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实验参考方法

Cell experiment [1]:
Cell lines	Peripheral blood mononuclear cells (PBMC)
Preparation Method	PBMC were isolated from fresh whole blood. PBMC were suspended at 2 x 10⁶ cells/well in 2 ml complete medium (RPMI-1640 supplemented with 2 mM L-glutamine, 10% FCS and 1% penicillin/streptomycin); 24-well non-treated culture plates were precoated with plate-bound 2 μg/ml anti-CD3 antibody and 2 μg/ml anti-CD28 antibody for 2.5 h at 37℃.
Reaction Conditions	PBMC were either plated as‘non-activated’ in non-coated wells or ‘activated’ in precoated wells. Plated cells were then treated with anti-PD-1 monoclonal antibody, pembrolizumab, at 2 μg/ml and were incubated for 24 h in a humidified incubator at 37℃ and 5% CO².
Applications	Pembrolizumab treatment could reduce PD-1 expression significantly in CD4⁺ T cells in non-activated and activated PBMC. Pembrolizumab treatment resulted in a significant reduction in PD-1 expression in both CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells.
Animal experiment ^[2]:
Animal models	NSG mice; Humanized NSG mice
Preparation Method	Humanized NSG mice were generated by intravenous injection of 105 human CD34⁺ (hCD34⁺) HPSCs into 3-week-old female NSG mice, 4 h post-140 cGy total body irradiation using the RS-2000 irradiator. Humanized NSG mice that had over 25% hCD45⁺ cells in the peripheral blood were considered as engrafted and humanized. Then patient-derived tumors were finely minced and loaded into 1-cc syringes with 14-gauge needles. Depending on the tumor model, 20–40 µl of homogenized tumor tissue was inoculated subcutaneously at the right flank of mice while under anesthesia.
Dosage form	10 mg/kg i.p. for the first dose, followed by 5 mg/kg, i.p. dosage on day 5, 10, 15, 20, and 25.
Applications	Pembrolizumab treatment significantly decreased PD^-1⁺ cell detection in hCD45⁺ cells in tumor models. But the percentage of PD^-L1⁺ cells in hCD45+ or hCD45^- cell populations was not affected by pembrolizumab treatment. Moreover, pembrolizumab treatment obviously delayed TNBC CDX growth compared with the vehicle control group and inhibited tumor growth in both TNBC and NSCLC PDX Onco-Humanized NSG models.
References: [1]. Toor SM, et al. In-vitro effect of pembrolizumab on different T regulatory cell subsets. Clin Exp Immunol. 2018 Feb;191(2):189-197. [2]. Wang M, et al. Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy. FASEB J. 2018 Mar;32(3):1537-1549.

产品描述

Pembrolizumab, an anti–programmed death-1 monoclonal antibody, has demonstrated clinically significant anti-tumor activity with acceptable safety in patients with advanced solid cancers and was approved by the U.S. FDA for the treatment of advanced melanoma, NSCLC, head and neck squamous cell cancer, and other malignant tumors.^[1]

In vitro study demonstrated that pembrolizumab had a greater effect on PD-1 expression in CD4+CD25- T cells which expressed most of the PD-1, and that are comprised of non-Treg and/or non-activated T cells. However, pembrolizumab did not affect the expression levels of Treg-related markers, including cytotoxic T lymphocyte antigen-4 (CTLA-4), CD15s, latency-associated peptide (LAP) and Ki-67 as well as the levels of FoxP3+/-Helios+/- Treg subsets in both cohorts.^[2]

In vivo study of pembrolizumab indicated that in Onco-Humanized NSG mice, pembrolizumab could inhibit tumor growth, not only in CDX but also in various PDX tumor models. Results showed that the efficacy of pembrolizumab is dependent on the engraftment of an adaptive human immune system in Onco-Humanized NSG mice, specifically hCD8+ T cells. Furthermore, pembrolizumab increased both CD4+ and CD8+ T-cell numbers in the blood of the 2 NSCLC Onco-Humanized NSG models but decreased both CD4+ and CD8+ T-cell numbers in the blood of the TNBC Onco-Humanized NSG model.^[3]

References:
[1]. Mo DC, et al. Safety and efficacy of pembrolizumab plus lenvatinib versus pembrolizumab and lenvatinib monotherapies in cancers: A systematic review. Int Immunopharmacol. 2021 Feb;91:107281.
[2]. Toor SM, et al. In-vitro effect of pembrolizumab on different T regulatory cell subsets. Clin Exp Immunol. 2018 Feb;191(2):189-197.
[3]. Wang M, et al. Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy. FASEB J. 2018 Mar;32(3):1537-1549.

