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

描述（由申请人提供）：I型干扰素（IFNa和IFN？）是有效的细胞生长调节剂，对许多人类癌症具有抑制作用。然而，它们的临床应用受到了限制，因为不能在肿瘤部位达到有效的IFN浓度而没有全身毒性。我们的目标是利用单克隆抗体的肿瘤靶向能力将IFN直接携带到癌症部位来克服这一限制。我们推测抗体-IFN融合蛋白可能是高效的癌症治疗剂，选择性地将IFN定位于肿瘤部位。我们最近成功地生产了靶向B细胞非霍奇金淋巴瘤表面表达的CD 20抗原的融合蛋白。含有鼠或人IFNa的抗CD 20-IFNa融合蛋白在体外和体内均表现出针对人CD 20+鼠淋巴瘤和人B细胞淋巴瘤的有效抗肿瘤作用。我们现在建议进一步表征和优化这些IFN融合蛋白，并将我们的研究扩展到包括与IFN？的融合，IFN？的效力比IFNa高10倍。为了实现这一目标，我们提出了以下具体目标：目标1：表征与鼠IFNa（mIFNa）或鼠IFN <$（mIFN <$）融合的抗CD 20在体外和体内对小鼠淋巴瘤的疗效，并确定其作用机制。A.表征融合蛋白对经工程改造以表达人CD 20的两种鼠B细胞淋巴瘤细胞系的体外活性，包括其抑制肿瘤细胞增殖、诱导细胞凋亡和介导抗体依赖性细胞毒性（ADCC）和补体依赖性细胞毒性（CDC）的能力。B。表征抗-CD 20-mIFNa和抗-CD 20-mIFN？对C3 H小鼠中38 C13-huCD 20和BALB/c huCD 20转基因小鼠中A20-huCD 20的体内效力。目标二：评价抗CD 20融合人IFN α（hIFN α）在体外和体内抗人淋巴瘤的疗效，并确定作用机制。A.使用代表各种组织学的人B细胞淋巴瘤细胞系，如目的1中所述，表征抗CD 20-hIFNa的体外活性。B。评价抗-CD 20-hIFNa对SCID小鼠中人B细胞淋巴瘤异种移植物的体内功效。C.体外测定抗CD 20-hIFNa对原发性人B细胞淋巴瘤标本的活性。目的3：构建抗CD 20与人IFN？的融合物，以尝试增加抗肿瘤效力。A.探索使用替代肽接头来优化融合蛋白的稳定性和IFN活性。B。表征最有前途的抗CD 20-hIFN？融合蛋白。目标4：探索使用淋巴瘤细胞存活途径的临床相关药理学抑制剂进一步增强抗CD 20-hIFNa/？疗效的可能性。A.当与mTOR（雷帕霉素或替西罗莫司）或PI 3激酶d（CAL-101）的抑制剂组合使用时，评价融合蛋白针对一组淋巴瘤细胞系和原代细胞的体外功效。B。评价融合蛋白与mTOR（替西罗莫司）或PI 3激酶d（CAL-120）抑制剂组合对人淋巴瘤异种移植物的体内功效。 公共卫生相关性：淋巴瘤仍然是一个主要的健康问题，在美国每年有超过66，000人死于这种疾病。在这项研究工作中，我们将进一步开发和表征新的治疗性蛋白质，即使在淋巴瘤对当前疗法有抗性的情况下，这些蛋白质也有很大的治疗淋巴瘤的希望。重要的是，我们正在开发的技术不仅限于治疗淋巴瘤，而且可以应用于治疗许多不同的癌症，包括黑色素瘤，前列腺癌，肾细胞癌和多发性骨髓瘤。

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.