Recombinant expression of a biomaterial-targeting BMP-2 containing a peptide tag

描述（由申请人提供）：生物疗法的出现，包括细胞、多肽和蛋白质，使医学能够解决许多未满足的临床需求。通常这些药物是非常有效的，专门激活内源性信号级联，以促进良好表征的生化和生理反应。例如，骨形态发生蛋白-2 （BMP-2）在近20年前被确定为成骨活性的强大启动剂，并已成为脊柱融合术中主要骨科产品的一部分（美敦力公司的InFuse）。尽管年销售额接近10亿美元，BMP-2已被证明可以在异位部位（如气管或食道）触发骨形成，如果不按照标签上的指示精确使用。在颈椎融合术中所谓的超说明书使用BMP-2已导致许多患者并发症，最终fda向医生发出警告，建议不要超说明书使用BMP-2。两个主要问题导致了这些并发症：1)在这些过程中使用了大剂量的BMP-2, 2)将药物添加到胶原蛋白海绵中，而BMP-2对胶原蛋白海绵只有有限的被动亲和力。这些因素是不可分割地联系在一起的，因为大剂量的应用是为了确保随着时间的推移，当蛋白质扩散离开递送部位时，治疗剂量得以保留。出于安全考虑，每年237,000例颈椎融合手术中约有7-10%使用BMP，而超过60%的腰椎融合手术使用BMP。允许在颈椎手术中使用BMP-2将使骨科市场的10亿美元增长近一倍。利用我们的核心技术，affinenergy开发了许多基于肽的连接基序，能够以高亲和力与一系列生物材料结合。可用肽连接物的靶标包括骨替代材料，如胶原蛋白和磷酸三钙。在这里，我们建议开发一种新的转基因重组生产含有肽连接体的BMP-2蛋白。所得分子既保留BMP-2活性，又比未修饰的BMP-2表现出更好的骨移植材料亲和力。我们相信该产品将解决目前未满足的BMP-2递送需求，确保治疗浓度的蛋白质随着时间的推移保持与递送基质的结合，从而减少所需的初始剂量。这一计划也恰逢其时，因为围绕BMP-2的大量知识产权将在未来2-3年内到期。拟建的研究项目将需要：1)构建包含编码序列中肽连接域的BMP-2表达载体；2)建立表达克隆，确保蛋白的可重复性生产；3)体外测试，确定肽标记BMP-2的活性及其对骨替代材料的亲和力。如果成功，第二阶段的工作将包括大规模的培养优化，在一个临界尺寸的缺陷动物模型中测试改良的BMP-2，以及一系列临床前测试。Affinergy认为，合作伙伴，如大型骨科公司，将抓住机会推销这种产品。

DESCRIPTION (provided by applicant): The emergence of biological therapeutics, including cells, peptides, and proteins, has allowed medical science to address a number of unmet clinical needs. Often these agents are extremely potent, specifically activating endogenous signaling cascades to promote well-characterized biochemical and physiological responses. For example, bone morphogenetic protein-2 (BMP-2) was identified almost 20 years ago as a powerful initiator of osteogenic activity, and has since become part of a leading orthopedic product used for spinal fusion procedures (InFuse from Medtronic). Despite annual sales of nearly $1B, BMP-2 has been shown to trigger bone formation at ectopic sites, such as the trachea or esophagus, when not used precisely as directed by its labeling. So-called off-label use of BMP-2 in cervical spinal fusion procedures has led to numerous patient complications, culminating in an FDA-issued warning to physicians advising against off-label use. Two major issues have led to these complications: 1) large doses of BMP-2 are used in these procedures and 2) the drug is added to a collagen sponge for which BMP-2 has only limited passive affinity. These factors are inextricably linked, in that large doses are applied to ensure that a therapeutic dose is retained over time as the protein diffuses away from the delivery site. Due to these safety concerns, BMP is used in approximately 7-10% of the 237,000 cervical fusion procedures performed annually, as opposed to over 60% of lumbar fusion procedures. Enabling the use of BMP-2 in cervical procedures would result in the near doubling of the $1B market for orthobiologics. Using our core technology, Affinergy has developed a number of peptide-based linker motifs, capable of binding to a range of biomaterials with high affinity. Targets for available peptide linkers include bone substitute materials such as collagen and tricalcium phosphate. Here, we propose the development of a novel transgene for the recombinant production of a BMP-2 protein containing a peptide linker. The resulting molecule will both retain BMP-2 activity and exhibit improved affinity for bone grafting materials over unmodified BMP-2. We believe this product will address both currently unmet needs for BMP-2 delivery by ensuring a therapeutic concentration of protein remains bound to the delivery matrix over time, thereby reducing the initial dosage required. This program is also well-timed, with the impending expiration of considerable intellectual property surrounding BMP-2 within the next 2-3 years. The proposed research program will require: 1) the construction of a BMP-2 expression vector containing the peptide-linker domains within the coding sequence; 2) the establishment of an expression clone to ensure the reproducible production of the protein and 3) in vitro testing to determine the activity of peptide-tagged BMP-2 as well as its affinity for bone substitute materials. When successful, Phase II work would include large-scale culture optimization, testing the modified BMP-2 in a critical-sized defect animal model, and a range of preclinical testing. Affinergy believes that partners, such as large orthopedics companies, would seize the opportunity to market this product. PUBLIC HEALTH RELEVANCE: The use of osteogenic proteins in spinal fusion has given rise to a $1B market in spine orthobiologics. While the use of these products continues to grow, certain indications are not able to employ these products due to a range of safety concerns. Here, we propose the incorporation of a peptide tag into the widely used orthobiologic, BMP-2. This peptide will contain a high affinity binding site for bone void filler materials currently used as carriers for BMP-2 in spinal fusion. We believe this product will (1) be restricted to the implantation site, and (2) allow for a smaller dose of BMP-2; these improvements mitigate BMP-2 safety issues such as ectopic bone formation. In addition, this product would enable a more wide-spread and safer use of BMP-2 in cervical spinal fusions, an indication in which orthobiologics are used in only 7-10% of cases because of the known increased incidence of adverse events when used in the cervical spine fusions. The use of Affinergy's proprietary peptides therefore provides effective leverage toward the near doubling of the orthobiologics market.