Pembrolizumab 是一种抗程序性死亡 1 单克隆抗体，已在晚期实体癌患者中证明具有临床显着的抗肿瘤活性和可接受的安全性，并已被美国 FDA 批准用于治疗晚期黑色素瘤、NSCLC、头颅和脑肿瘤。颈部鳞状细胞癌等恶性肿瘤。^[1]

体外研究表明，派姆单抗对 CD4+CD25-T 细胞中的 PD-1 表达有更大的影响，这些细胞表达大部分 PD-1，并且由非 Treg 和/或未激活的 T 细胞组成。然而，pembrolizumab 不影响 Treg 相关标志物的表达水平，包括细胞毒性 T 淋巴细胞抗原 4 (CTLA-4)、CD15s、潜伏相关肽 (LAP) 和 Ki-67 以及 FoxP3+/- Helios+/- 两个队列中的 Treg 子集。^[2]

pembrolizumab 的体内研究表明，在 Onco-Humanized NSG 小鼠中，pembrolizumab 可以抑制肿瘤生长，不仅在 CDX 中，而且在各种 PDX 肿瘤模型中。结果表明，pembrolizumab 的疗效取决于适应性人类免疫系统在 Onco-Humanized NSG 小鼠中的植入，特别是 hCD8+ T 细胞。此外，pembrolizumab 增加了 2 个 NSCLC Onco-Humanized NSG 模型血液中的 CD4+ 和 CD8+ T 细胞数量，但减少了 TNBC Onco-Humanized NSG 模型血液中的 CD4+ 和 CD8+ T 细胞数量。^{[ 3]}

Chemical Properties

Cas No.	1374853-91-4	SDF
别名	派姆单抗,帕博利珠单抗,Lambrolizumab; MK-3475
分子式		分子量	146266.23
溶解度		储存条件	Store at -80°C
General tips	请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。
Shipping Condition	评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。

溶解性数据

制备储备液
		1 mg	5 mg	10 mg
	1 mM	0.0068 mL	0.0342 mL	0.0684 mL
	5 mM	0.0014 mL	0.0068 mL	0.0137 mL
	10 mM	0.0007 mL	0.0034 mL	0.0068 mL

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体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。

注意：1. 首先保证母液是澄清的；
2. 一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。

Research Update

Pembrolizumab (Keytruda)

The programmed cell death protein 1 (PD1) is one of the checkpoints that regulates the immune response. Ligation of PD1 with its ligands PDL1 and PDL2 results in transduction of negative signals to T-cells. PD1 expression is an important mechanism contributing to the exhausted effector T-cell phenotype. The expression of PD1 on effector T-cells and PDL1 on neoplastic cells enables tumor cells to evade anti-tumor immunity. Blockade of PD1 is an important immunotherapeutic strategy for cancers. Pembrolizumab (Keytruda) is a humanized monoclonal anti-PD1 antibody that has been extensively investigated in numerous malignancies. In melanoma refractory to targeted therapy, pembrolizumab induced overall response rates (ORRs) of 21-34%. It was superior to another immune checkpoint inhibitor ipilimumab (Yervoy) in stage III/IV unresectable melanoma. In refractory non-small cell lung cancer (NSCLC), pembrolizumab induced ORRs of 19-25%. Based on these results, pembrolizumab was approved by the USA FDA for the treatment of advanced melanoma and NSCLC. Tumor cell PDL1 expression may be a valid response predictor. Molecular analysis also showed that tumors with high gene mutation burdens, which might result in the formation of more tumor-related neo-antigens, had better responses to pembrolizumab. In malignancies including lymphomas and other solid tumors, preliminary data showed that ORRs of around 20-50 % could be achieved. Adverse events occurred in up to 60% of patients, but grade 3/4 toxicities were observed in <10% of cases. Immune-related adverse events including thyroid dysfunction, hepatitis and pneumonitis are more serious and may lead to cessation of treatment.

