liver of animals in the CDE Group presents a large number of tumors (<b>C</b>). Tumors in Group CDE+MVC were less numerous and much smaller than those in the previous Group (<b>D</b>). Scale bar for A–D = 1 cm. Histological images were stained with hematoxylin-eosin (<b>E–H</b>), with the fluorescent TUNEL technique (<b>I–L</b>), anti Ki67 (<b>M–P</b>), or with anti-CCR5 antibody (Q–T). The first 2 Groups; Control and MVC (<b>E,F</b>) displayed a normal liver morphology. The liver of the CDE Group had numerous atypic cells and frank tumors (<b>G</b>). Animals treated with MVC had intermediate characteristics (<b>H</b>). Scale bar for E–H = 100 µm. The TUNEL technique detected few apoptotic cells in the liver of animals belonging to control Groups (<b>I,J</b>) but the number increased in animals treated with CDE (<b>K</b>) and was reduced by treatment with MVC (<b>L</b>). Scale bar for I–L = 200 µm. The proliferation marker Ki67 detected few cells in control animals (<b>M,N</b>) and great numbers of positive cells in the CDE Group (<b>O</b>). The number of proliferating cells was intermediate in the CDE+MVC Group (<b>P</b>). CCR5 expression was not detected in control Groups (<b>Q,R</b>) but was found in macrophages (arrowheads), HSC (arrows), and other cell types in the CDE (<b>S</b>) and CDE+MVC (<b>T</b>) Groups. Scale bar for M–T = 100 µm.</p