用高亲和力免疫调节化合物 CC-220 靶向 Cereblon：一种治疗 B 细胞恶液质的新型治疗剂,Blood

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用高亲和力免疫调节化合物 CC-220 靶向 Cereblon：一种治疗 B 细胞恶液质的新型治疗剂
Blood ( IF 21.0 ) Pub Date : 2012-11-16 , DOI: 10.1182/blood.v120.21.1055.1055
Peter H Schafer ₁ , Emily Rychak ₂ , Derek Mendy ₂ , Anastasia Parton ₂ , Lori Capone ₁ , Antonia Lopez-Girona ₂ , Jilly F Evans ₃ , Tom O Daniel ₂ , Rajesh Chopra ₂

Affiliation

摘要 1055 背景：Cereblon (CRBN) 是 E3 泛素连接酶复合物的一个组成部分，包括 CUL4A、DDB1 和 ROC-1，被发现是沙利度胺 (Thalomid®)、来那度胺 (Revlimid®) 和泊马度胺。CC-220 是一种新型免疫调节化合物，其效力增强，目前正在开发用于治疗免疫疾病。分析了 CC-220 对 CRBN 结合、泛素化和细胞增殖的影响。方法：在竞争性测定中使用沙利度胺类似物缀合的珠子进行与 CRBN 的结合研究。来自人类 U266 多发性骨髓瘤 (MM) 细胞的内源性 CRBN 通过将细胞提取物与不同浓度的 CC-220 或泊马度胺作为阳性对照一起孵育来测量。将与沙利度胺酸类似物偶联的亲和珠与 U266 提取物一起孵育，并在对珠进行充分洗涤后，洗脱结合的蛋白质。通过定量 CRBN 免疫印迹测定确定 CRBN 与沙利度胺偶联的亲和珠的结合。在 HEK293T 细胞中测量 CRBN 泛素化，这些细胞用氨基末端 His-生物素标记的 CRBN 构建体转染，然后与化合物预孵育 1 小时，然后用 MG132 蛋白酶体抑制剂处理（以阻止泛素化蛋白质的降解）。裂解细胞并处理以通过 SDS-PAGE 和使用抗泛素抗体的免疫印迹分析测量 CRBN 泛素化。在来那度胺敏感性和难治性多发性骨髓瘤细胞中进行细胞增殖研究。来那度胺耐药或敏感的 H929 MM 细胞系用 CC-220 处理 5 天，然后通过 7-氨基放线菌素 D（7-AAD）染色评估细胞增殖和活力。使用固定化抗 CD3 抗体在细胞培养物中刺激 2 天，在纯化的原代人 T 细胞中测量 T 细胞共刺激，并通过 ELISA 测量细胞因子分泌。通过在 B 细胞分化因子重组人 IL-2 (20 U/mL)、IL-10 (50 ng/mL) 存在下培养，测量正常供体外周血单核细胞的免疫球蛋白 M 和 G（IgG 和 IgM）产生)、IL-15 (10 ng/mL)、His 标记的 CD40 配体 (50 ng/mL)、多组氨酸小鼠 IgG1 抗体 (5 μg/mL) 和 ODN 2006-人类 TLR9 配体 (10 μg/mL) 4天，然后是 IL-2、IL-10、IL-15 和 IL-6 (50 ng/mL)，再持续 3 天。IgM和IgG通过ELISA测量。结果：在竞争性 CRBN 结合研究中，使用 3 μM 浓度的泊马度胺预培养导致与亲和珠结合的 CRBN 减少约 50%，而浓度为 0.1 μM 的 CC-220 导致类似的 CRBN 结合。转染的 HEK293T 细胞中的 CRBN 泛素化研究产生以下效力：CC-220 IC50 = 0.19 μM；来那度胺 IC50 = 12.9 μM；和泊马度胺 IC50 = 21.6 μM。CC-220 抑制增殖的 IC50 值从亲本 H929 细胞系中的 0.01 μM 和 DMSO 处理的亚克隆中的 0.04 μM 转变为来那度胺抗性亚克隆中的 0.51–1.58 μM。用 CC-220 处理 H929 细胞 24 小时后，细胞周期（S 期）明显减少 50%。在 48 小时，CC-220 降低存活蛋白和成视网膜细胞瘤蛋白 (pRB) 的表达，并增加细胞周期蛋白依赖性激酶抑制剂 p27 的表达。CC-220 共刺激 T 细胞产生 IL-2，EC50 约为 0.29 nM，而泊马度胺为 10 nM。CC-220 抑制 IgM 和 IgG 的产生，IC50 分别为 0.35 和 2.1 nM，而泊马度胺的 IC50 为 17 nM 和 63 nM。结论：结果表明，CC-220 与 CRBN 的结合亲和力比泊马度胺高约 30 倍，并在该系统中以比泊马度胺高约 110 倍的效力抑制 CRBN 泛素化。CC-220 在共刺激 T 细胞产生 IL-2 方面比泊马度胺强约 34 倍，在抑制免疫球蛋白生成方面比泊马度胺强 30 至 48 倍。总之，CC-220 是一种新型高亲和力 CRBN 配体，其细胞效力比泊马度胺高 1 或 2 个数量级，目前正在开发用于治疗免疫疾病，包括涉及 B 细胞恶液质的疾病。披露：Schafer：Celgene：就业、股权。Rychak：Celgene：就业、股权。Mendy：Celgene 公司：就业、股权。Parton：Celgene Corp：就业，股权。Capone：Celgene 公司：就业、股权。Lopez-Girona：Celgene 公司：就业、股权。Daniel：Celgene 公司：就业。Chopra：Celgene 公司：就业、股权。包括那些涉及 B 细胞恶液质的疾病。披露：Schafer：Celgene：就业、股权。Rychak：Celgene：就业、股权。Mendy：Celgene 公司：就业、股权。Parton：Celgene Corp：就业，股权。Capone：Celgene 公司：就业、股权。Lopez-Girona：Celgene 公司：就业、股权。Daniel：Celgene 公司：就业。Chopra：Celgene 公司：就业、股权。包括那些涉及 B 细胞恶液质的疾病。披露：Schafer：Celgene：就业、股权。Rychak：Celgene：就业、股权。Mendy：Celgene 公司：就业、股权。Parton：Celgene Corp：就业，股权。Capone：Celgene 公司：就业、股权。Lopez-Girona：Celgene 公司：就业、股权。Daniel：Celgene 公司：就业。Chopra：Celgene 公司：就业、股权。

