Novel Tumor-Targeted, Engineered IL-2 Variant (IL2v)-Based Immunocytokines For Immunotherapy Of Cancer
Novel Tumor-Targeted, Engineered IL-2 Variant (IL2v)-Based Immunocytokines For Immunotherapy Of Cancer
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用于癌症免疫治疗的新型肿瘤靶向、工程化 IL-2 变体 (IL2v) 免疫细胞因子
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发表时间：
2013
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通讯作者：
P. Umaña
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作者：
Inja Waldhauer;V. Nicolini;C. Dunn;A. Freimoser;G. Danny;O. Boerman;T. Nayak;S. Herter;E. V. Puijenbroek;O. Ast;T. Hofer;R. Hosse;Sabine Lang;S. Neumann;H. Kettenberger;M. Neubauer;Ingo H Gorr;D. Tuerck;S. Evers;C. Gerdes;V. Levitsky;M. Bacac;E. Moessner;P. Umaña
介绍IL-2治疗可以导致癌症患者的持久反应，但与显著的毒性相关。已知的基于IL-2的免疫细胞因子由于设计上的各种限制而没有进入关键的临床试验；特别是两个野生型IL-2部分与抗体的融合并保留了基于免疫细胞因子的FcgR结合。这种设计的结果是：1)高亲和力与免疫细胞上IL-2raβγ的Pm亲和力结合，损害肿瘤靶向，诱导快速全身清除和较短的半衰期；2)高亲和力表达于肺血管内皮细胞，有助于肺毒性；3)Treg优先激活免疫效应器。在这里，我们描述了一类新的单体肿瘤靶向免疫细胞因子，其中单个已取消CD25结合的工程化IL-2变异体(IL2v)与带有异源二聚体Fc-部分的抗体的C末端融合。FcγR和C1q结合被一种新的Fc突变完全取消。肿瘤靶向性选用人抗CEA(GA504，CEA-IL2v)或FAP(GA501，FAP-IL2v)高亲和力抗体。CEA-IL2v识别人癌胚抗原(CEA)的膜近端表位，并优先与膜结合的CEA结合，而不是脱落CEA。
方法以重组蛋白CEA和FAP-IL2v作为重组蛋白，通过检测P-STAT5的激活、细胞增殖、对Fas诱导的细胞凋亡敏感性、活化标志物的表达和细胞因子的释放来检测它们在效应细胞上的活性。将曲妥珠单抗与西妥昔单抗联合应用于SCID和完全免疫C57BL/6小鼠，分析其安全性、药代动力学(PK)、药效学和抗肿瘤效果。采用SPECT显像技术，在Balb/c小鼠原位同基因肾癌模型中进行了肿瘤靶向性研究。
结果FAP和CEA-IL2v完全不与CD2 5结合，但保留了IL-Rβγ结合，并与相应的抗原、成纤维细胞和肿瘤细胞表现出PM结合亲和力。由于取消了与CD25的结合，这些分子不会优先激活Treg。与野生型IL-2免疫细胞因子相比，IL2v处理效应细胞降低了它们对Fas介导的凋亡(也称为激活诱导的细胞死亡)的敏感性。IL-2RFAP-βγ和CEA-IL2v对NK、CD_4~+和CD_8~+T细胞的激活标志、细胞增殖和细胞因子释放均有一定的激活作用。此外，CEA-IL2v和FAP-IL2v与ADCC活性抗体联合应用可增强NK细胞的细胞毒活性。对完全免疫活性小鼠的作用机制研究表明，这些分子强烈地扩张和激活NK、CD8+T细胞和gdT细胞(高达100倍)，并使外周血、淋巴组织和肿瘤中的CD8+T细胞强烈偏向CD8+T细胞。在C57BL/6小鼠中，CEA-和FAP-IL2v尽管暴露和循环半衰期比类似的基于IL-2的免疫细胞因子更高，但仍表现出更好的安全性。放射性标记FAP-IL2v的MicroSPECT/CT显像显示，在同种异体原位RencA模型中，FAP介导的肿瘤靶向性好，正常组织摄取低，淋巴组织蓄积低，而类似的基于IL-2的免疫细胞因子显示优先靶向淋巴组织。在荷瘤小鼠的研究表明，FAP-IL2v和CEA-IL2v在同基因模型中的抗肿瘤效果呈剂量依赖关系。在转基因人CD16A的SCID小鼠异种移植模型中的其他研究表明，CEA-IL2v显著增强了ADCC竞争性抗体(包括曲妥珠单抗和西妥昔单抗)介导的抗肿瘤效果和/或存活率。
结论与经典的IL-2免疫细胞因子相比，CEA-IL2v和FAP-IL2v具有更好的安全性、PK和肿瘤靶向性，但由于取消了CD25结合、单价和高亲和力的肿瘤靶向性，缺乏对Tregs的优先诱导。它们通过IL-2Rβγ在外周和肿瘤微环境中保持激活和扩大NK和CD8+效应T细胞的能力。这些数据支持了他们对癌症免疫治疗的进一步非临床和临床研究。CEA-IL2v的临床试验预计将于2014年进行。
披露：克莱恩：罗氏Glycart AG：就业。沃尔德豪尔：罗氏：就业。尼科里尼：罗氏：就业。邓恩：罗氏：就业。Freimoser-Grundschber：罗氏：就业。丹尼：罗氏：研究基金。布尔曼：罗氏：研究基金。纳亚克：罗氏：就业。赫特：罗氏：就业。范·普金布鲁克：罗氏：就业。AST：罗氏：就业。霍弗：罗氏：就业。霍斯：罗氏：就业。朗：罗氏：就业。诺伊曼：罗氏：就业。凯滕伯格：罗氏：就业。诺伊鲍尔：罗氏：就业。戈尔：罗氏：就业。图尔克：罗氏：就业。埃弗斯：罗氏：就业。格德斯：罗氏：就业。莱维茨基：罗氏：就业。巴卡奇：罗氏：就业。莫斯纳：罗氏：就业。乌玛纳：罗氏：就业，股权所有权。
Introduction IL-2 therapy can lead to durable responses in cancer patients, but is associated with significant toxicity. None of the known IL-2-based immunocytokines has yet progressed to pivotal clinical trials due to various constraints in their design; in particular, the fusion of two wild-type IL-2 moieties to the antibody and retained FcgR binding of IgG-based immunocytokines. This design results in 1) high affinity binding with pM affinity to IL-2Raβγ on immune cells compromising tumor targeting and inducing rapid systemic clearance and short half-life; 2) high affinity for CD25 (IL-2Ra) expressed on pulmonary vascular endothelium contributing to pulmonary toxicity; and 3) preferential activation of Tregs over immune effectors. Here we describe a novel class of monomeric tumor-targeted immunocytokines where a single, engineered IL-2 variant (IL2v) with abolished CD25 binding is fused to the C-terminus of an antibody with a heterodimeric Fc-part. FcγR and C1q binding is completely abolished by a novel Fc mutation. For tumor targeting, human(-ized) high affinity antibodies against CEA (GA504, CEA-IL2v) or FAP (GA501, FAP-IL2v) were selected. CEA-IL2v recognizes a membrane proximal epitope of human carcinoembryonic antigen (CEA) and binds preferentially to membrane-bound CEA, but not shed CEA.