Updated Analysis of KEYNOTE-024: Pembrolizumab Versus Platinum-Based Chemotherapy for Advanced Non-Small-Cell Lung Cancer With PD-L1 Tumor Proportion Score of 50% or Greater

Purpose: In the randomized, open-label, phase III KEYNOTE-024 study, pembrolizumab significantly improved progression-free survival and overall survival (OS) compared with platinum-based chemotherapy in patients with previously untreated advanced non-small-cell lung cancer (NSCLC) with a programmed death ligand 1 tumor proportion score of 50% or greater and without EGFR/ALK aberrations. We report an updated OS and tolerability analysis, including analyses adjusting for potential bias introduced by crossover from chemotherapy to pembrolizumab.
Patients and methods: Patients were randomly assigned to pembrolizumab 200 mg every 3 weeks (for up to 2 years) or investigator's choice of platinum-based chemotherapy (four to six cycles). Patients assigned to chemotherapy could cross over to pembrolizumab upon meeting eligibility criteria. The primary end point was progression-free survival; OS was an important key secondary end point. Crossover adjustment analysis was done using the following three methods: simplified two-stage method, rank-preserving structural failure time, and inverse probability of censoring weighting.
Results: Three hundred five patients were randomly assigned (pembrolizumab, n = 154; chemotherapy, n = 151). At data cutoff (July 10, 2017; median follow-up, 25.2 months), 73 patients in the pembrolizumab arm and 96 in the chemotherapy arm had died. Median OS was 30.0 months (95% CI, 18.3 months to not reached) with pembrolizumab and 14.2 months (95% CI, 9.8 to 19.0 months) with chemotherapy (hazard ratio, 0.63; 95% CI, 0.47 to 0.86). Eighty-two patients assigned to chemotherapy crossed over on study to receive pembrolizumab. When adjusted for crossover using the two-stage method, the hazard ratio for OS for pembrolizumab versus chemotherapy was 0.49 (95% CI, 0.34 to 0.69); results using rank-preserving structural failure time and inverse probability of censoring weighting were similar. Treatment-related grade 3 to 5 adverse events were less frequent with pembrolizumab compared with chemotherapy (31.2% v 53.3%, respectively).
Conclusion: With prolonged follow-up, first-line pembrolizumab monotherapy continues to demonstrate an OS benefit over chemotherapy in patients with previously untreated, advanced NSCLC without EGFR/ALK aberrations, despite crossover from the control arm to pembrolizumab as subsequent therapy.

Pemetrexed plus platinum with or without pembrolizumab in patients with previously untreated metastatic nonsquamous NSCLC: protocol-specified final analysis from KEYNOTE-189

Background: In the phase III KEYNOTE-189 study (NCT02578680), pembrolizumab plus pemetrexed and platinum-based chemotherapy (pemetrexed-platinum) significantly improved overall survival (OS) and progression-free survival (PFS) in patients with previously untreated metastatic nonsquamous non-small-cell lung cancer (NSCLC) versus placebo plus pemetrexed-platinum. We report updated efficacy outcomes from the protocol-specified final analysis, including outcomes in patients who crossed over to pembrolizumab from pemetrexed-platinum and in patients who completed 35 cycles (?2 years) of pembrolizumab.
Patients and methods: Eligible patients were randomized 2 : 1 to receive pembrolizumab 200 mg (n = 410) or placebo (n = 206) every 3 weeks (for up to 35 cycles, ?2 years) plus four cycles of pemetrexed (500 mg/m²) and investigators' choice of cisplatin (75 mg/m²) or carboplatin (area under the curve 5 mg·min/ml) every 3 weeks, followed by pemetrexed until progression. Patients assigned to placebo plus pemetrexed-platinum could cross over to pembrolizumab upon progression if eligibility criteria were met. The primary endpoints were OS and PFS.
Results: After a median follow-up of 31.0 months, pembrolizumab plus pemetrexed-platinum continued to improve OS [hazard ratio (HR), 0.56; 95% confidence interval (CI), 0.46-0.69] and PFS (HR, 0.49; 95% CI, 0.41-0.59) over placebo plus pemetrexed-platinum regardless of programmed death-ligand 1 expression. Objective response rate (ORR) (48.3% versus 19.9%) and time to second/subsequent tumor progression on next-line treatment (PFS2; HR, 0.50; 95% CI, 0.41-0.61) were improved in patients who received pembrolizumab plus pemetrexed-platinum. Eighty-four patients (40.8%) from the placebo plus pemetrexed-platinum group crossed over to pembrolizumab on-study. Grade 3-5 adverse events occurred in 72.1% of patients receiving pembrolizumab plus pemetrexed-platinum and 66.8% of patients receiving placebo plus pemetrexed-platinum. Fifty-six patients completed 35 cycles (?2 years) of pembrolizumab; ORR was 85.7% and 53 (94.6%) were alive at data cut-off.
Conclusions: Pembrolizumab plus pemetrexed-platinum continued to show improved efficacy outcomes compared with placebo plus pemetrexed-platinum, with manageable toxicity. These findings support first-line pembrolizumab plus pemetrexed-platinum in patients with previously untreated metastatic nonsquamous NSCLC.

