SMCC-DM1

Maytansinoids; Tubulin

SMCC-DM1 抑制 HCC1954 和 MDA-MB-468 细胞增殖，IC50 值分别为 17.2 和 49.9 nM[1]。

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抗体-药物偶联物是一种靶向抗癌药物，由细胞毒性药物共价连接到单克隆抗体上，具有肿瘤抗原特异性活性。一旦结合到靶细胞表面抗原上，就必须对缀合物进行加工，以释放一种活性形式的药物，这种药物可以到达细胞内的靶标。在这里，研究人员使用生物学和生物化学方法来更好地定义抗体-美坦素缀合物的这一过程。特别是，研究人员检测了含有二硫连接体(huC242-SPDB-DM4)或硫醚连接体(huC242-SMCC-DM1)的huC242-maytansinoid偶联物在细胞中的代谢命运。结合细胞周期分析和溶酶体抑制剂，我们发现溶酶体处理对于抗体-美坦素缀合物的活性是必需的，而不管连接体是什么。我们还鉴定和表征了这些偶联物释放的美坦素分子，并测量了它们的释放速度与细胞周期停滞的动力学相比较。这两种结合物在溶酶体中有效降解，产生由完整的美坦素类药物和连接赖氨酸组成的代谢物。赖氨酸加合物是硫醚缀合物的唯一代谢物。然而，由二硫化物连接的共轭物产生的赖氨酸代谢物被还原并s -甲基化，产生亲脂性和强细胞毒性的代谢物s -甲基- dm4。这些发现提供了对抗体-美坦素缀合物的作用机制的深入了解，更具体地说，确定了一种生化机制，可以解释用二硫化物连接的缀合物观察到的显著增强的抗肿瘤功效。[3]

接下来在携带已建立的CD138阳性MOLP-8人MM细胞的SCID小鼠中评估nBT062-SPDB-DM4、nBT0662-SMCC-DM1和nBT062-SPP-DM1的体内功效。单次静脉注射免疫偶联物可在耐受性良好的浓度下引起显著的剂量依赖性肿瘤生长抑制和肿瘤消退，稳定的体重证明了这一点。nBT062-SPDB-DM4是在该模型中测试的最具活性的缀合物。此外，每周给药nBT062-SMCC-DM1（六剂13.8μg/kg）在给药期间完全阻断了肿瘤生长[2]。

Kadcyla®（T-DM1）是一种用于治疗HER2+乳腺癌症的抗体驱动药物偶联物（ADC），已于2013年获得美国食品药品监督管理局（FDA）的批准。作为一种随机赖氨酸共轭的ADC，由于DAR分布不均匀，它在DAR控制和PK方面存在困难。由于细胞毒素DM1的疏水性，它还在结合过程中引起聚集。T-DM1，SMCC-DM1中的连接药物是疏水性的，并且在缀合溶液中需要一定百分比的有机溶剂如DMA，这限制了有机溶剂兼容装置中的制造过程并增加了额外的成本。为了解决这些问题，基于Caddick及其同事的工作，开发了一种位点特异性缀合方法，包括抗体的完全还原和与桥状缀合物药物的完全缀合，以获得与DAR 4的位点定向抗体-药物缀合物。用SMCC-DM1和不同长度的亲水性聚乙二醇（PEG）部分组装桥状偶联物。通过在连接药物的侧链中应用PEG部分，可以减少结合中使用的有机溶剂。当PEG长度为约26个单位时，在缀合中不再需要有机溶剂。减少偶联中有机溶剂的量也可以减少偶联过程中聚集的发生。此外，文中还讨论了所设计的共轭器的共轭构型。所得ADC的结合亲和力没有显示出显著降低，并且基于细胞的测定和动物研究已经显示出与T-DM1的可比结果[1]。

