Bifunctional Antibodies for Melanoma Therapy

描述（由申请人提供）：该申请利用单克隆抗体3E10，这是一种罕见的狼疮衍生的自身抗体，可以穿透人体细胞而无毒。3E10为开发双特异性抗体和抗体-肽融合蛋白提供了一个独特的平台，可以针对黑色素瘤治疗的细胞内因子进行定制。3E10 scFv片段（由3E10重链和轻链的可变区域组成的重组单链蛋白，由短连接域连接）可以渗透到细胞和细胞核中，并通过重组融合传递蛋白质货物。我们将通过利用人类黑色素瘤的两个公认的弱点来验证这一假设，即这种双功能分子可以合理设计，专门针对黑色素瘤癌细胞：(1)癌基因成瘾（由于激活或过度表达的癌基因，如MDM2）或(2)合成致命性（由于黑色素瘤细胞DNA修复能力的畸变）。在初步工作中，我们已经证明了3E10 scFv在小鼠体内系统给药后能够渗透到人类实体瘤异种移植物的癌细胞细胞核中。为了实现我们的目标，我们组建了一个研究小组。Glazer， Sweasy和Weisbart)在癌症治疗，DNA修复和抗体技术方面具有互补的专业知识。这项提议还利用了耶鲁大学皮肤癌孢子项目的PI Ruth Halaban博士从患者样本中直接建立的大量原发性黑色素瘤细胞群。孢子收集了300多个黑色素瘤样本，从中建立了100多个细胞培养物。Halaban博士将为本提案的工作提供该集合，以测试在临床相关的人类黑色素瘤培养背景下所提出的双功能抗体的功效。在Aim 1中，我们将针对与MDM2过表达相关的人类黑素瘤中的癌基因成瘾。一种由3E10 scFv与MDM2抗体（3G5）的scFv片段结合而成的双功能抗体已经被制备出来。MDM2是p53的负调控因子，MDM2过表达的黑色素瘤对p53有依赖性。我们有初步证据表明，破坏MDM2/p53与3E10-3G5抗体的相互作用会导致细胞凋亡并抑制黑色素瘤的生长。在Aim 2中，我们将3E10 scFv与DNA修复因子衍生的肽结合，BRCA2的伴侣和定位器（PALB2），我们已经发现可以通过阻断这些蛋白之间的关键相互作用来破坏PALB2/RAD51 DNA修复复合物。这种PALB2肽被预测对在碱基切除修复（BER）或同源依赖修复（HDR）两种DNA修复途径中任何一种异常的人类癌细胞具有合成致死性。通过结合抗体-抗原或蛋白-蛋白相互作用的特异性与细胞穿透3E10 scFv片段的独特特性，我们希望这项工作能够提供原理证明，为开发基于抗体的试剂来选择性破坏细胞内通路提供一个新的平台。

DESCRIPTION (provided by applicant): This application takes advantage of monoclonal antibody 3E10, an unusual lupus-derived auto-antibody that can penetrate human cells without toxicity. 3E10 provides a unique platform for developing bi-specific antibodies and antibody-peptide fusion proteins that can be tailored to target intracellular factors for melanoma therapy. The 3E10 scFv fragment (a recombinant single-chain protein composed of the variable regions of the 3E10 heavy and light chains joined by a short linking domain) can penetrate into cells and nuclei and can deliver protein cargoes through recombinant fusion. We will test the hypothesis that such bi-functional molecules can be rationally designed to specifically target melanoma cancer cells by exploiting two well established vulnerabilities in human melanoma: (1) oncogene addiction (due to activated or over-expressed oncogenes such as MDM2) or (2) synthetic lethality (due to aberrations in DNA repair capacity in the melanoma cells). In preliminary work, we have already demonstrated the ability of 3E10 scFv to penetrate into cancer cell nuclei within human solid tumor xenografts in mice following systemic administration. To achieve our goals, we have assembled a team of investigators (Drs. Glazer, Sweasy and Weisbart) with complementary expertise in cancer therapy, DNA repair, and antibody technology. This proposal also takes advantage of a large collection of primary melanoma cell populations established directly from patient samples by Dr. Ruth Halaban, the PI of the Yale SPORE in Skin Cancer. The SPORE collection includes over 300 melanoma samples, from which over 100 cell cultures have been established. Dr. Halaban will make this collection available for the work of this proposal to test the efficacy of the proposed bi-functional antibodies in the context of clinically relevant human melanoma cultures. In Aim 1, we will target oncogene addiction in human melanomas linked to over-expression of MDM2. A bi-functional antibody with 3E10 scFv combined with the scFv fragment of an antibody to MDM2 (3G5) has already been produced. MDM2 is a negative regulator of p53, and melanomas with over-expression of MDM2 have addiction to this factor. We have preliminary evidence that disrupting MDM2/p53 interactions with the 3E10-3G5 antibody causes apoptosis and inhibits melanoma growth. In Aim 2, we will combine 3E10 scFv with a peptide derived from the DNA repair factor, Partner and Localizer of BRCA2 (PALB2), that we have discovered can disrupt PALB2/RAD51 DNA repair complexes by blocking key interactions between these proteins. This PALB2 peptide is predicted to be synthetically lethal to human cancer cells with abnormalities in either of two DNA repair pathways, base excision repair (BER) or homology-dependent repair (HDR). We expect this work to provide proof-of-principle to establish a novel platform for developing antibody- based reagents to selectively disrupt intracellular pathways, by combining the specificity of antibody-antigen or protein-protein interactions with the unique properties of the cell-penetrating 3E10 scFv fragment.