(T1330-01-01) ST266 Amnion-Derived Mutipotent Cell Secretome for Prevention and Treatment of Necrotizing Enterocolitis

Purpose: Necrotizing Enterocolitis (NEC) is a rare devastating gastro-intestinal disease, with high mortality, primarily affecting premature ( < 28 weeks), very low birthweight infants¹. NEC etiology is partially attributed to amniotic fluid deprivation in the GI tract of the fetus which is known to attenuate inflammatory cytokine production activated by TLR4².  We investigated the anti-inflammatory, anti-apoptotic and regenerative properties of the ST266 amnion-derived multipotent cell secretome in mouse and piglet models of NEC³.

Methods: Secretome production: cGMP ST266 was produced by Noveome Biotherapeutics from full-term donated placentas obtained from scheduled caesarian section deliveries.  Selected amnion-derived multipotent progenitor cells were separated from the placenta, and cultured in proprietary media³. The cells were discarded and the supernatant ST266 secretome was filtered and collected. The ST266 proteins were characterized by quantitative and bioassay analysis. In vivo NEC studies: All mice and piglet studies were approved by Johns Hopkins University protocols following the Guide for Care and Use of Laboratory Animals.  NEC mouse and piglet models were conducted over 5 days. Experimental NEC was induced in 7-8 day old C57/Bl6  neonatal mouse pups by gavage feeding of formula and bacterial stock cultured from stool of a severe NEC infant⁴. Mice were randomly allocated into NEC or NEC plus ST266 treatment groups. ST266 was administered to treated mice by the intraperitoneal (IP) or the oral route. White Yorkshire sows prematurely delivered approximately 1 kg piglets at 95% gestation by cesarean section. NEC was induced by feeding formula supplemented with enteric bacteria made from an infant with surgical NEC⁵. Piglets were randomly divided into NEC and NEC plus ST266 treatment groups. ST266 was administered to piglets via the IP route. Treatment effects were assessed by NEC severity on deidentified, hematoxylin and eosin (H&E) stained sections of the ileum. Ileal tissue was harvested for analysis of proinflammatory cytokine gene expression by qRT-PCR⁶. RNA-seq analysis of mouse intestinal epithelium were analyzed and gene enrichment analysis was performed to include biological process, chemical component, and molecular function.

Results: NEC mice not treated with ST266 exhibited gross appearance of the gastrointestinal tract, including patchy intestinal edema, bowel dilation, and air within the wall of the bowel (pneumatosis intestinalis). H&E staining of the intestine revealed ileal mucosa disruption and loss of the crypt-villus architecture. First, an NEC mouse prevention study showed small intestines of IP or orally ST266 treated mice displaying pink and healthy gross appearance with intact histological intestinal mucosal. ST266 treated mice also yielded significantly reduced terminal ileum proinflammatory Tnfα, IL-1β cytokines and 3-nitrotyrosine compared to NEC controls. The second study tested ST266 treatment in established NEC. Mice were induced with NEC for 3 days without ST266 treatment. Two days of oral or IP ST266 significantly reduced the severity of NEC compared with mice who had untreated NEC. Treated mice showed improved gross and histological appearance of the terminal ileum, with reduced expression of Tnfα and IL-1β(Figure1). Thus, ST266 prevented and treated NEC in mice. We then examined ST266 prevention of NEC in premature piglets whose size and intestinal architecture closely matches the human premature infant. Piglet small intestine induced to develop NEC exhibited marked edema and inflammation and persistent bloody diarrhea. In contrast, ST266 administration via the intraperitoneal route at 0.5 mL or 1.0 mL/piglet/day significantly reduced the severity of NEC in a dose-dependent manner(Figure 2). The small intestine of ST266 treated piglets was architecturally more intact, resulted in improved histological appearance of the terminal ileal mucosa, reduced Tnfα and IL-1β, and reduced oxidative-mucosal injury. ST266 administration to piglets restored the intestinal barrier integrity. The 5-day piglet model showed ST266 reduced NEC mortality from 38% (5/13) in untreated animals, to 9% (1/11) in ST266 treated piglets. Additional studies demonstrated that ST266 mechanistically inhibited TLR4 signaling, shown to be a key pathway in NEC etiology^4,6. Transcriptome analysis of intestine in mice who were subjected to experimental NEC in the presence or absence of ST266 were found to regulate pathways clustered into gut remodeling, immune responses, gut differentiation and energy metabolism. Thus, ST266 altered the transcriptional profile of the intestinal mucosa.

Conclusion: These results showed that ST266 secretome administration derived from amniotic cells can prevent and/or treat NEC in mouse and piglet models. ST266 reduced piglet mortality, with no observable ST266 toxicity. They showed that the anti-inflammatory effects of ST266 are partially mediated by TLR4 inhibition. ST266 altered the expression of genes that were involved in gut remodeling, immune responses, gut differentiation, and energy metabolism. There is no current means of treating NEC in babies which are responsible for 20% of all neonatal intensive care costs in the US⁷. These findings raise the possibility that ST266 could be useful in the prevention or treatment of clinical NEC⁶ and has resulted in Noveome obtaining Rare Pediatric Disease and Orphan Disease designations from the FDA.

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4. Zhou Q, Niño DF, Yamaguchi Y, Wang S, Fulton WB, Jia H, Lu P, Prindle T Jr, Pamies D, Morris M, Chen LL, Sodhi CP, Hackam DJ. Necrotizing enterocolitis induces T lymphocyte-mediated injury in the developing mammalian brain. Sci Transl Med. 2021 Jan 6;13(575):eaay6621.
5. Sodhi CP, Wipf P, Yamaguchi Y, Fulton WB, Kovler M, Niño DF, Zhou Q, Banfield E, Werts AD, Ladd MR, Buck RH, Goehring KC, Prindle T Jr, Wang S, Jia H, Lu P, Hackam DJ. The human milk oligosaccharides 2'-fucosyllactose and 6'-sialyllactose protect against the development of necrotizing enterocolitis by inhibiting toll-like receptor 4 signaling. Pediatr Res. 2021 Jan;89(1):91-101.
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Figure 1. After a 2 day induction of NEC in neonatal mice, ST266 treatment by the oral or intraperitoneal routes significantly reduced severity of NEC and the inflammatory cytokine TNFa. H&E staining of the gastrointestinal tract showed NEC untreated controls had disrupted, disorganized membrane and villi structures in contrast to ST266 treated NEC mice which exhibited intact gastrointestinal membrane and villi structure for bith oral and ip administration routes.


Figure 2. ST266 reduced the mortality and the inflammatory cytokine TNf-a in 1 kilogram, NEC premature piglets. Gross gastrointestinal structure was preserved without pneumatosis in ST266 treated piglets. H&E staining showed intact gastrointestinal membrane and villi which contrasted with the disrupted structures in untreated NEC piglets.

Acknowledgements: David Hackam is supported by the Office of Extramural Research, National Institutes of Health Grant R35GM141956, and support by a Sponsored Research Grant from Noveome Biotherapeutics.