Importance  The success of immunotherapy with checkpoint inhibitors is not replicated in most cases of colorectal cancer; therefore, different strategies are urgently required. The oncofetal antigen 5T4 is expressed in more than 90% of cases of metastatic colorectal cancer (mCRC). Preliminary data using modified vaccinia Ankara–5T4 (MVA-5T4) in mCRC demonstrated that it safely induced serologic and T-cell responses.

Objective  To determine whether antitumor immunity in mCRC could be increased using MVA-5T4, metronomic low-dose cyclophosphamide, or a combination of both treatments.

Design, Setting, and Participants  In this randomized clinical trial, 55 patients with inoperable mCRC and prior stable disease after standard chemotherapy were enrolled at a single center and randomized to watch and wait (n = 9), cyclophosphamide treatment only (n = 9), MVA-5T4 only (n = 19), and a combination of MVA-5T4 and cyclophosphamide (n = 18). Patients were enrolled and treated from July 9, 2012, through February 8, 2016, and follow-up was completed on December 13, 2016. Data were analyzed based on intention to treat.

Interventions  Patients randomized to a cyclophosphamide group received 50 mg twice daily on treatment days 1 to 7 and 15 to 21. Patients randomized to a MVA-5T4 group received an intramuscular injection at a dose of 1 × 10⁹ 50% tissue culture infectious dose on treatment days 22, 36, 50, 64, 78, and 106.

Main Outcomes and Measures  The predefined primary end point was the magnitude of anti-5T4 immune responses (5T4-specific T-cell and antibody levels) generated at treatment week 7. Secondary end points included analysis of the kinetics of anti-5T4 responses, progression-free survival (PFS), and overall survival (OS).

Results  Fifty-two patients (38 men and 14 women; mean [SD] age, 64.2 [10.1] years) were included in the study analysis. The 5T4-specific antibody immune responses were significantly increased in the MVA-5T4 (83.41 [36.09] relative units [RU]; P = .02) and combination treatment (65.81 [16.68] RU; P = .002) groups compared with no treatment (20.09 [7.20] RU). Cyclophosphamide depleted regulatory T cells in 24 of 27 patients receiving MVA-5T4, independently prolonging PFS (5.0 vs 2.5 months; hazard ratio [HR], 0.48; 95% CI, 0.21-1.11; P = .09). MVA-5T4 doubled baseline anti-5T4 responses in 16 of 35 patients, resulting in significantly prolonged PFS (5.6 vs 2.4 months; HR, 0.21; 95% CI, 0.09-0.47; P < .001) and OS (20.0 vs 10.3 months; HR, 0.32; 95% CI, 0.14-0.74; P = .008). No grade 3 or 4 adverse events were observed.

Conclusions and Relevance  This initial randomized clinical immunotherapy study demonstrates a significant survival benefit in mCRC. Prior depletion of regulatory T cells by cyclophosphamide did not increase immune responses generated by MVA-5T4 vaccination; however, cyclophosphamide and MVA-5T4 each independently induced beneficial antitumor immune responses, resulting in prolonged survival without toxic effects. Larger clinical trials are planned to further validate these data.

Trial Registration  isrctn.org Identifier: ISRCTN54669986

重要性  在大多数结直肠癌病例中，检查点抑制剂免疫治疗的成功是无法复制的；因此，迫切需要不同的战略。癌胚抗原 5T4 在超过 90% 的转移性结直肠癌 (mCRC) 病例中表达。在 mCRC 中使用改良牛痘 Ankara-5T4 (MVA-5T4) 的初步数据表明，它可以安全地诱导血清学和 T 细胞反应。

目的  确定使用 MVA-5T4、节拍式低剂量环磷酰胺或两种治疗的组合是否可以增加 mCRC 的抗肿瘤免疫力。

设计、设置和参与者  在这项随机临床试验中，55 名在标准化疗后无法手术且既往疾病稳定的患者被纳入一个中心并随机观察和等待（n = 9），仅使用环磷酰胺治疗（n = 9） ，仅 MVA-5T4 (n = 19)，以及 MVA-5T4 和环磷酰胺的组合 (n = 18)。患者于 2012 年 7 月 9 日至 2016 年 2 月 8 日入组并接受治疗，随访于 2016 年 12 月 13 日完成。根据治疗意向分析数据。

干预措施随机分配到环磷酰胺组的患者在第 1 至 7 天和第 15 至 21 天接受 50 mg，每天两次。随机分配到 MVA-5T4 组的患者接受 1 × 10   ⁹ 50% 组织培养感染剂量的肌肉注射治疗第 22、36、50、64、78 和 106 天。

主要结果和措施  预定义的主要终点是治疗第 7 周产生的抗 5T4 免疫反应（5T4 特异性 T 细胞和抗体水平）的大小。次要终点包括分析抗 5T4 反应的动力学、进展- 无生存期 (PFS) 和总生存期 (OS)。

结果  52 名患者（38 名男性和 14 名女性；平均 [SD] 年龄，64.2 [10.1] 岁）被纳入研究分析。与无治疗组相比，MVA-5T4（83.41 [36.09] 相对单位 [RU]； P  = .02）和联合治疗组（65.81 [16.68] RU；P  = .002）组的 5T4 特异性抗体免疫应答显着增加。治疗（20.09 [7.20] RU）。接受 MVA-5T4 的 27 名患者中有 24 名使用环磷酰胺耗尽了调节性 T 细胞，从而独立地延长了 PFS（5.0 与 2.5 个月；风险比 [HR]，0.48；95% CI，0.21-1.11；P  = .09）。MVA-5T4 在 35 名患者中的 16 名患者中使基线抗 5T4 反应翻了一番，导致 PFS 显着延长（5.6 个月 vs 2.4 个月；HR，0.21；95% CI，0.09-0.47；P < .001) 和 OS（20.0 与 10.3 个月；HR，0.32；95% CI，0.14-0.74；P  = .008）。未观察到 3 级或 4 级不良事件。

结论和相关性  这项初步的随机临床免疫治疗研究表明 mCRC 具有显着的生存获益。环磷酰胺对调节性 T 细胞的先前消耗不会增加 MVA-5T4 疫苗接种产生的免疫反应；然而，环磷酰胺和 MVA-5T4 各自独立地诱导有益的抗肿瘤免疫反应，从而延长存活时间而没有毒性作用。计划进行更大规模的临床试验以进一步验证这些数据。

试用注册  isrctn.org 标识符：ISRCTN54669986