Methods CEA- and FAP-IL2v were produced as recombinant proteins and their activity tested oneffector cells by assessing the activation of P-STAT5, cell proliferation, sensitivity to Fas-induced apoptosis, expression of activation markers and cytokine release upon treatment. Safety, pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor efficacy were analyzed in SCID and fully immunocompetent C57Bl/6 mice as single agent and in combination with trastuzumab and cetuximab. Tumor targeting was investigated in the orthotopic syngeneic Renca renal cell cancer tumor model in Balb/c mice by SPECT imaging.
Results FAP- and CEA-IL2v completely lack binding to CD25, but retain IL-Rβγ binding, and show pM binding affinity to respective antigens, FAP on fibroblasts and CEA on tumor cells. As consequence of abolished binding to CD25 these molecules do not preferentially activate Tregs. The treatment of effector cells with IL2v reduces their sensitivity for Fas-mediated apoptosis (also known as activation induced cell death) as compared to wild-type IL-2 based immunocytokine. IL-2Rβγ bioactivity was retained and FAP- and CEA-IL2v activate NK, CD4+ and CD8+ T cells as shown by induction of activation markers, cell proliferation and cytokine release. Furthermore, CEA-IL2v and FAP-IL2v enhanced the cytotoxic activity of NK cells when combined with ADCC-competent antibodies. Mechanism of action studies in fully immunocompetent mice showed that the molecules strongly expand and activate NK, CD8+ T cells and gd T cells (up to 100-fold) and skew the CD4:CD8 ratio strongly towards CD8+ T cells in the peripheral blood, lymphoid tissues, and in the tumor. In C57Bl/6 mice, CEA- and FAP-IL2v demonstrate improved safety despite a higher exposure and circulatory half-life than the analogous IL-2 based immunocytokine. MicroSPECT/CT imaging with radioactively labeled FAP-IL2v revealed good FAP-mediated tumor targeting in the orthotopic syngeneic Renca model with low normal tissue uptake and low accumulation in lymphoid tissues, contrary to analogous IL-2 based immunocytokine that showed preferential targeting to lymphoid tissue. Studies in tumor-bearing mice showed dose-dependent anti-tumor efficacy of FAP-IL2v and CEA-IL2v in syngeneic models. Additional studies in xenograft models in SCID mice transgenic for human CD16A showed that CEA-IL2v strongly enhances the antitumor efficacy and/or survival mediated by ADCC-competentantibodies, including trastuzumab and cetuximab.
Conclusion CEA- and FAP-IL2v demonstrate superior safety, PK and tumor targeting, while lacking preferential induction of Tregs due to abolished CD25 binding, monovalency and high-affinity tumor-targeting as compared to classical IL-2-based immunocytokines. They retain capacity to activate and expand NK and CD8+ effector T cells through IL-2Rβγ in the periphery and the tumor microenvironment. These data support their further nonclinical and clinical investigation for immunotherapy of cancer. Clinical trials with CEA-IL2v are foreseen in 2014.
Disclosures: Klein: Roche Glycart AG: Employment. Waldhauer: Roche: Employment. Nicolini: Roche: Employment. Dunn: Roche: Employment. Freimoser-Grundschober: Roche: Employment. Danny: Roche: Research Funding. Boerman: Roche: Research Funding. Nayak: Roche: Employment. Herter: Roche: Employment. Van Puijenbroek: Roche: Employment. Ast: Roche: Employment. Hofer: Roche: Employment. Hosse: Roche: Employment. Lang: Roche: Employment. Neumann: Roche: Employment. Kettenberger: Roche: Employment. Neubauer: Roche: Employment. Gorr: Roche: Employment. Tuerck: Roche: Employment. Evers: Roche: Employment. Gerdes: Roche: Employment. Levitsky: Roche: Employment. Bacac: Roche: Employment. Moessner: Roche: Employment. Umana: Roche: Employment, Equity Ownership.