Immunophysiological Mechanisms in the Biological Therapy of Cancer

癌症免疫治疗新方法的成功开发需要理解复杂的、相互依赖的早期先天反应元素的活动，以及随后强大的适应性免疫反应。我们正在采用几种新方法来最大限度地提高宿主产生有效抗肿瘤反应的能力。这些方法包括通过连接CD40来优化抗原呈递能力，CD40是一种TNF超家族受体，可作为树突状细胞和巨噬细胞的有效触发因子，在先天和适应性反应之间提供关键接口。当与IL-2或IL-15联合使用时，激动剂CD40抗体刺激树突状细胞的效力增强，并且抗CD40激动剂与IL-2或IL-15联合使用对小鼠转移性肾癌的抗肿瘤活性增强。我们分析了发生在肾脏、肺和肝脏的原发肿瘤中所含的白细胞，它们分别是肿瘤转移的原发和继发部位。在用IL-2/抗cd40或IL-15/抗cd40治疗荷瘤小鼠后，我们发现巨噬细胞和T细胞的募集似乎是抗肿瘤反应的关键介质。一旦出现在发展中的肿瘤部位，这些细胞能够产生许多不同的可溶性介质，如干扰素γ (IFNg)、一氧化氮和VEGF，它们可能影响肿瘤的进展。通过调节这些细胞类型在肿瘤内积累的能力，我们正在确定这些细胞在原发性肿瘤进展和转移到远处器官中所起的作用。我们的数据表明，单独抗cd40与与IL-2联合使用介导的生物学效应存在巨大的机制差异，包括IFNg和控制肿瘤负荷的一氧化氮表达的协同上调。迄今为止，我们已经证明IL-2/抗cd40诱导增强的抗肿瘤反应依赖于效应CD8+ T细胞浸润已建立的肿瘤，以及伴随的ifng依赖性降低CD4+/FoxP3+调节性T细胞（Tregs）、髓源性抑制细胞（MDSC）和肿瘤微环境中Th2趋化因子的表达。这些结果可能有助于解释抗cd40作为单一药物的有限临床疗效，因为它不能特异性地从肿瘤微环境中去除Tregs和MDSC，它们表明抗cd40与其他选定的免疫药物（如IL-2）联合使用可能更有益。我们正在进行的工作是研究肿瘤微环境中调节性T细胞和MDSC选择性损失的机制。我们的数据表明，IL-2/抗cd40诱导Fas在这些细胞群上的表达，以及表达CD8+ T细胞和其他白细胞的Fas配体浸润原发肿瘤。这一过程引起fas依赖性的Tregs和MDSC细胞死亡或凋亡。我们的数据表明，fas介导的Treg和MDSC去除对IL-2/抗CD40联合治疗的抗肿瘤效果至关重要。此外，他们建议免疫治疗策略，如IL-2/抗CD40，靶向Tregs和MDSC对fas介导的细胞死亡的易感性，有望进一步发展为癌症治疗策略。我们当前和未来的研究旨在阐明对观察到的aCD40生物学效应至关重要的细胞和分子事件，以及抗cd40与合理选择的分子靶向药物互补使用的潜力。在一种这样的方法中，我们将抗cd40与阿斯利康开发的ATP竞争性mTOR药物AZD8055结合起来。这一策略的基本原理有两个方面。首先，mTOR抑制是目前RCC的主要临床靶点。我们还假设AZD8055与aCD40抗体联合使用可以通过直接杀死肿瘤并随后将肿瘤相关抗原释放到抗原提呈细胞和同步调节体内免疫细胞功能来诱导更有效的抗肿瘤作用。我们最近发表的研究结果表明，在同基因小鼠转移性肾细胞癌（RCC）模型中，AZD8055和aCD40通过激活巨噬细胞和dc，并在肿瘤微环境中诱导强烈的Th1免疫反应，协同肿瘤消退。我们还将IL-2与IL-12联合使用的早期临床前研究结果转化为人体临床试验。我们已经在NIH临床研究中心完成了IL-12/pulse IL-2的I期临床试验。Jon Wigginton和John Janik（现在在BMS工作）。IL-2在第1天和第9天每8小时给药一次，IL-12在每35天周期的第2、4、6、10、12和14天每天静脉给药一次。确定最大耐受剂量为600,000 IU/kg IL-2和300 ng/kg IL-12。大多数患者出现预期的临床副作用，如发热、寒战、头痛、疲劳、缺氧、低血压、肌痛、恶心、呕吐和口炎、中性粒细胞减少和淋巴细胞减少。毒性通常在每个治疗周期结束后迅速消退。与随后的剂量相比，第一次IL-12给药后ifn - γ的产生达到峰值。有趣的是，在治疗第二周注射IL-12后，IL-2部分恢复了ifn - γ的产生，IL-18水平和IL-12受体（CD212Beta1）在IL-12/脉冲IL-2治疗后均增加。许多患者个体肿瘤病变消退，2例RCC患者实现了客观的部分缓解。虽然IL-2部分克服了IL-12引起的快速反应和一些病变的消退，但总的客观有效率并没有比IL-12或IL-2作为单一药物提高。这种效应的一种可能解释是，外源性全身细胞因子管理无法达到肿瘤微环境中重建局部免疫成分所需的水平，这一假设构成了我们目前使用IL-2与抗CD40联合使用的部分基础，在其在肿瘤微环境中的多种作用中，诱导CD40阳性白细胞产生IL-12。

Successful development of new approaches for the immunotherapy of cancer requires an understanding of complex, interdependent activities of early innate response elements with subsequent powerful adaptive immune responses. We are taking several novel approaches to maximize the host's ability to mount an effective antitumor response. These approaches include optimizing antigen presenting capability through the ligation of CD40, a TNF superfamily receptor that serves as a potent trigger for dendritic cells and macrophages, which provide a key interface between innate and adaptive responses. The potency of dendritic cell stimulation by agonist CD40 antibodies is enhanced when used in conjunction with IL-2 or IL-15 and the combination of agonist anti-CD40 plus either IL-2 or IL-15 shows enhanced antitumor activity against metastatic kidney cancer in mice. We have analyzed leukocytes contained within the primary tumors that develop in the kidney as well as those in the lungs and livers, which serve as primary and secondary sites of tumor metastases, respectively. After treating tumor-bearing mice with IL-2/anti-CD40 or IL-15/anti-CD40, we have identified the recruitment of macrophages and T cells that appear to be critical mediators of anti-tumor responses. Once present at the developing tumor site, these cells are capable of producing many different soluble mediators, such as interferon gamma (IFNg), nitric oxide, and VEGF that may influence tumor progression. By regulating the ability of these cell types to accumulate within tumors, we are identifying the role that these cells play during both primary tumor progression and metastasis to distant organs. Our data have illustrated the potential for dramatic mechanistic differences in biological effects mediated by anti-CD40 alone versus its use in combination with IL-2 that includes the synergistic upregulation of IFNg and nitric oxide expression that controls tumor burden. To date, we have shown IL-2/anti-CD40 induces enhanced antitumor responses that depend on the infiltration of established tumors by effector CD8+ T cells and a concomitant IFNg-dependent reduction in