生长抑制试验和增殖试验。如前所述（16），通过测量3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化染料吸光度来评估nBT062-SMCC-DM1、nBT0662-SPDB-DM4、nBT0062-SPP-DM1和地塞米松对MM细胞系、PBMC和BM基质细胞（BMSC）生长的生长抑制作用。一个抗体分子附着在它上面～3.5个分子DM4。抗体的分子量不会因DM4分子的附着而显著增加。[2]
评估了nBT062-SPDB-DM4介导近端CD138阴性细胞的抗原依赖性旁观者杀伤的能力。将CD138阳性的MM OPM2细胞（1×10-4/孔）和CD138阴性的Namalwa细胞（3×10-3/孔）分别接种或混合在96孔圆底板中，并暴露于nBT062-SPDB-DM4 120小时。然后使用WST-8试剂评估细胞活力。为了评估免疫偶联物在BM环境中对MM细胞的生长抑制作用，在药物存在或不存在的情况下，将MM细胞（2×104/孔）在BMSC（1×104/孔）包被的96孔板（Costar）中培养48小时。DNA合成通过[3H]胸苷摄取来测量，在48小时培养的最后8小时添加[3H]胸腺嘧啶核苷（0.5μCi/孔）。所有实验一式四份。[2]
细胞周期分析。将MM细胞（1×106）与试剂或不与试剂一起孵育，用PBS洗涤，在−20°C下暴露于70%乙醇30分钟使其透化，在室温下与碘化丙啶（50μg/mL）在含有20单位/mL RNase a的0.5 mL PBS中孵育30分钟，并通过流式细胞术分析DNA含量。

Green fluorescent protein–positive human MM xenograft mouse model and SCID-hu mouse model.[2]
OPM1 cells were transfected with green fluorescent protein (OPM1GFP+) using a lentiviral vector, as previously described. CB17 SCID mice (48-54 days old) were purchased from Charles River Laboratories. All animal studies were conducted according to protocols approved by the Animal Ethics Committee of the Dana-Farber Cancer Institute. Mice were inoculated s.c. with 5 × 106 OPM1GFP+ MM cells in 100 μL RPMI 1640. When tumors became palpable, mice were assigned into the treatment group receiving 200 μg conjugate per mouse via tail vein injection weekly or the control group receiving vehicle alone. Caliper measurements of the longest perpendicular tumor diameters were done every alternate day to estimate the tumor volume using the following formula representing the three-dimensional volume of an ellipse: 4 / 3 × (width / 2)2 × (length / 2). Animals were sacrificed when tumors reached 2 cm or when moribund. Survival was evaluated from the first day of treatment until death. Tumor growth was evaluated using caliper measurements from the first day of treatment until day of sacrifice, day 10 for control, and day 21 for the nBT062-SPDB-DM4 treatment group. Mice were monitored by whole-body fluorescence imaging using Illumatool Bright Light System LT-9900 (Lightools Research) after shaving the tumor area. The images were captured with a Canon IXY digital 700 camera. Ex vivo analysis of tumor image was captured with a LEICA DM IL microscope connected to the LEICA DFC300 FX camera at 40 units/0.60.
Human fetal long bones were implanted into CB17 SCID mice (SCID-hu), as previously described (18). Briefly, 4 wk after bone implantation, 2.5 × 106 INA-6 cells in a final volume of 100 μL of RPMI 1640 were injected directly into the human BM cavity in the SCID-hu mice. An increase in the levels of soluble human IL-6 receptor (shuIL-6R), which is released by INA-6 cells, was used as a parameter of MM cell growth and burden of disease in SCID-hu mice. Mice developed measurable serum shuIL-6R ∼4 wk after INA-6 cell injection and then received 0.176 mg conjugate or vehicle control via tail vein injection weekly for 7 wk. After treatments, blood samples were collected and assayed for shuIL-6R levels by an ELISA.

[1]. Site-specific and hydrophilic ADCs through disulfide-bridged linker and branched PEG. Bioorg Med Chem Lett. 2018 May 1;28(8):1363-1370.

[2]. The monoclonal antibody nBT062 conjugated to cytotoxic Maytansinoids has selective cytotoxicity against CD138-positive multiple myeloma cells in vitro and in vivo. Clin Cancer Res. 2009 Jun 15;15(12):4028-37. doi: 10.1158/1078-0432.CCR-08-2867.

[3]. Antibody-maytansinoid conjugates are activated in targeted cancer cells by lysosomal degradation and linker-dependent intracellular processing. Cancer Res. 2006 Apr 15;66(8):4426-33.

