LXR[1]
Cancer immunotherapy is restricted to immune resistance caused by immunosuppressive tumor microenvironment. Pyroptosis involved in antitumor immunotherapy as a new schedule is prospective to reverse immunosuppression. Herein, acidic tumor microenvironment (TME)-evoked MRC nanoparticles (MRC NPs) co-delivering immune agonist RGX-104 and photosensitizer chlorine e6 (Ce6) are reported for pyroptosis-mediated immunotherapy. RGX-104 remodels TME by transcriptional activation of ApoE to regress myeloid-derived suppressor cells' (MDSCs) activity, which neatly creates foreshadowing for intensifying pyroptosis. Considering Ce6-triggered photodynamic therapy (PDT) can strengthen oxidative stress and organelles destruction to increase immunogenicity, immunomodulatory-photodynamic MRC nanodrugs will implement an aforementioned two-pronged strategy to enhance gasdermin E (GSDME)-dependent pyroptosis. RNA-seq analysis of MRC at the cellular level is introduced to first elucidate the intimate relationship between RGX-104 acting on LXR/ApoE axis and pyroptosis, where RGX-104 provides the prerequisite for pyroptosis participating in antitumor therapy. Briefly, MRC with favorable biocompatibility tackles the obstacle of hydrophobic drugs delivery, and becomes a powerful pyroptosis inducer to reinforce immune efficacy. MRC-elicited pyroptosis in combination with anti-PD-1 blockade therapy boosts immune response in solid tumors, successfully arresting invasive metastasis and extending survival based on remarkable antitumor immunity. MRC may initiate a new window for immuno-photo pyroptosis stimulators augmenting pyroptosis-based immunotherapy.[2] Adv Healthc Mater . 2022 Nov;11(21):e2201233.

肿瘤免疫治疗仅限于免疫抑制肿瘤微环境引起的免疫抵抗。焦亡参与抗肿瘤免疫治疗作为一种新的方案，有望逆转免疫抑制。本文报道了酸性肿瘤微环境(TME)诱发的MRC纳米颗粒(MRC NPs)共同递送免疫激动剂RGX-104和光敏剂氯e6 (Ce6)用于焦热介导的免疫治疗。RGX-104通过转录激活ApoE来重塑TME，从而降低髓源性抑制细胞(MDSCs)的活性，这为强化焦亡埋下了伏笔。考虑到ce6触发的光动力疗法(PDT)可以增强氧化应激和细胞器破坏以增加免疫原性，免疫调节光动力MRC纳米药物将实施上述双管齐下的策略来增强气凝胶蛋白E (GSDME)依赖性焦亡。引入细胞水平MRC的RNA-seq分析，首次阐明作用于LXR/ApoE轴的RGX-104与焦亡之间的密切关系，其中RGX-104为焦亡参与抗肿瘤治疗提供了前提条件。简而言之，MRC具有良好的生物相容性，解决了疏水药物递送的障碍，成为一种强大的焦亡诱导剂，增强免疫功效。mrc诱导的焦亡联合抗pd -1阻断治疗可增强实体瘤的免疫应答，成功阻止侵袭性转移，并基于显著的抗肿瘤免疫延长生存期。MRC可能为免疫光热亡刺激剂增加基于热亡的免疫治疗开启一个新的窗口。[2]Adv Healthc Mater . 2022 Nov;11(21):e2201233.