CD4+/FoxP3+ regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSC) and Th2 chemokine expression within the tumor-microenvironment. These results may help to explain the limited clinical efficacy of anti-CD40 as a single agent based on its inability to remove Tregs and MDSC specifically from within the tumor microenvironment and they suggest that anti-CD40 may be more beneficial in combination with other selected immune agents, such as IL-2. Our ongoing work is investigating the mechanisms underlying the selective loss of regulatory T cells and MDSC within the tumor microenvironment. Our data indicates that IL-2/anti-CD40 induces the expression of Fas on these cell populations, as well as the infiltration of primary tumors by Fas ligand expressing CD8+ T cells and other leukocytes. This process elicits Fas-dependent cell death, or apoptosis, of Tregs and MDSC. Our data demonstrates the critical importance of Fas-mediated Treg and MDSC removal towards the anti-tumor efficacy of IL-2/anti CD40 combination therapy. Furthermore, they suggest that immunotherapeutic strategies, such as IL-2/anti CD40, that target the susceptibility of Tregs and MDSC to Fas-mediated cell death hold promise for further development as cancer treatment strategies. Our current and future studies seek to clarify the cellular and molecular events critical for the observed biological effects of aCD40, and the potential for complementary use of anti-CD40 with rationally selected molecularly targeted agents. In one such approach, we have combined anti-CD40 with an ATP competitive mTOR drug, AZD8055, developed by AstraZeneca. The rationale for this strategy is two-fold. First, mTOR inhibition represents is currently a leading clinical target for RCC. We also hypothesized that combining AZD8055 with aCD40 antibody would induce more efficient antitumor effects by a combination of direct tumor killing and subsequent release of tumor-associated antigens to antigen presenting cells and coincident modulation of immune cell functions in vivo. The results of our recently published study show that in a syngeneic mouse metastatic renal cell carcinoma (RCC) model, AZD8055 and aCD40 synergize for tumor regression by activating macrophages and DCs and inducing strong Th1 immune responses in the tumor microenvironment. We have also translated our earlier preclinical findings using IL-2 in combination with IL-12 to human clinical trials. We have completed a phase I clinical trial of IL-12/pulse IL-2 at the NIH Clinical Research Center via collaboration with Drs. Jon Wigginton and John Janik (now at BMS). IL-2 was administered every 8 hours on days 1 and 9 and intravenous IL-12 was administered once daily on days 2, 4, 6, 10, 12 and 14 of each 35 day cycle of therapy. The maximum tolerated dose was determined to be 600,000 IU/kg IL-2 and 300 ng/kg IL-12. Anticipated clinical side effects of fever, chills, headache, fatigue, hypoxia, hypotension, myalgias, nausea, vomiting and stomatitis, neutropenia and lymphopenia were seen in most patients. Toxicities generally resolved promptly upon completion of each cycle of therapy. IFN-gamma production peaked after the first IL-12 administration in comparison with subsequent doses. Interestingly, IL-2 partially rescued IFN-gamma production following IL-12 injection in the second week of therapy, and IL-18 levels and IL-12 receptor (CD212Beta1) both increased following IL-12/pulse IL-2 therapy. Regression of individual tumor lesions was seen in many patients, and two patients with RCC achieved objective partial responses. Although IL-2 partially overcame IL-12 induced tachyphylaxis and induced regression of some lesions, the overall objective response rate was not improved over IL-12 or IL-2 as single agents. One possible explanation for this effect could be the inability of exogenous systemic cytokine administration to achieve levels needed in the tumor microenvironment to re-structure local immune components, and this hypothesis formed part of the basis for our current use of IL-2 in combination with anti-CD40 which, among its diverse effects in the tumor microenvironment, induces IL-12 production by CD40 positive leukocytes.