Antibody-interferon fusion proteins for treatment of B-cell malignancies

描述(申请人提供)：I型干扰素(IFNA和干扰素)是细胞生长的有效调节剂，对许多人类癌症有抑制作用。然而，它们的临床应用一直受到限制，因为无法在没有全身毒性的情况下在肿瘤部位达到有效的干扰素浓度。我们的目标是克服这一限制，利用单抗的肿瘤靶向能力将IFN直接携带到癌症部位。我们推测，抗体-干扰素融合蛋白可能是一种高效的肿瘤治疗药物，选择性地将干扰素定位于肿瘤部位。我们最近成功地生产了针对B细胞非霍奇金淋巴瘤表面表达的CD20抗原的融合蛋白。含有鼠或人IFNA的抗CD20-IFNA融合蛋白在体内外对人CD20小鼠淋巴瘤和人B细胞淋巴瘤均显示出较强的抗肿瘤作用。我们现在建议进一步鉴定和优化这些干扰素融合蛋白，并扩大我们的研究范围，以包括与干扰素的融合，干扰素的效力是IFNA的10倍。为实现这一目标，我们提出了以下具体目标：目的1：鉴定抗CD20抗体与小鼠干扰素(MIFNa)或小鼠干扰素(MIFN)在体内外对小鼠淋巴瘤的抑制作用，并确定其作用机制。A.鉴定融合蛋白对两种表达人CD20的小鼠B细胞淋巴瘤细胞株的体外活性，包括它们抑制肿瘤细胞增殖、诱导细胞凋亡、介导抗体依赖的细胞毒(ADCC)和补体依赖的细胞毒(CDC)的能力。B.鉴定抗CD20-mIFNa和抗CD20-m干扰素对C3H小鼠38C13-huCD20和BALB/c huCD20转基因小鼠的体内效应。目的：评价抗CD20抗体与人干扰素(HIFNa)融合后在体内外对人淋巴瘤的治疗效果，并探讨其作用机制(S)。A.使用代表不同组织结构的人B细胞淋巴瘤细胞系，鉴定抗CD20-hIFNa的体外活性。B.评价抗CD20-hIFNa对SCID小鼠人B细胞淋巴瘤移植瘤的体内疗效。C.检测抗CD20-hIFNa对原代人B细胞淋巴瘤标本的体外杀伤活性。目的3：构建抗CD20抗体与人干扰素融合，以提高其抗肿瘤活性。A.探索使用替代的多肽连接物来优化融合蛋白的稳定性和干扰素活性。B.鉴定最有希望的抗CD20-h干扰素融合蛋白。目的：探讨利用淋巴瘤细胞生存通路的临床相关药物抑制物进一步增强抗CD20-hIFNa/？疗效的可能性。A.评估融合蛋白与mTOR(雷帕霉素或坦西罗莫斯)或PI3激酶d(CAL-101)抑制剂联合使用时对一组淋巴瘤细胞株和原代细胞的体外疗效。B.评估融合蛋白与mTOR(替西罗莫司)或PI3激酶d(CAL-120)抑制剂联合使用对人淋巴瘤异种移植瘤的体内疗效。 公共卫生相关性：淋巴瘤仍然是一个主要的健康问题，美国每年有超过6.6万人死于这种疾病。在这项研究中，我们将进一步开发和表征新的治疗蛋白，即使在淋巴瘤对目前的治疗方法耐药的情况下，这些蛋白也显示出巨大的治疗前景。重要的是，我们正在开发的技术不仅限于淋巴瘤的治疗，还可以应用于许多不同癌症的治疗，包括黑色素瘤、前列腺癌、肾癌和多发性骨髓瘤。

DESCRIPTION (provided by applicant): The type I interferons (IFNa and IFN¿) are potent regulators of cell growth with inhibitory effects against many human cancers. However, their clinical use has been limited by inability to achieve effective concentrations of IFN at sites of tumor without systemic toxicity. Our goal is to overcome this limitation using the tumor-targeting ability of monoclonal antibodies to carry IFNs directly to cancer sites. We hypothesize that antibody-IFN fusion proteins could be highly effective cancer therapeutic agents, selectively localizing IFN to sites of tumor. We have recently succeeded in producing fusion proteins targeting the CD20 antigen expressed on the surface of B cell non-Hodgkin lymphomas. Anti-CD20-IFNa fusion proteins containing murine or human IFNa exhibited potent anti-tumor effects against a human CD20+ murine lymphoma and human B cell lymphoma both in vitro and in vivo. We now propose to further characterize and optimize these IFN fusion proteins and to extend our studies to include fusion with IFN¿, which is up to 10-fold more potent than IFNa. To achieve this, we propose the following Specific Aims: Aim 1: Characterize the efficacy of anti-CD20 fused with murine IFNa (mIFNa) or murine IFN¿ (mIFN¿) against mouse lymphomas in vitro and in vivo and determine their mechanisms of action. A. Characterize the in vitro activity of fusion proteins against two murine B cell lymphoma cell lines engineered to express human CD20, including their ability to inhibit tumor cell proliferation, induce apoptosis, and mediate antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cellular cytotoxicity (CDC). B. Characterize the in vivo efficacy of anti-CD20-mIFNa and anti-CD20-mIFN¿ against 38C13-huCD20 in C3H mice and A20-huCD20 in BALB/c huCD20-transgenic mice. Aim 2: Evaluate the efficacy of anti-CD20 fused with human IFNa (hIFNa) against human lymphomas in vitro and in vivo and determine the mechanism(s) of action. A. Using human B cell lymphoma cell lines representing various histologies, characterize the in vitro activity of anti-CD20-hIFNa as in Aim 1. B. Evaluate the in vivo efficacy of anti-CD20-hIFNa against human B cell lymphoma xenografts in SCID mice. C. Measure the activity of anti-CD20-hIFNa against primary human B cell lymphoma specimens in vitro. Aim 3: Construct anti-CD20 fusions with human IFN¿ in an attempt to increase anti-tumor potency. A. Explore the use of alternative peptide linkers to optimize the stability and IFN activity of the fusion protein. B. Characterize the most promising anti-CD20-hIFN¿ fusion proteins. Aim 4: Explore the possibility of further enhancing anti-CD20-hIFNa/¿ efficacy using clinically-relevant pharmacologic inhibitors of lymphoma cell survival pathways. A. Evaluate the in vitro efficacy of the fusion proteins against a panel of lymphoma cell lines and primary cells when used in combination with inhibitors of mTOR (rapamycin or temsirolimus) or PI3 kinase d (CAL-101). B. Evaluate the in vivo efficacy of the fusion proteins against human lymphoma xenografts in combination with inhibitors of mTOR (temsirolimus) or PI3 kinase d (CAL-120). PUBLIC HEALTH RELEVANCE: Lymphoma remains a major health problem with over 66,000 individuals in the United States succumbing each year from the disease. In this research effort we will further develop and characterize novel therapeutic proteins that show great promise for the treatment of lymphoma even in cases where the lymphoma is resistant to current therapies. Importantly the technology that we are developing is not limited to the treatment of lymphoma, but can be applied to the treatment of many different cancers including melanoma, prostate, renal cell carcinoma and multiple myeloma.