Bifunctional Immunostimulatory Antibodies for Treatment of Breast Cancer

描述(申请人提供)：癌症通过产生转化生长因子-�(转化生长因子-�)来逃避免疫监视，转化生长因子-转化生长因子是一种多功能细胞因子，通过促进上皮-间充质转化和抑制免疫反应促进肿瘤的进展、侵袭和转移。转化生长因子-�直接减弱免疫效应细胞(NK细胞和CD8+T细胞)的激活和细胞毒功能，促进调节性T细胞(Tregs)的发育和功能，调节性T细胞是表达叉头盒P3(FoxP3+)的免疫抑制T细胞的一个亚群。转化生长因子-�诱导FoxP3表达，上调细胞毒性T淋巴细胞抗原-4(CTLA-4)，这是一种抑制T细胞激活的分子。我们的研究表明，转化生长因子-�和CTLA-4在免疫系统中起着协同但多余的刹车作用，并表明这些免疫检查点的取消是对抗Treg和释放针对肿瘤细胞的先天和获得性免疫反应所必需的。为了推进这一策略，我们开发了两种新的双功能免疫刺激抗体来对抗靶肿瘤细胞微环境中的转化生长因子-�：1.抗�-转化生长因子受体RII：一种双功能抗体，通过将转化生长因子受体II的胞外结构域序列与抗�重链抗体(西妥昔单抗)融合在一起来隔离转化生长因子-GFR。2.抗CTLA-4-TG-�RII：一种双功能抗体，通过融合到抗CTLA-4抗体重链C末端的胞外结构域序列来隔离转化生长因子-�-�。大约15%-25%的乳腺癌患者患有三阴性乳腺癌(TNBC)，这是一种侵袭性的治疗难治性亚型，缺乏激素受体和HER2/neu。尽管80%的TNBC过度表达表皮生长因子受体(EGFR)，但它们对西妥昔单抗不起作用。我们的研究表明，转化生长因子-�是肿瘤对西妥昔单抗耐药的关键决定因素。由于TnBC表现出转化生长因子-�活性的信号--EMT和Treg的浸润，它们可能成为靶向的双功能免疫刺激抗体的理想靶点，以对抗转化生长因子-�介导的免疫耐受。具体目的是：1.评价抗EGFR-转化生长因子�RII对荷瘤小鼠的抗肿瘤效果：(A)检测抗EGFR-转化生长因子�RII的抗肿瘤效果是否优于西妥昔单抗，并确定其作用机制；(B)检测抗EGFR-转化生长因子�RII能否逆转人CD34+细胞重组NOG小鼠的免疫耐受，促进T细胞介导的肿瘤消退。2.评价抗CTLA-4-转化生长因子�RII对人CD34+细胞构建的荷瘤NOG小鼠的抗肿瘤作用：(A)检测抗CTLA-4-转化生长因子�RII是否能拮抗Tregs并激活抗肿瘤免疫；(B)比较抗CTLA-4-转化生长因子�RII与伊匹单抗或抗转化生长因子-�的抗肿瘤作用。影响：该项目寻求通过新型靶向双功能免疫刺激抗体对抗肿瘤诱导的免疫耐受来推进TNBC和其他常见癌症的治疗。

DESCRIPTION (provided by applicant): Cancers circumvent immune surveillance via production of transforming growth factor-� (TGF-�), a multifunctional cytokine that facilitates tumor progression, invasion, and metastasis via promotion of epithelial- mesenchymal transition (EMT) and suppression of immune responses. TGF-� directly attenuates the activation and cytotoxic function of immune effector cells (NK cells and CD8+ T cells), and promotes the development and function of regulatory T cells (Tregs), a sub-population of immunosuppressive CD4+ T cells that express forkhead box P3 (FoxP3+). TGF-� induces expression of FoxP3, which upregulates cytotoxic T lymphocyte antigen-4 (CTLA-4), a molecule that restrains the activation of T cells. Our studies demonstrate that TGF-� and CTLA-4 function as cooperative, yet redundant, brakes on the immune system, and indicate that the abrogation of these immune checkpoints is required to counteract Tregs and unleash innate and adaptive immune responses against tumor cells. To advance this strategy, we have developed two novel bifunctional immunostimulatory antibodies to counteract TGF-� in the microenvironment of the targeted tumor cell or Treg: 1. Anti-EGFR-TGF�RII: A bifunctional antibody that sequesters TGF-� via an extracellular domain sequence of TGF Receptor II fused to the C-terminus of the heavy chain of anti-EGFR antibody (Cetuximab). 2. Anti-CTLA4-TG�RII: A bifunctional antibody that sequesters TGF-� via an extracellular domain sequence of TGF-� Receptor II fused to the C-terminus of the heavy chain of anti-CTLA-4 antibody (Ipilimumab). Approximately 15-25% of patients with breast cancer have triple negative breast cancer (TNBC), an aggressive treatment-refractory subtype that lacks hormone receptors and HER2/neu. Although 80% of TNBC overexpress epidermal growth factor receptor (EGFR), they do not respond to cetuximab. Our studies demonstrate that TGF-� is a key determinant of the resistance of cancers to cetuximab. Since TNBC exhibit EMT and Treg infiltration, the tell-tale signs of TGF-� activity, they may be ideal targets for targeted bifunctional immunostimulatory antibodies that counteract TGF-�-mediated immune tolerance. The Specific Aims are: I. Evaluate the antitumor efficacy of anti-EGFR-TGF�RII in TNBC tumor-bearing mice: (A) Examine whether anti-EGFR-TGF�RII has superior antitumor efficacy compared to cetuximab, and determine the role of ADCC in its mechanism of action; (B) Examine whether anti-EGFR-TGF�RII can reverse immune tolerance and enhance T cell-mediated tumor regression in NOG mice reconstituted with human CD34+ cells. II. Evaluate the antitumor efficacy of anti-CTLA-4-TGF�RII in TNBC-bearing NOG mice reconstituted with human CD34+ cells: (A) Examine whether anti-CTLA-4-TGF�RII counteracts Tregs and activates antitumor immunity; (B) Compare the antitumor efficacy of anti-CTLA-4-TGF�RII with that of Ipilimumab or anti-TGF-�. IMPACT: The project seeks to advance the treatment of TNBC and other common cancers by counteracting tumor-induced immune tolerance via novel targeted bifunctional immunostimulatory antibodies.