A Randomized, Placebo-Controlled Trial of Pembrolizumab Plus Chemotherapy in Patients With Metastatic Squamous NSCLC: Protocol-Specified Final Analysis of KEYNOTE-407

Introduction: In the randomized KEYNOTE-407 study (ClinicalTrials.gov, NCT02775435), pembrolizumab plus carboplatin and paclitaxel/nab-paclitaxel (chemotherapy) significantly improved overall survival (OS) and progression-free survival (PFS) compared with placebo plus chemotherapy in patients with previously untreated metastatic squamous NSCLC. We report updated efficacy outcomes from the protocol-specified final analysis and, for the first time, progression on next line of treatment.
Methods: Eligible patients were randomized to chemotherapy plus either pembrolizumab (n = 278) or placebo (n = 281). After positive results from the second interim analysis, patients still receiving placebo could cross over to pembrolizumab monotherapy at the time of confirmed progressive disease. The primary end points were OS and PFS. PFS-2 (time from randomization to progression on next-line treatment/death, whichever occurred first) was an exploratory end point.
Results: After median (range) follow-up of 14.3 (0.1-31.3) months, pembrolizumab plus chemotherapy continued to exhibit a clinically meaningful improvement over placebo plus chemotherapy in OS (median, 17.1 mo [95% confidence interval (CI): 14.4?19.9] versus 11.6 mo [95% CI: 10.1?13.7]; hazard ratio [HR], 0.71 [95% CI: 0.58?0.88]) and PFS (median, 8.0 mo [95% CI: 6.3?8.4] versus 5.1 mo [95% CI: 4.3?6.0]; HR, 0.57 [95% CI: 0.47?0.69]). PFS-2 was longer for patients randomized to first-line pembrolizumab plus chemotherapy (HR, 0.59 [95% CI: 0.49?0.72]). Grade 3 to 5 adverse events occurred in 74.1% and 69.6% of patients receiving pembrolizumab plus chemotherapy and placebo plus chemotherapy, respectively.
Conclusions: Pembrolizumab plus chemotherapy continued to exhibit substantially improved OS and PFS in patients with metastatic squamous NSCLC. The PFS-2 outcomes support pembrolizumab plus chemotherapy as a standard first-line treatment in patients with metastatic squamous NSCLC.

Pembrolizumab versus Ipilimumab in Advanced Melanoma

Background: The immune checkpoint inhibitor ipilimumab is the standard-of-care treatment for patients with advanced melanoma. Pembrolizumab inhibits the programmed cell death 1 (PD-1) immune checkpoint and has antitumor activity in patients with advanced melanoma.
Methods: In this randomized, controlled, phase 3 study, we assigned 834 patients with advanced melanoma in a 1:1:1 ratio to receive pembrolizumab (at a dose of 10 mg per kilogram of body weight) every 2 weeks or every 3 weeks or four doses of ipilimumab (at 3 mg per kilogram) every 3 weeks. Primary end points were progression-free and overall survival.
Results: The estimated 6-month progression-free-survival rates were 47.3% for pembrolizumab every 2 weeks, 46.4% for pembrolizumab every 3 weeks, and 26.5% for ipilimumab (hazard ratio for disease progression, 0.58; P<0.001 for both pembrolizumab regimens versus ipilimumab; 95% confidence intervals [CIs], 0.46 to 0.72 and 0.47 to 0.72, respectively). Estimated 12-month survival rates were 74.1%, 68.4%, and 58.2%, respectively (hazard ratio for death for pembrolizumab every 2 weeks, 0.63; 95% CI, 0.47 to 0.83; P=0.0005; hazard ratio for pembrolizumab every 3 weeks, 0.69; 95% CI, 0.52 to 0.90; P=0.0036). The response rate was improved with pembrolizumab administered every 2 weeks (33.7%) and every 3 weeks (32.9%), as compared with ipilimumab (11.9%) (P<0.001 for both comparisons). Responses were ongoing in 89.4%, 96.7%, and 87.9% of patients, respectively, after a median follow-up of 7.9 months. Efficacy was similar in the two pembrolizumab groups. Rates of treatment-related adverse events of grade 3 to 5 severity were lower in the pembrolizumab groups (13.3% and 10.1%) than in the ipilimumab group (19.9%).
Conclusions: The anti-PD-1 antibody pembrolizumab prolonged progression-free survival and overall survival and had less high-grade toxicity than did ipilimumab in patients with advanced melanoma. (Funded by Merck Sharp & Dohme; KEYNOTE-006 ClinicalTrials.gov number, NCT01866319.).

Pembrolizumab

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