Prevention of Proliferative Vitreoretinopathy by HC-HA/PTX3

HC-HA/PTX 3预防视网膜变性的实验研究 总结 增生性玻璃体视网膜病变（PVR）的特征是在视网膜表面形成膜 在孔源性视网膜脱离（RRD）之后，在此期间RPE细胞分散到玻璃体中 它们失去接触抑制并暴露于多种生长因子和细胞因子的腔。这 病理环境促进RPE细胞向成纤维细胞或肌成纤维细胞的增殖和EMT， 玻璃体内膜这些PVR膜对视网膜施加牵引力，并成为视网膜的主要结构。 RRD手术后失败的原因。尽管进行了额外的手术干预， 扶贫在初次RRD手术中预防PVR可提高视力成功率。可惜 以前使用不同试剂的尝试都没有成功。 使用体外RPE细胞培养模型，我们已经报道了以下接触抑制扰动 在RPE细胞中，EGF和FGF-2上调，而TGF-β 1下调经典Wnt信号。 在不可逆瘢痕形成中，TGF-β 1促进典型的TGF-β/Smad/ZEB信号传导 EMT阶段我们已经成功地从羊膜（AM）中纯化和表征了HC-HA/PTX 3。 并报道说，这种独特的基质负责AM的抗炎，抗瘢痕形成和抗- 血管生成治疗作用。HC-HA/PTX 3通过五聚蛋白3（PTX 3）与HC-HA之间的紧密结合形成。 HC-HA，由共价连接至重链1（HCl）的高分子量透明质酸（HA）组成 通过肿瘤坏死因子刺激基因6（TSG-6）的催化作用，产生α-胰蛋白酶间抑制剂（II）。 通过第一阶段的支持，我们已经证明了HC-HA/PTX 3可以成为一种新型的"生物制剂"， 通过在上述体外模型中抑制增殖和EMT来抑制PVR，该体外模型已被优化以更好地 在细胞密度、生长因子刺激和测量方面模拟PVR的体内病理过程 方法.我们已开发了作为生产过程中控制所需的效价测定 来自不同供体的HC-HA/PTX 3，证明了安全性（即，缺乏细胞毒性）和HC- HA/PTX 3在宽剂量范围内的抗肿瘤作用，证明了HC-HA/PTX 3而不是HA在抑制肿瘤细胞增殖方面的功效。 由ARPE-19细胞和原代人RPE细胞引起的增殖和凝胶收缩，并描绘了 HC-HA/PTX 3抑制上述Wnt和TGF-β信号传导的作用模式。这些 这些成就使我们能够在第二阶段申请中提出扩大HC-HA/PTX 3的生产规模 通过将来自同一供体的AM和脐带（UC）组合（目的1），以建立释放标准和 通过可重现的GMP生产（目的2），确定HC-HA/PTX 3制剂的稳定性，并确定 在我们最近建立的兔PVR模型中玻璃体内注射HC-HA/PTX 3的安全性和有效性（目的3）。 总的来说，我们希望收集必要和充分的临床前数据，以便向FDA提交IND申请 因此，公司可以通过部署这种新型生物制剂来抓住独特的商机， 治疗这种严重的视网膜致盲疾病的全球需求尚未得到满足。

Prevention of Proliferative Vitreoretinopathy by HC-HA/PTX3 Summary Proliferative vitreoretinopathy (PVR) is characterized by membranes that develop on the surface of the retina after rhegmatogenous retinal detachments (RRD), during which time RPE cells are dispersed into the vitreous cavity where they lose contact inhibition and are exposed to multiple growth factors and cytokines. This pathological setting promotes proliferation and EMT of RPE cells to fibroblasts or myofibroblasts that produce intravitreal membranes. These PVR membranes exert tractional forces on the retina and become the leading cause of failure after RRD surgery. Despite additional surgical interventions, the visual outcome still remains poor. Prevention of PVR during the initial RRD surgery could improve the visual success rate. Unfortunately, all previous attempts using different agents have been unsuccessful. Using an in vitro RPE cell culture model, we have reported that following perturbation of contact inhibition of RPE cells, EGF and FGF-2 upregulate while TGF-β1 downregulates canonical Wnt signaling in the proliferative phase, but TGF-β1 promotes canonical TGF-β/Smad/ZEB signaling in the irreversible scarring phase of EMT. We have successfully purified and characterized HC-HA/PTX3 from amniotic membrane (AM) and have reported that this unique matrix is responsible for AM's anti-inflammatory, anti-scarring and anti- angiogenic therapeutic actions. HC-HA/PTX3 is formed by tight association between pentraxin 3 (PTX3) and HC-HA, which consists of high molecular weight hyaluronic acid (HA) covalently linked to heavy chain 1 (HC1) of inter-α-trypsin inhibitor (I I) through the catalytic action of tumor necrosis factor-stimulated gene-6 (TSG-6). Through Phase I support, we have proven the concept that HC-HA/PTX3 can be a novel “biologic” to prevent PVR by inhibiting proliferation and EMT in the aforementioned in vitro model that has been optimized to better mimic in vivo pathological processes of PVR regarding cell density, growth factor stimulation, and measurement methods. We have developed the potency assay that is required as an in-process control of the manufacturing of HC-HA/PTX3 from different donors, demonstrated the safety (i.e., lack of cytotoxicity) and the efficacy of HC- HA/PTX3 over a wide range of doses, demonstrated the efficacy of HC-HA/PTX3, but not HA, in inhibiting proliferation and gel contraction caused by both ARPE-19 cells and primary human RPE cells, and delineated the mode of action of HC-HA/PTX3 in inhibiting the aforementioned Wnt and TGF-β signaling. These accomplishments allow us to propose in this Phase II application to scale up the manufacturing of HC-HA/PTX3 by combining AM and umbilical cord (UC) from the same donor (Aim 1), to establish the release criteria and the stability of the HC-HA/PTX3 formulation via reproducible GMP manufacturing (Aim 2), and to determine the safety and efficacy of intravitreal injection of HC-HA/PTX3 in our recently-established rabbit PVR model (Aim 3). Collectively, we would like to gather necessary and sufficient pre-clinical data for an IND submission to the FDA so that the Company can capture a unique business opportunity by deploying this novel biologic to fulfill the unmet global need of treating this severe retinal blinding disease.