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在Transwell MDSC分化实验中，将骨髓细胞与B16F10黑色素瘤细胞和GM-CSF共培养6天，在第3天添加 RGX-104（2 µM）可显著减少第6天时Gr-1^highCD11b⁺细胞（粒细胞样MDSC）的数量，效果与不添加癌细胞组相当。
在体外，用 RGX-104（1 µM 或 2 µM）处理从荷瘤小鼠分离的MDSC，可显著降低MDSC存活率并增加裂解caspase-3⁺ MDSC的比例，表明其诱导了细胞凋亡。

当给患有可见肿瘤的小鼠口服 RGX-104（每天 100 毫克/公斤）时，可以有效抑制多种癌症类型的生长。 RGX-104 和抗 PD-1 的组合被发现比单独使用任何一种药物更有效。值得注意的是，在抗 PD-1 治疗的基础上接受 RGX-104 治疗的小鼠具有良好的耐受性，并且没有表现出明显的损伤 [1]。

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口服 RGX-104（50 mg/kg/天、100 mg/kg/天 via 饲料，或80 mg/kg/天 via 腹腔注射）能显著抑制多种同源和异种移植小鼠模型的肿瘤生长，包括B16F10黑色素瘤、GL261胶质母细胞瘤、Lewis肺癌（LLC）、MC38结肠癌以及免疫缺陷小鼠体内的人SKOV3卵巢癌。
RGX-104 治疗降低了荷瘤小鼠肿瘤、脾脏和外周血中粒细胞样（G-MDSC）和单核细胞样（M-MDSC）髓源性抑制细胞的数量。
RGX-104 引起的MDSC减少与肿瘤浸润细胞毒性T淋巴细胞（CTL）的活化增强相关，表现为IFN-γ⁺Granzyme B⁺ CD8⁺ T细胞和PD-1⁺ CD8⁺ T细胞频率升高。
在使用gp100特异性Pmel CD8⁺ T细胞的过继性T细胞治疗模型中，联合使用 RGX-104 增强了抗肿瘤活性并提高了小鼠生存率。
在B16F10黑色素瘤和LLC模型中，RGX-104 与抗PD-1抗体联合治疗显示出优于任一单药的治疗效果，即使在Gvax诱导的MDSC积累背景下也是如此。
在一项涉及癌症患者的1期临床试验中，口服 RGX-104 治疗（28天周期中，每天一次，连续21天）显著降低了6名患者中5人的循环粒细胞样MDSC水平（中位减少86%）和单核细胞样MDSC水平。
患者接受 RGX-104 治疗后，循环CD8⁺ T细胞（尤其是PD-1⁺ 亚群）的活化增加，通过GITR表达进行测定。

MDSC体外增殖试验[1]
骨髓源性抑制细胞如前所述从荷瘤小鼠脾组织中分离。10万个细胞在聚l -赖氨酸包被板上四次镀。以1uM Abequolixron (RGX-104)或DMSO为载体处理3小时后，用4% PFA固定细胞15分钟，染色前用1X PBS洗涤3次。兔单克隆抗ki67抗体(1:400稀释)在4℃下过夜。细胞用Alexa Fluor 488二抗(1:200稀释，Invitrogen)在室温下孵育1小时，用DAPI反染(1:100稀释)，用延长金贴载。为了分析Ki67阳性细胞的百分比，使用蔡司Axio Imager荧光显微镜在20倍放大下对每个重复的5个视野进行成像。使用CellProfiler软件进行图像分析。

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MDSC黏附试验[1]
骨髓来源的抑制细胞从荷瘤小鼠脾组织中分离出来。10万个细胞分三次镀在聚l -赖氨酸包被板上。细胞用1uM Abequolixron (RGX-104)或DMSO作为载体处理2小时，以300 rpm振荡30分钟。之后，用4% PFA固定细胞15分钟，用1X PBS洗涤3次，用DAPI反染，用延长金贴壁。为了进行分析，使用蔡司Axio Imager荧光显微镜对每个重复的10个视场进行20倍放大成像。使用CellProfiler软件确定剩余细胞的数量。

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体外MDSC细胞凋亡测定[1]
从WT、LXRαβ−/−、ApoE−/−或LRP8−/−小鼠中分离小鼠脾脏，匀浆形成单细胞悬液。细胞用1X ACK裂解缓冲液裂解并清除红细胞。使用髓源性抑制细胞分离试剂盒从细胞悬浮液中分离MDSCs。将分离的MDSCs涂于载玻片上，按指定浓度和时间用Abequolixron (RGX-104)或小鼠重组ApoE处理。然后用Cleaved Caspase-3抗体对样品进行染色。