Purpose: We investigated the antitumor effect of murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives against multiple myeloma (MM) cells in vitro and in vivo. Experimental design: We examined the growth inhibitory effect of BT062-SPDB-DM4, BT062-SMCC-DM1, and BT062-SPP-DM1 against MM cell lines and primary tumor cells from MM patients. We also examined in vivo activity of these agents in murine MM cell xenograft model of human and severe combined immunodeficient (SCID) mice bearing implant bone chips injected with human MM cells (SCID-hu model). Results: Anti-CD138 immunoconjugates significantly inhibited growth of MM cell lines and primary tumor cells from MM patients without cytotoxicity against peripheral blood mononuclear cells from healthy volunteers. In MM cells, they induced G(2)-M cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. Nonconjugated nBT062 completely blocked cytotoxicity induced by nBT062-maytansinoid conjugate, confirming that specific binding is required for inducing cytotoxicity. Moreover, nBT062-maytansinoid conjugates blocked adhesion of MM cells to bone marrow stromal cells. The coculture of MM cells with bone marrow stromal cells protects against dexamethasone-induced death but had no effect on the cytotoxicity of immunoconjugates. Importantly, nBT062-SPDB-DM4 and nBT062-SPP-DM1 significantly inhibited MM tumor growth in vivo and prolonged host survival in both the xenograft mouse models of human MM and SCID-hu mouse model. Conclusion: These results provide the preclinical framework supporting evaluation of nBT062-maytansinoid derivatives in clinical trials to improve patient outcome in MM.[2]

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Researchers report that the huC242-maytansinoid conjugate, huC242-SMCC-DM1, with a “noncleavable” linker containing a thioether bond was at least as potent in vitro as the selected conjugate, huC242-SPDB-DM4, which has a “cleavable” linker containing a disulfide bond. This was surprising because huC242-SMCC-DM1 displayed significantly lower in vivo activity in multiple xenograft tumor models. To investigate this conundrum, we undertook a series of experiments to elucidate the mechanism of cell killing by the conjugates. The results delineate an activation process, for both conjugates, that requires lysosomal degradation of the antibody component of the conjugate. However, distinct maytansinoid metabolites produced by intracellular processing of huC242-SPDB-DM4 were identified and characterized, providing a likely mechanism for its superior antitumor efficacy.[3]

C51H66CLN5O16S

1072.6116528511

1071.391

C, 57.11; H, 6.20; Cl, 3.30; N, 6.53; O, 23.87; S, 2.99

1228105-51-8

92131096

White to off-white solid powder

1.4±0.1 g/cm3

1.626

3.28

282.83

2

17

15

74

2250

8

ClC1C(=CC2CC(C)=CC=CC([C@]3(C[C@@H]([C@@H](C)C4[C@](C)(C(CC(N(C)C=1C=2)=O)OC([C@H](C)N(C)C(CCSC1CC(N(C1=O)CC1CCC(C(=O)ON2C(CCC2=O)=O)CC1)=O)=O)=O)O4)OC(N3)=O)O)OC)OC |t:7,9|

IADUWZMNTKHTIN-IOBAKXROSA-N

InChI=1S/C51H66ClN5O16S/c1-27-10-9-11-37(69-8)51(67)25-35(70-49(66)53-51)28(2)45-50(4,72-45)38(24-42(61)55(6)33-21-31(20-27)22-34(68-7)44(33)52)71-47(64)29(3)54(5)39(58)18-19-74-36-23-43(62)56(46(36)63)26-30-12-14-32(15-13-30)48(65)73-57-40(59)16-17-41(57)60/h9-11,21-22,28-30,32,35-38,45,67H,12-20,23-26H2,1-8H3,(H,53,66)/b11-9-,27-10+/t28-,29+,30?,32?,35+,36?,37-,38-,45?,50+,51+/m1/s1

2,5-dioxopyrrolidin-1-yl 4-((3-((3-(((2S)-1-(((14S,16S,33S,2R,4R,10E,12Z,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-1-oxopropan-2-yl)(methyl)amino)-3-oxopropyl)thio)-2,5-dioxopyrrolidin-1-yl)methyl)cyclohexane-1-carboxylate

DM1-SMCC; DM1 SMCC; DM1SMCC; DM1-SMCC; SCHEMBL20153009; IADUWZMNTKHTIN-MLSWMBHTSA-N; 1613362-80-3; SMCC-DM1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。

Ship with dry ice.

DMSO : ~16.67 mg/mL (~15.54 mM)

配方 1 中的溶解度: 2 mg/mL (1.86 mM) 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; 超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 20.0 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 mg/mL (1.86 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), 澄清溶液; 超声助溶.
例如，若需制备1 mL的工作液，可将 100 μL 20.0mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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: ≥ 2 mg/mL (1.86 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 20.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 4 中的溶解度: 10% DMSO+ 40% PEG300+ 5% Tween-80+ 45% saline: 2 mg/mL (1.86 mM)

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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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。