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Targeting Cereblon with the High Affinity Immunomodulatory Compound CC-220: A Novel Therapeutic Agent for B Cell Dyscrasias

Abstract 1055 Background: Cereblon (CRBN) is a component of the E3 ubiquitin ligase complex including CUL4A, DDB1, and ROC-1, and was found to be the molecular binding target of thalidomide (Thalomid®), lenalidomide (Revlimid®), and pomalidomide. CC-220 is a novel immunomodulatory compound developed with increased potency and is currently in development for the treatment of immune conditions. The effect of CC-220 on CRBN binding, ubiquitination, and cell proliferation was profiled. Methods: Binding studies to CRBN were conducted using thalidomide analog-conjugated beads in a competitive assay. Endogenous CRBN from human U266 multiple myeloma (MM) cells was measured by incubating cell extracts with varying concentrations of either CC-220 or pomalidomide as a positive control. Affinity beads coupled to a thalidomide acid analog were incubated with the U266 extracts and, after extensive washing of the beads, the bound proteins were eluted. CRBN binding to the thalidomide-coupled affinity beads was determined by quantitative CRBN immunoblot determination. CRBN ubiquitination was measured in HEK293T cells, which were transfected with an amino-terminal His-biotin-tagged CRBN construct, then preincubated with compounds for one hour followed by treatment with the MG132 proteasome inhibitor (to arrest degradation of ubiquitinated proteins). Cells were lysed and processed to measure CRBN ubiquitination by SDS-PAGE and immunoblot analysis using an anti-ubiquitin antibody. Cell proliferation studies were conducted in lenalidomide-sensitive and -refractory multiple myeloma cells. Lenalidomide-resistant or -sensitive H929 MM cell lines were treated with CC-220 for 5 days, and then cell proliferation and viability were assessed by 7-aminoactinomycin D (7-AAD) staining. T-cell costimulation was measured in purified primary human T cells stimulated using immobilized anti-CD3 antibody in cell culture for 2 days, and cytokine secretion was measured by ELISA. Immunoglobulin M and G (IgG and IgM) production was measured from normal donor peripheral blood mononuclear cells by culturing in the presence of the B cell differentiation factors recombinant human IL-2 (20 U/mL), IL-10 (50 ng/mL), IL-15 (10 ng/mL), His-tagged CD40 Ligand (50 ng/mL), polyHistidine mouse IgG1 antibody (5 μg/mL), and ODN 2006-Human TLR9 ligand (10 μg/mL) for 4 days, followed by IL-2, IL-10, IL-15, and IL-6 (50 ng/mL) for an additional 3 days. IgM and IgG were measured by ELISA. Results: In the competitive CRBN binding studies, preincubation with pomalidomide at a concentration of 3 μM resulted in approximately 50% less CRBN bound to the affinity beads, while CC-220 at a concentration of 0.1 μM resulted in similar CRBN binding. CRBN ubiquitination studies in the transfected HEK293T cells resulted in the following potencies: CC-220 IC50 = 0.19 μM; lenalidomide IC50 = 12.9 μM; and pomalidomide IC50 = 21.6 μM. The IC50 value for inhibition of proliferation by CC-220 shifted from 0.01 μM in the parental H929 cell line and 0.04 μM in the DMSO-treated subclone to 0.51–1.58 μM in the lenalidomide-resistant subclones. A 50% decrease in cell cycle (S-phase) was evident after 24 hours of treatment of H929 cells with CC-220. At 48 hours, CC-220 decreased expression of survivin and retinoblastoma protein (pRB) and increased expression of the cyclin-dependent kinase inhibitor p27. CC-220 costimulated IL-2 production by T cells with an EC50 of approximately 0.29 nM, compared with 10 nM for pomalidomide. CC-220 inhibited IgM and IgG production with an IC50 of 0.35 and 2.1 nM, respectively, compared to 17 nM and 63 nM for pomalidomide. Conclusions: The results indicate that CC-220 binds to CRBN with approximately 30-fold higher affinity than pomalidomide, and inhibits CRBN ubiquitination with approximately 110-fold greater potency than pomalidomide in this system. CC-220 is approximately 34-fold more potent than pomalidomide for costimulating IL-2 production by T cells, and is 30- to 48-fold more potent than pomalidomide for inhibiting immunoglobulin production. In summary, CC-220 is a novel high affinity CRBN ligand with cellular potencies 1 or 2 orders of magnitude greater than that of pomalidomide, and is currently in development for the treatment of immune conditions, including those involving B cell dyscrasias. Disclosures: Schafer:Celgene: Employment, Equity Ownership. Rychak:Celgene: Employment, Equity Ownership. Mendy:Celgene Corp.: Employment, Equity Ownership. Parton:Celgene Corp: Employment, Equity Ownership. Capone:Celgene Corp: Employment, Equity Ownership. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Daniel:Celgene Corporation: Employment. Chopra:Celgene Corp: Employment, Equity Ownership.

更新日期：2012-11-16

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