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将骨髓细胞与 B16F10 黑色素瘤细胞和 GM-CSF 一起培养 6 天。第 3 天，将 RGX-104 (2 μM) 添加到培养物中。第 6 天通过流式细胞术评估每 50 mL 培养液中 Gr-1high CD11b+ 细胞的平均数量[1]。

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体外MDSC凋亡实验：使用特异性分离试剂盒从荷瘤小鼠脾脏分离MDSC。将分离的MDSC铺板，并用指定浓度（如1 µM, 2 µM）和时间（如3小时, 6小时）的 RGX-104 或对照处理。然后固定细胞并用抗裂解caspase-3抗体染色，以量化凋亡细胞。
体外MDSC分化实验：从小鼠股骨分离骨髓细胞，培养于孔板底部。将B16F10黑色素瘤细胞置于Transwell插入物中。培养物中添加GM-CSF。第3天，加入 RGX-104（2 µM）或对照。第6天，收集基底室细胞，用CD11b和Gr-1染色，并通过流式细胞术分析以量化MDSC群体。
T细胞抑制实验：从初始小鼠分离CD8⁺ T细胞，用增殖染料标记，并与从对照或 RGX-104 处理的荷瘤小鼠分离的MDSC在CD3/CD28激活珠和IL-2存在下共培养。24小时后，通过流式细胞术评估T细胞活化（IFN-γ产生）和增殖（染料稀释）。
[1]

Animal/Disease Models: NOD SCID or RAG mice injected with 1×106 SKOV3 ovarian cancer cells[1].
Doses: 100 mg/kg.
Route of Administration: Oral administration daily for about 60 days.
Experimental Results: Robustly suppressed tumor growth and progression.

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Mice[1] B16F10 cancer cells are subcutaneously injected into C57BL/6 mice. Following tumor growth to 5-10 mm3 in volume, mice are fed either control chow, chow supplemented with GW3965 (100 mg/kg), or chow supplemented with RGX-104 (100 mg/kg)[1].

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RGX-104 was administered either through formulated drug chow at 100mg/kg/day or 50mg/kg/day or delivered via intraperitoneal injection (80mg/kg/day) in a vehicle suspension consisting of corn oil and ethanol (2.5% by volume) as indicated in each figure. Control cohorts were treated with either normal chow (Purina 5001) or with vehicle consisting of corn oil and ethanol (2.5% by volume), respectively. Tumor measurements were taken on the days indicted throughout the course of the experiment with calipers. For survival analysis, mice were euthanized when total tumor burden approached IACUC guidelines with a tumor burden exceeding 1,500 mm3 in volume. For the relevant experiments, anti-PD-1 mAb (clone RMP1-14) or a control isotype-matched antibody was administered at 10mg/kg intraperitoneally on days 3, 6, and 9 post-tumor injection. Gvax was generated as previously described and administered at high frequency (every 3 days) during the experiments as indicated.[1]

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For most tumor growth studies, cancer cells were suspended in PBS, mixed 1:1 with Matrigel, and injected subcutaneously into the flanks of 6-8 week old mice. Once tumors reached a specified volume (e.g., 5–10 mm³, 30–40 mm³), mice were randomized into treatment groups.
RGX-104 was administered orally via drug-formulated chow at doses of 50 mg/kg/day or 100 mg/kg/day. In some experiments, it was administered via intraperitoneal injection (80 mg/kg/day) in a vehicle consisting of corn oil with 2.5% ethanol by volume.
Control groups received normal chow or the corn oil/ethanol vehicle via intraperitoneal injection.
Tumor dimensions (length and width) were measured periodically with calipers, and volume was calculated.
For combination therapy with anti-PD-1, the antibody was administered intraperitoneally at 10 mg/kg on specified days (e.g., days 3, 6, and 9 post-tumor injection).
For adoptive T cell therapy experiments, CD8⁺ T cells were isolated from Pmel-1 TCR transgenic mice and transferred via retro-orbital injection into tumor-bearing recipients, which also received a gp100 peptide vaccine.

