Targeting CCL24 in Inflammatory and Fibrotic Diseases: Rationale and Results from Three CM-101 Phase 1 Studies.

BACKGROUND: Overexpression of C-C motif chemokine ligand 24 (CCL24) is associated with inflammatory and fibrotic diseases, including primary sclerosing cholangitis (PSC), systemic sclerosis, metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH). CM-101 is a humanized monoclonal antibody that neutralizes CCL24 to attenuate inflammation and fibrosis in preclinical models. Here we report the results from two Phase 1a studies investigating the safety and tolerability of intravenous (IV) and subcutaneous (SC) CM-101 in healthy participants, and in one Phase 1b study of IV and SC CM-101 in patients with MASLD without evidence of MASH. METHODS: In each dose group (0.75 mg/kg, 2.5 mg/kg, 5.0 mg/kg, and 10.0 mg/kg) of the single-center, double-blind, placebo-controlled Phase 1a IV study, healthy volunteers were randomized 3:1 to receive a single IV infusion of CM-101 or placebo. In another Phase 1a, single-center, double-blind placebo-controlled study, healthy volunteers were randomized 3:1 to receive a single SC injection of CM-101 5.0 mg/kg or placebo. In the multicenter, double-blind, placebo-controlled Phase 1b MASLD study, patients with MASLD without evidence of MASH were randomized 3:1 to receive the following: cohort 1, IV CM-101 2.5 mg/kg or placebo, and cohort 2, SC CM-101 5.0 mg/kg or placebo every three weeks for 12 weeks. The primary endpoints (for all these studies) were safety, tolerability, and serum pharmacokinetic parameters of CM-101. RESULTS: In each study, adverse events were rare and mild to moderate. The CM-101 pharmacokinetics profile was typical of a monoclonal antibody, with a terminal half-life of approximately 19 days when given IV and approximately 17 days when given as SC injection. In patients with MASLD without evidence of MASH, CM-101 was associated with decreased serum levels of inflammatory, fibrotic, and collagen turnover biomarkers. CONCLUSIONS: In healthy volunteers and patients with MASLD without evidence of MASH, IV and SC CM-101 was well tolerated at doses ranging from 0.75 mg/kg to10.0 mg/kg and engaged its target (i.e., CCL24), indicating therapeutic potential in treating inflammatory and fibrotic diseases. CLINICAL TRIAL RETROSPECTIVELY REGISTRATION: NCT06025851, NCT06037577, and NCT06044467. Date of registration: September 2023.

BND-22, a first-in-class humanized ILT2-blocking antibody, promotes antitumor immunity and tumor regression.

BACKGROUND: Cancer immunotherapy has revolutionized cancer treatment. However, considering the limited success of immunotherapy to only some cancer types and patient cohorts, there is an unmet need for developing new treatments that will result in higher response rates in patients with cancer. Immunoglobulin-like transcript 2 (ILT2), a LILRB family member, is an inhibitory receptor expressed on a variety of immune cells including T cells, natural killer (NK) cells and different myeloid cells. In the tumor microenvironment, binding of class I MHC (in particular HLA-G) to ILT2 on immune cells mediates a strong inhibitory effect, which manifests in inhibition of antitumor cytotoxicity of T and NK cells, and prevention of phagocytosis of the tumor cells by macrophages. METHODS: We describe here the development and characteristics of BND-22, a novel, humanized monoclonal antibody that selectively binds to ILT2 and blocks its interaction with classical MHC I and HLA-G. BND-22 was evaluated for its binding and blocking characteristics as well as its ability to increase the antitumor activity of macrophages, T cells and NK cells in various in vitro, ex vivo and in vivo systems. RESULTS: Collectively, our data suggest that BND-22 enhances activity of both innate and adaptive immune cells, thus generating robust and comprehensive antitumor immunity. In humanized mice models, blocking ILT2 with BND-22 decreased the growth of human tumors, hindered metastatic spread to the lungs, and prolonged survival of the tumor-bearing mice. In addition, BND-22 improved the antitumor immune response of approved therapies such as anti-PD-1 or anti-EGFR antibodies. CONCLUSIONS: BND-22 is a first-in-human ILT2 blocking antibody which has demonstrated efficient antitumor activity in various preclinical models as well as a favorable safety profile. Clinical evaluation of BND-22 as a monotherapy or in combination with other therapeutics is under way in patients with cancer. TRIAL REGISTRATION NUMBER: NCT04717375.

A blocking monoclonal antibody to CCL24 alleviates liver fibrosis and inflammation in experimental models of liver damage.

