Administration of intratumoral immunocytokine to activate immune rejection of spontaneous canine melanoma

恶性黑色素瘤是退伍军人的严重健康问题，如果发生转移性黑色素瘤，通常无法治愈 转移到远处的地方。令人兴奋和令人印象深刻的免疫检查点封锁数据展示了 免疫系统在一些转移性黑色素瘤患者中产生持久反应的能力 更改了护理1的标准。有效的免疫检查点阻断治疗似乎需要 激活针对多种黑色素瘤抗原的抗黑色素瘤T细胞，包括患者- 独特的新抗原。犬恶性黑色素瘤为研究黑色素瘤提供了良好的临床前模型 免疫疗法，因为它类似于在完整免疫状态下自发发生的人类黑色素瘤 并通过淋巴管或血管转移至区域淋巴结、肺、肝、 大脑和肾脏。与这一应用密切相关的是，GD2二唾液酸神经节苷脂(GD2)在人类 和犬黑色素瘤2-4。因此，我们建议瘤内注射GD2反应性hu14.18-IL2 免疫细胞因子(IC)(IT-IC)单独及与其他治疗方法在临床前小鼠中的协同作用 在伴有黑色素瘤的狗身上建立模型，将注射的肿瘤转化为有效的原位肿瘤 疫苗接种5。我们的中心假设是IT-IC联合局部放射治疗(RT)对犬的影响 黑色素瘤可以诱导T细胞对黑色素瘤的反应。此外，我们假设这种反应可以是 通过免疫检查点封锁放大，该方案是安全和耐受性良好的。这些 假设将通过达到以下目的来检验：目标1)确定毒性、免疫原性和评估 单独和局部RT联合IT注射hu14.18-IL2对局部晚期卵巢癌犬的抗肿瘤活性 转移性黑色素瘤；目的2)评估IT交付hu14.18后的局部和全身抗肿瘤活性 IL-2联合RT和抗-PD1免疫检查点阻断局部晚期卵巢癌的实验研究 转移性黑色素瘤；以及目标3)识别生物标记物，以便将免疫治疗策略与IT相结合 局部晚期或转移性黑色素瘤犬体内的hu14.18-IL2。标准临床评估 毒性和耐受性的评估，以及临床明显疾病的测量和成像是目标1的一部分 2.连续的血液样本和肿瘤活检将允许进行详细的组织学和免疫学评估 在目标3中，确定抗肿瘤活性的机制并确定组织学结果是否 在我们的小鼠模型中看到的伴随免疫耐受在狗身上也存在。探索性实验室研究 将：i)评估免疫治疗前后周围和肿瘤细胞环境的变化，ii) 利用新型免疫监测确定犬黑色素瘤免疫应答的候选生物标志物 接受IT-IC，以及iii)评估肿瘤内和外周血T细胞受体的多样性和克隆性 曲目。这项研究使用自发性犬黑色素瘤来测试一种新的RT IT-IC免疫检查点 封锁方案已被证明能够长期、有效地消除大面积局部 以及荷瘤小鼠远端已建立的黑色素瘤病变。这项犬类试验的结果可能 使这些概念能够在包括退伍军人在内的人类患者身上快速转化为临床测试， 晚期黑色素瘤。这种方法在临床上是有利的，因为它涉及的药物/治疗是 目前可用或在临床测试中，它可以很容易地作为一种现成的治疗方法在#年提供 美国各地的退伍军人医院。此外，它通过针对有效和患者进行个性化治疗- 特定的新抗原。最后，RT IT-IC免疫检查点阻断可以纳入治疗 几乎任何其他恶性肿瘤，包括退伍军人中其他常见的恶性肿瘤。唯一的要求是 因为这种策略是针对被靶向的恶性肿瘤具有肿瘤反应性的单抗(MAb)， 与hu14.18类似，它既可以被表示为IC，也可以存在IC。

Malignant melanoma is a serious health issue for Veterans, and metastatic melanoma is usually incurable if it metastasizes to distant sites. Exciting and impressive data with immune checkpoint blockade demonstrate the ability of the immune system to produce durable responses in some metastatic melanoma patients and have changed the standard of care1. Effective treatment with immune checkpoint blockade seems to require activation of anti-melanoma T cells that are specific for a wide variety of melanoma antigens including patient- unique neoantigens. Canine malignant melanoma provides an excellent preclinical model to study melanoma immunotherapy as it is similar to human melanoma occurring spontaneously in the setting of an intact immune system and with metastasis occurring via lymphatics or blood vessels to regional lymph nodes, lungs, liver, brain, and kidney. Germane to this application, the GD2 disialoganglioside (GD2) is expressed in both human and canine melanoma2-4. We therefore propose intratumoral (IT) injection of the GD2-reactive hu14.18-IL2 immunocytokine (IC) (IT-IC) alone, and in combination with other therapies synergistic in preclinical murine models, in companion dogs with melanoma to convert the injected tumor into an effective in situ tumor vaccine5. Our central hypothesis is that IT-IC in combination with local radiation therapy (RT) in canine melanoma can induce a T cell response to melanoma. Further, we hypothesize that this response can be amplified with immune checkpoint blockade, and that this regimen is safe and well tolerated. These hypotheses will be tested by achieving the following: Aim 1) Determine toxicity, immunogenicity, and assess antitumor activity of IT delivery of hu14.18-IL2 alone and with local RT in dogs with locally advanced or metastatic melanoma; Aim 2) Evaluate local and systemic antitumor activity following IT delivery of hu14.18- IL2 combined with RT and immune checkpoint blockade with anti-PD1 in dogs with locally advanced or metastatic melanoma; and Aim 3) Identify biomarkers to inform combination immunotherapy strategies with IT delivery of hu14.18-IL2 in dogs with locally advanced or metastatic melanoma. Standard clinical assessments of toxicity and tolerance, as well as measurement and imaging of clinically evident disease, are part of Aims 1 and 2. Serial blood samples and tumor biopsies will allow for detailed histologic and immunologic assessments in Aim 3 to determine mechanisms of antitumor activity and to determine whether histologic findings of concomitant immune tolerance seen in our murine model are also present in the dog. Exploratory lab studies will: i) evaluate alterations in the cellular milieu in the periphery and tumor before and after immunotherapy, ii) utilize novel immune monitoring to identify a candidate biomarker of response for dogs with melanoma receiving IT-IC, and iii) assess the diversity and clonality of intratumoral and peripheral blood T cell receptor repertoire. This study uses spontaneous canine melanoma to test a novel RT + IT-IC + immune checkpoint blockade regimen already proven to induce long lasting, potent and effective elimination of large local and distant established melanoma lesions in tumor-bearing mice5. The results from this canine trial could enable rapid translation into clinical testing of these concepts in human patients, including Veterans, with advanced melanoma. This approach is clinically advantageous as it involves agents/treatments that are currently available or in clinical testing, and it could be readily made available as an “off the shelf” therapy in Veterans Hospitals throughout the USA. Moreover, it personalizes treatment by targeting potent and patient- specific neoantigens. Finally, RT + IT-IC + immune checkpoint blockade could be incorporated into treatment of virtually any other malignancy, including other common malignancies in Veterans. The only requirement for this strategy is having a tumor-reactive monoclonal antibody (mAb) for the malignancy being targeted, analogous to hu14.18, that could either be formulated as an IC or for which an IC exists.