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¿）是细胞生长的有效调节剂，对许多人类癌症具有抑制作用。然而，它们的临床应用受到限制，因为无法在肿瘤部位达到有效浓度的IFN而不产生全身毒性。我们的目标是利用单克隆抗体的肿瘤靶向能力来克服这一限制，将ifn直接携带到癌症部位。我们假设抗体-IFN融合蛋白可能是非常有效的癌症治疗剂，选择性地将IFN定位到肿瘤部位。我们最近成功地生产了靶向表达在B细胞非霍奇金淋巴瘤表面的CD20抗原的融合蛋白。含有小鼠或人IFNa的抗CD20-IFNa融合蛋白在体外和体内均显示出对人CD20+小鼠淋巴瘤和人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，并扩展我们的研究，包括与IFN融合，其效力比IFNa高10倍。为了实现这一目标，我们提出了以下具体目标：目的1：表征抗cd20与小鼠IFNa （mIFNa）或小鼠IFN¿（mIFN¿）融合在体外和体内对抗小鼠淋巴瘤的功效，并确定其作用机制。A.描述融合蛋白对两种表达人CD20的小鼠B细胞淋巴瘤细胞系的体外活性，包括它们抑制肿瘤细胞增殖、诱导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cd20 - mifna和抗cd20 - mifn¿对C3H小鼠38C13-huCD20和BALB/c hucd20转基因小鼠A20-huCD20的体内作用。目的2：在体外和体内评价抗cd20融合人IFNa （hIFNa）治疗人淋巴瘤的疗效，并确定其作用机制。A.使用代表不同组织学的人B细胞淋巴瘤细胞系，表征抗cd20 - hifna的体外活性，如Aim 1所示。B.评估抗cd20 - hifna在SCID小鼠体内对人B细胞淋巴瘤异种移植物的治疗效果。C.体外测定抗cd20 - hifna对人原代B细胞淋巴瘤标本的活性。目的3：构建抗cd20融合物与人IFN¿，试图提高抗肿瘤效力。A.探索使用替代肽连接物来优化融合蛋白的稳定性和IFN活性。B.描述最有希望的抗cd20 - hifn融合蛋白。目的4：探索利用淋巴瘤细胞存活途径的临床相关药物抑制剂进一步增强抗cd20 - hifna /¿疗效的可能性。当与mTOR抑制剂（雷帕霉素或替西莫司）或pi3k激酶d （CAL-101）联合使用时，评估融合蛋白对一组淋巴瘤细胞系和原代细胞的体外疗效。B.评估融合蛋白与mTOR（替西莫司）或PI3激酶d （CAL-120）抑制剂联合抗人淋巴瘤异种移植物的体内疗效。

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.