BACKGROUND & AIMS: C-C motif chemokine ligand 24 (CCL24) is a chemokine that regulates inflammatory and fibrotic activities through its receptor, C-C motif chemokine receptor (CCR3). The aim of the study was to evaluate the involvement of the CCL24-CCR3 axis in liver fibrosis and inflammation and to assess the potential of its blockade, by a monoclonal anti-CCL24 antibody, as a therapeutic strategy for non-alcoholic steatohepatitis (NASH) and liver fibrosis. METHODS: Expression of CCL24 and CCR3 was evaluated in liver biopsies and blood samples. CCL24 involvement in NAFLD/NASH pathogenesis was assessed in Ccl24 knockout mouse using the methionine-choline deficient (MCD) diet experimental model. Antifibrotic and anti-inflammatory effects of CM-101 were tested in the MCD and STAM mouse models and in the thioacetamide (TAA) model in rats. Liver enzymes, liver morphology, histology and collagen deposition, as well as fibrosis- and inflammation-related protein expression were assessed. Activation of hepatic stellate cells (HSCs) was evaluated in the human LX2 cell line. RESULTS: Patients with NASH and advanced NAFLD exhibited significant expression of both CCL24 and CCR3 in liver and blood samples. In the experimental MCD-diet model, Ccl24 knockout mice showed an attenuated liver damage response compared to wild-type mice, exhibiting reduced histological NAFLD activity scores and fibrosis, as well as lower levels of liver enzymes. Blocking CCL24 using CM-101 robustly reduced liver damage in 3 experimental animal models (MCD, STAM and TAA), as demonstrated by attenuation of liver fibrosis and NAFLD activity score. Furthermore, blocking CCL24 by CM-101 significantly inhibited CCL24-induced HSC motility, α-SMA expression and pro-collagen I secretion. CONCLUSION: Our results reveal that blocking CCL24 significantly attenuates liver fibrosis and inflammation and may have a potential therapeutic effect in patients with NASH and/or liver fibrosis. LAY SUMMARY: CCL24 is a chemokine that regulates inflammation and fibrosis. It was found to be significantly expressed in patients with non-alcoholic steatohepatitis, in whom it regulates profibrotic processes in the liver. Herein, we show that blockade of CCL24 using a monoclonal antibody robustly attenuated liver fibrosis and inflammation in animal models, thus suggesting a potential therapeutic role for an anti-CCL24 agent.

Pivotal trial with plant cell-expressed recombinant glucocerebrosidase, taliglucerase alfa, a novel enzyme replacement therapy for Gaucher disease.

Taliglucerase alfa (Protalix Biotherapeutics, Carmiel, Israel) is a novel plant cell-derived recombinant human ß-glucocerebrosidase for Gaucher disease. A phase 3, double-blind, randomized, parallel-group, comparison-dose (30 vs 60 U/kg body weight/infusion) multinational clinical trial was undertaken. Institutional review board approvals were received. A 9-month, 20-infusion trial used inclusion/exclusion criteria in treatment-naive adult patients with splenomegaly and thrombocytopenia. Safety end points were drug-related adverse events: Ab formation and hypersensitivity reactions. Primary efficacy end point was reduction in splenic volume measured by magnetic resonance imaging. Secondary end points were: changes in hemoglobin, hepatic volume, and platelet counts. Exploratory parameters included biomarkers and bone imaging. Twenty-nine patients (11 centers) completed the protocol. There were no serious adverse events; drug-related adverse events were mild/moderate and transient. Two patients (6%) developed non-neutralizing IgG Abs; 2 other patients (6%) developed hypersensitivity reactions. Statistically significant spleen reduction was achieved at 9 months: 26.9% (95% confidence interval [CI]: -31.9, -21.8) in the 30-unit dose group and 38.0% (95% CI: -43.4, -32.8) in the 60-unit dose group (both P < .0001); and in all secondary efficacy end point measures, except platelet counts at the lower dose. These results support safety and efficacy of taliglucerase alfa for Gaucher disease.

A plant-derived recombinant human glucocerebrosidase enzyme--a preclinical and phase I investigation.

UNLABELLED: Gaucher disease is a progressive lysosomal storage disorder caused by the deficiency of glucocerebrosidase leading to the dysfunction in multiple organ systems. Intravenous enzyme replacement is the accepted standard of treatment. In the current report, we evaluate the safety and pharmacokinetics of a novel human recombinant glucocerebrosidase enzyme expressed in transformed plant cells (prGCD), administered to primates and human subjects. Short term (28 days) and long term (9 months) repeated injections with a standard dose of 60 Units/kg and a high dose of 300 Units/kg were administered to monkeys (n = 4/sex/dose). Neither clinical drug-related adverse effects nor neutralizing antibodies were detected in the animals. In a phase I clinical trial, six healthy volunteers were treated by intravenous infusions with escalating single doses of prGCD. Doses of up to 60 Units/kg were administered at weekly intervals. prGCD infusions were very well tolerated. Anti-prGCD antibodies were not detected. The pharmacokinetic profile of the prGCD revealed a prolonged half-life compared to imiglucerase, the commercial enzyme that is manufactured in a costly mammalian cell system. These studies demonstrate the safety and lack of immunogenicity of prGCD. Following these encouraging results, a pivotal phase III clinical trial for prGCD was FDA approved and is currently ongoing. TRIAL REGISTRATION: ClinicalTrials.gov NCT00258778.

Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher's disease using a plant cell system.

