Targeting EMP2 for the treatment of triple negative breast cancer with novel anti-EMP2 Granzyme B immunoconjugates

项目摘要/摘要 免疫结合物的设计是将高度细胞毒剂集中输送到特定的靶细胞，使用 单克隆抗体，潜在地提高了该试剂的治疗指数。为此，我们建议 用抗EMP2 IgG1抗体运送细胞毒蛋白有效载荷。EMP2，或上皮膜蛋白-2， 是一种生物标记物，在所检查的大多数浸润性乳腺癌肿瘤(63%；n=97)中高度表达 与健康乳腺上皮相比，超过75%的三阴性乳腺上皮有EMP2高表达 乳腺癌原发灶和转移灶中均可见EMP2的病例 损伤。由于工程重组抗体为癌症诊断和治疗带来了巨大的希望，我们已经 精心组建了一个以加州大学洛杉矶分校和德克萨斯大学MD安德森癌症中心为中心的研究团队 中心研制与人丝氨酸蛋白酶颗粒酶B融合的重组抗EMP2抗体 心理治疗。加州大学洛杉矶分校的研究小组此前已经表明，针对EMP2的全长IgG1促进了细胞凋亡 在体外和体内都有一些EMP2阳性的肿瘤，但由于新的数据提示抗EMP2的IgG1 可以迅速内化，我们假设它可能作为一种新的候选药物的偶联。对.的使用 颗粒酶B(Grazyme B，GRB)作为一种免疫结合物，被认为是一种神奇的子弹，一旦被输送到 在细胞质中，它激活了细胞的凋亡途径，几乎没有诱导免疫原性。圣战者组织 实验室利用人GRB作为产生重组细胞死亡诱导融合的有效载荷 蛋白质，并清楚地表明，含有GrB的融合结构具有令人印象深刻和高度 通过抗体或生长因子细胞靶向传递到细胞质时的选择性细胞毒作用 承运人。 在这项提案中，明确的目标是促进抗EMP2抗体以及GRB作为一种 为临床检测细胞毒有效载荷，我们将研制并测定两种抗EMP2的IgG1-Grb的疗效 共轭关系。因此，具体目标是设计和测试与抗EMP2相关的GRB的有效性 用于细胞毒性的IgG1以及开始创建临床前包以了解其功能亲和力和 稳定性。当裸露抗体在人和老鼠之间发生交叉反应时， 免疫结合物，包括药代动力学和最大耐受量，也将被确定。vt.给出 它在一些妇科肿瘤中高表达，包括卵巢和子宫内膜，这些研究将 将EMP2定位为女性癌症的可行靶点非常重要。考虑到对新毒素的需求，它也会 免疫原性低，有助于将颗粒酶B定位在抗体结合毒素的前沿。

PROJECT SUMMARY/ABSTRACT Immunoconjugates are designed to focus the delivery of highly cytotoxic agents to specific target cells using monoclonal antibodies, potentially improving the therapeutic index of the agent. To this end, we propose to deliver a cytotoxic protein payload with an anti-EMP2 IgG1 antibody. EMP2, or epithelial membrane protein-2, is a biomarker that is highly expressed in the majority (63%; n=97) of invasive breast cancer tumors examined compared to healthy mammary epithelium, and high EMP2 expression is observed in over 75% of triple negative breast cancer cases examined where EMP2 is observed in both the primary tumor as well as in metastatic lesions. As engineered recombinant antibodies hold great promise for cancer diagnostics and therapy, we have carefully assembled a research team centered at UCLA and The University of Texas MD Anderson Cancer Center to develop recombinant anti-EMP2 antibodies fused to the human serine protease granzyme B for therapy. The group at UCLA has previously shown that a full length IgG1 against EMP2 promotes apoptosis both in vitro and in vivo in a number of EMP2 positive tumors, but as new data suggests that the anti-EMP2 IgG1 can internalize rapidly, we hypothesize that it may serve as a novel candidate for drug conjugation. The use of Granzyme B (GrB) as an immunoconjugate has been proposed as a “magic bullet” as, once delivered to the cytoplasm of a cell, it activates apoptosis pathways with little to no induction of immunogenicity. The Mohamedali lab has utilized human GrB as an effective payload for the generation of recombinant cell death-inducing fusion proteins and has clearly demonstrated that GrB-containing fusion constructs have impressive and highly selective cytotoxic effects when delivered to the cytoplasm by either antibody or growth factor cell targeting carriers. In this proposal, with the explicit goal of advancing the science of anti-EMP2 antibodies as well as GrB as a cytotoxic payload for clinical testing, we will create and determine the efficacy of two anti-EMP2 IgG1- GrB conjugates. Accordingly, the specific aims propose to design and test the efficacy of GrB linked to an anti-EMP2 IgG1 for cytotoxicity as well as start creating a preclinical package to understand its functional affinity and stability. As the naked antibody cross-reacts between human and mouse, the toxicity profile of the immunoconjugate, including pharmacokinetics and maximum tolerated dosage, will also be determined. Given its high expression in a number of gynecological tumors including ovarian and endometrial, these studies will be important to position EMP2 as a viable target for cancers in women. It will also, given the need for new toxins with low immunogenicity, help position granzyme B in the forefront of antibody linked toxins.