In the Phase 1 clinical trial, RGX-104 was reported to be well tolerated in the first six evaluable patients, with no dose-limiting toxicities observed.
Treatment was not associated with significant changes in key hematologic parameters (total white blood cell, absolute neutrophil, absolute lymphocyte, and absolute monocyte counts) within the first two weeks, with changes normalizing over the 4-week cycle.

[1]. LXR/ApoE Activation Restricts Innate Immune Suppression in Cancer. Cell. 2018 Feb 8;172(4):825-840.e18.

Abequolixron is an orally bioavailable agonist of the nuclear receptor liver X receptor beta (LXRbeta; NR1H2; LXR-b), with potential immunomodulating and antineoplastic activities. Upon oral administration, abequolixron selectively targets and binds to LXRbeta, thereby activating LXRbeta-mediated signaling, leading to the transcription of certain tumor suppressor genes and the downregulation of certain tumor promoter genes. This particularly activates the expression of apolipoprotein E (ApoE), a tumor suppressor protein, in tumor cells and certain immune cells. This activates the innate immune system, resulting in depletion of immunosuppressive myeloid-derived suppressor cells (MDSCs), tumor cells and endothelial cells in the tumor microenvironment. This reverses immune evasion, enhances anti-tumor immune responses and inhibits proliferation of tumor cells. LXRbeta, a member of the oxysterol receptor family, which is in the nuclear receptor family of transcription factors, plays a key role in cholesterol transport, glucose metabolism and the modulation of inflammatory responses; activation of LXRbeta suppresses tumor cell invasion, angiogenesis, tumor progression, and metastasis in a variety of tumor cell types. The expression of the ApoE protein becomes silenced in human cancers as they grow, become invasive, and metastasize; ApoE silencing is related to reduced survival in cancer patients. The LXR-ApoE pathway regulates the ability of cancers to evade the immune system and recruit blood vessels.

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RGX-104 is an investigational LXRβ agonist undergoing a multicenter Phase 1 a/b clinical trial (NCT02922764) in patients with advanced solid cancers or lymphomas.
Its anti-tumor and immunomodulatory effects are mediated through activation of the LXR/ApoE axis. LXR activation transcriptionally upregulates ApoE, which in turn promotes MDSC apoptosis via the LRP8 receptor, leading to reduced innate immune suppression and enhanced cytotoxic T cell activity.
The study presents RGX-104 as a first-in-class therapeutic strategy to target MDSCs and reverse immune evasion in cancer.

C34H33CLF3NO3

596.078939199448

595.21

C, 68.51; H, 5.58; Cl, 5.95; F, 9.56; N, 2.35; O, 8.05

610318-54-2

RGX-104 hydrochloride;610318-03-1

10218693

White to off-white solid powder

6.3

49.8Ų

1

7

13

42

783

1

C[C@H](CCOC1=CC=CC(=C1)CC(=O)O)N(CC2=C(C(=CC=C2)C(F)(F)F)Cl)CC(C3=CC=CC=C3)C4=CC=CC=C4

ZLJZDYOBXVOTSA-XMMPIXPASA-N

InChI=1S/C34H33ClF3NO3/c1-24(18-19-42-29-16-8-10-25(20-29)21-32(40)41)39(22-28-15-9-17-31(33(28)35)34(36,37)38)23-30(26-11-4-2-5-12-26)27-13-6-3-7-14-27/h2-17,20,24,30H,18-19,21-23H2,1H3,(H,40,41)/t24-/m1/s1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.17 mg/mL (3.64 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 21.7 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.08 mg/mL (3.49 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 0.83 mg/mL (1.39 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 8.3 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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