Gaucher's disease, a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy using recombinant GCD (Cerezyme) expressed in Chinese hamster ovary (CHO) cells. As complex glycans in mammalian cells do not terminate in mannose residues, which are essential for the biological uptake of GCD via macrophage mannose receptors in human patients with Gaucher's disease, an in vitro glycan modification is required in order to expose the mannose residues on the glycans of Cerezyme. In this report, the production of a recombinant human GCD in a carrot cell suspension culture is described. The recombinant plant-derived GCD (prGCD) is targeted to the storage vacuoles, using a plant-specific C-terminal sorting signal. Notably, the recombinant human GCD expressed in the carrot cells naturally contains terminal mannose residues on its complex glycans, apparently as a result of the activity of a special vacuolar enzyme that modifies complex glycans. Hence, the plant-produced recombinant human GCD does not require exposure of mannose residues in vitro, which is a requirement for the production of Cerezyme. prGCD also displays a level of biological activity similar to that of Cerezyme produced in CHO cells, as well as a highly homologous high-resolution three-dimensional structure, determined by X-ray crystallography. A single-dose toxicity study with prGCD in mice demonstrated the absence of treatment-related adverse reactions or clinical findings, indicating the potential safety of prGCD. prGCD is currently undergoing clinical studies, and may offer a new and alternative therapeutic option for Gaucher's disease.

A truncated IFN-regulatory factor-8\IFN consensus sequence-binding protein acts as dominant-negative, interferes with endogenous protein-protein interactions and leads to apoptosis of immune cells.

IFN consensus sequence-binding protein (ICSBP) is a member of the IFN-regulatory factors (IRF) and is thus also called IRF-8. Its expression is restricted to hematopoietic cells and IRF-8\ICSBP(-/-) mice are defective in myeloid cell differentiation. This factor exerts its transcriptional activity through interaction with other transcription factors, which leads to either repression or activation. In this paper, we describe the use of a dominant-negative (DN) mutant of IRF-8\ICSBP designed to serve as a molecular tool to dissociate the role of the various protein-protein interactions. This DN-ICSBP is truncated at the DNA-binding domain and can still associate with other factors, but the heterocomplexes produced are incapable of binding to the DNA. We show that the DN-ICSBP is able to compete for the interaction of IRF-8\ICSBP with either IRF or non-IRF members such as PU.1. Accordingly, this DN construct was able to inhibit the PU.1-dependent expression of the IgLlambda in the plasmacytoma cell line J558L. However, stable expression of this DN-ICSBP led to apoptosis of only hematopoietic cells. The data suggests that DN-ICSBP can form heterocomplexes with an as-yet unidentified survival factor for hematopoietic cells.

The eukaryote chaperonin CCT is a cold shock protein in Saccharomyces cerevisiae.

The eukaryotic Hsp60 cytoplasmic chaperonin CCT (chaperonin containing the T-complex polypeptide-1) is essential for growth in budding yeast, and mutations in individual CCT subunits have been shown to affect assembly of tubulin and actin. The present research focused mainly on the expression of the CCT subunits, CCTalpha and CCTbeta, in yeast (Saccharomyces cerevisiae). Previous studies showed that, unlike most other chaperones, CCT in yeast does not undergo induction following heat shock. In this study, messenger ribonucleic acid (mRNA) and protein levels of CCT subunits following exposure to low temperatures, were examined. The Northern blot analysis indicated a 3- to 4-fold increase in mRNA levels of CCTalpha and CCTbeta genes after cold shock at 4 degrees C. Interestingly, Western blot analysis showed that cold shock induces an increase in the CCTalpha protein, which is expressed at 10 degrees C, but not at 4 degrees C. Transfer of 4 degrees C cold-shocked cells to 10 degrees C induced a 5-fold increase in the CCTalpha protein level. By means of fluorescent immunostaining and confocal microscopy, we found CCTalpha to be localized in the cortex and the cell cytoplasm of S. cerevisiae. Localization of CCTalpha was not affected at low temperatures. Co-localization of CCT and filaments of actin and tubulin was not observed by microscopy. The induction pattern of the CCTalpha protein suggests that expression of the chaperonin may be primarily important during the recovery from low temperatures and the transition to growth at higher temperatures, as found for other Hsps during the recovery phase from heat shock.

ICSBP/IRF-8 transactivation: a tale of protein-protein interaction.

Interferon (IFN) consensus sequence binding protein (ICSBP) is a member of a family of transcription factors termed IFN regulatory factors (IRF) and is also called IRF-8. Its expression is restricted mainly to cells of the immune system, and it plays a key role in the maturation of macrophages. ICSBP exerts its activity through the formation of different DNA-binding heterocomplexes. The interacting partner dictates a specific DNA recognition sequence, thus rendering ICSBP dual transcriptional activity, that is, repression or activation. Accordingly, such DNA elements were identified at the promoter regions of target genes that manifest macrophage action. A specific module (IRF association domain [IAD]) within ICSBP and a PEST domain located on the interacting partners mediate this association. Thus, ICSBP serves as an excellent prototype, demonstrating how a small subset of transcription factors can regulate gene expression in a spatial, temporal, and delicate tuning through combinatorial protein-protein interactions on different enhanceasomes.