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【肿瘤免疫治疗】Anti-CD73 的蛋白标志物检测金标准

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Cell旗下《Trends in Cancer》发表了一篇题为“Anti-CD73 in Cancer Immunotherapy: Awakening New Opportunities”的综述。这一综述讨论了CD73与肿瘤发生、发展以及扩散之间的关系,强调了这一分子作为药物靶标的潜在价值,并表示CD73有望成为个性化癌症治疗中的新生物标志物。

 

近年来,癌症免疫治疗的飞速发展使科学界对肿瘤生物学和免疫学有了更好的了解。肿瘤微环境是一个动态的niche,包括了癌细胞、免疫细胞、成纤维细胞、成肌纤维细胞、细胞因子、血管和细胞外基质。肿瘤往往处在缺氧的条件下,且环境中葡萄糖和其它营养物质也很缺乏。

 

癌症免疫疗法

为了生存,癌细胞在这样的环境下会重组它们的代谢机制。其中,最新的证据表明,调整嘌呤代谢是非常关键的一步,尤其是增加ecto-5′-nucleotidase(CD73)的表达。这种降解酶能够使AMP脱去磷酸,从而会导致肿瘤微环境中免疫抑制和pro-angiogenic adenosine halo的产生,促进癌症的发生和发展。

临床前研究表明,靶向CD73能够产生良好的抗肿瘤作用,且将CD73阻断治疗与其它免疫分子调节剂联合(如CTLA-4抗体、PD-1抗体)是一种极具吸引力的选择。作者们表示,尽管还有很长的路要走,但是通过CD73抗体的发展,抗CD73治疗有望成为抗癌领域的新型生物疗法。

 

细胞外代谢途径中CD73的结构和作用

此外,该综述总结了以下四大趋势

       第一,免疫系统对肿瘤细胞的识别和抑制有至关重要的作用。第一代免疫疗法已经使用了数十年,在一些肿瘤类型中取得了成功。然而,大部分免疫疗法依然缺乏实质性效果或特异性,由此引发的副作用限制了这些疗法的临床使用。

       第二,在过去的几年里,研究者们对免疫系统与肿瘤之间的复杂关系有了更好的理解,鉴定出了监管两者关系的关键分子,包括CTLA-4、PD-1和PD-L1等。这些发现使癌症免疫疗法再次走到科学前沿。

       第三,新型免疫疗法与传统化疗和靶向治疗之间的互补性表明了联合治疗可能带来协同效应。

      第四,尽管CTLA-4、PD-1和PD-L1抑制已经在临床中成功应用,但科学家们也在积极研究其它的检查点通路。在这些通路中,ecto-5′-nucleotidase (CD73)在驱动癌症免疫逃逸中发挥了关键的作用,因此,可能成为发展新型抗癌免疫疗法极具潜力的靶点。


Targeting CD73 in the tumor microenvironment with MEDI9447

MedImmune, LLC, Gaithersburg, MD, USA;

Astrazeneca, Gaithersburg, MD, USA;

Macrogenics, MacroGenics, Inc., Rockville, MD, USA


ABSTRACT

MEDI9447 is a human monoclonal antibody that is specific for the ectoenzyme CD73 and currently undergoing Phase I clinical trials. Here we show that MEDI9447 is a potent inhibitor of CD73 ectonucleotidase activity, with wide ranging immune regulatory consequences.

MEDI9447 results in relief from adenosine monophosphate (AMP)-mediated lymphocyte suppression in vitro and inhibition of mouse syngeneic tumor growth in vivo. In contrast with other cancer immunotherapy agents such as checkpoint inhibitors or T-cell agonists,

MEDI9447 drives changes in both myeloid and lymphoid infiltrating leukocyte populations within the tumor microenvironment of mouse models. Changes include significant alterations in a number of tumor micro-environmental subpopulations including increases in CD8C effector cells and activated macrophages. Furthermore, these changes correlate directly with responder and non-responder subpopulations within animal studies using syngeneic tumors.

 

Combination data showing additive activity between MEDI9447 and anti-PD-1 antibodies using human cells in vitro and mouse tumor models further demonstrate the potential value of relieving adenosinemediated immunosuppression. Based on these data, a Phase I study to test the safety, tolerability, and clinical activity of MEDI9447 in cancer patients was initiated (NCT02503774).


Mixed leukocyte reactions

PBMC were isolated fresh from healthy donors or from frozen  healthy leukopak aliquots.

For fresh PBMCs, blood from healthy normal donors was collected into 8 mL BD Vacutainer  CPTTM  Cell preparation tubes then centrifuged for 25 min at 2,700 £  g with no break. PBMCs were collected and then washed three times with AIM-V assay medium (Gibco). Red blood cells were lysed using Red Blood Cell Lysing Buffer (Gibco). PBMCs were prepared from leukopaks (All Cells) by collecting into 1-L bottles and dilution with wash buffer (PBS containing 2% fetal bovine serum). The PBMCs were then isolated by layering over LSM Separation Medium (MP Biomedicals) in 50 mL Sepmate Tubes (Stem Cell Technologies). The tubes were centrifuged at 1,200 £  g for 10 min.

 

The cells were collected and then washed by centrifugation three times with wash buffer at 300 £  g for 8 min each wash. Red blood cells were lysed using Red Blood Cell Lysing Buffer (Gibco). PBMCs were re-suspended in Cell Freezing Medium (Gibco) and frozen at ¡ 80 C. For the MLR, PBMCs were re-suspended in assay medium. PBMCs from two donors were mixed together 1:1 in AIM-V assay medium and plated into 96-well U-bottom plates (Costar) at a density of 200,000 cells per well per donor in 100 m L. MEDI9447 and an isotype control antibody were diluted in serum-free AIMV medium and then added to the cells for 72– 96 h. Brightfi eld images of the plates were taken daily at 2.5£  magnifi cation using the Cellomics ArrayScan instrument (Thermo Fischer). Plates were then centrifuged at 1,200£ g for 3 min. Supernatant was harvested and then assayed for cytokine secretion using the human TH1/TH2 multiplex ELISA (Meso Scale Discovery) according to the manufacturer’ s instructions.

 

Meso Scale Discovery 提供了小鼠,人,猴子等多种高灵敏度多因子检测试剂盒:

 

物种 品名 特点 货号
Human TH1/TH2 10-Plex Tissue Culture Kit IFN-γ, IL-1β, IL-10, IL-12p70, IL-13, IL-2, IL-4, IL-5, IL-8, TNF-α | Human 适合检测超高浓度 K15010B-1
U-PLEX TH1/TH2 Combo (hu) SECTOR (1 PL) IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, IL-13, TNF-α | Human 全球第一样本检出率 K15010K-1
Human TH1/TH2 7-Plex Tissue Culture Kit IL-10, IL-13, IL-2, IL-4, IL-5, IL-12p70, IFN-γ | Human 适合检测超高浓度 K15011B-1
小鼠 Mouse TH1/TH2 9-Plex Tissue Culture Kit IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12/IL-23p40, KC/GRO, TNF-α, IL-12 Total | Mouse 适合检测超高浓度 K15013B-1
V-PLEX Plus Human Cytokine 30-Plex Kit Eotaxin, Eotaxin-3, GM-CSF, IFN-γ, IL-10, IL-12p70, IL-12/IL-23p40, IL-13, IL-15, IL-16, IL-17A, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, TARC, TNF-α, TNF-β, VEGF-A, IL-8 (HA) | Human 30因子筛选 K15054G-1
小鼠 U-PLEX TH1/TH2 Combo (ms) SECTOR (1 PL) IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12p70, IL-13, KC/GRO, TNF-α | Mouse 全球第一样本检出率 K15071K-1
猴子 U-PLEX TH1/TH2 Combo (NHP) SECTOR (1 PL) IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, TNF-α | Non-human primate 全球第一样本检出率 K15080K-1

 

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