【作者】 杨敏；

【导师】 卢斌峰；

【作者基本信息】 苏州大学 ， 免疫学， 2020， 博士

【摘要】 第一部分 TIM-3在肿瘤微环境中的表达及靶向TIM-3抗肿瘤作用机制背景:近年来,肿瘤的免疫治疗取得了令人瞩目的革命性临床效果。尽管针对细胞毒性T淋巴细胞抗原4(CTLA-4)和程序性死亡受体1(PD-1)的免疫检查点阻断为策略的肿瘤免疫治疗在临床上取得了巨大的成功,但大部分癌症患者尚未从这种新型疗法中获益。如何提高免疫检查点抑制剂对肿瘤治疗的响应率是当前肿瘤治疗的重大课题。除了 PD-1和CTLA-4外,还有多种免疫调节分子在肿瘤浸润淋巴细胞(TIL)中表达。以这些分子作为靶点的免疫治疗有望成为改善现有免疫治疗的有效途径。研究发现,T细胞免疫球蛋白和粘蛋白结构-3(TIM-3)在感染,同种免疫,自身免疫,移植免疫以及肿瘤免疫小鼠模型中介导免疫耐受。之前的研究发现TIM-3在非小细胞肺癌(NSCLC)患者的调节性T细胞(Treg)中高表达,并且CD4+T细胞中TIM-3的表达比例较高,与癌症患者的临床病理参数不良相关。另外大量的数据显示TIM-3在肿瘤组织中特异性表达。因此,靶向TIM-3可能成为进一步改进当前免疫疗法的新策略。尽管大量实验数据显示TIM-3在体内具有免疫抑制功能,但具体机制尚不明确。为了有效靶向TIM-3用于肿瘤免疫治疗,需要对TIM-3介导的免疫抑制进行进一步深入的机制研究。本项研究将为其他免疫检查点阻断剂新型联合治疗的合理设计提供思路。目的:研究免疫检查点分子TIM-3在肿瘤微环境中的表达分布及其表达意义;探讨TIM-3单克隆抗体联合PD-1单克隆抗体在多种小鼠肿瘤模型中的治疗作用及作用机制;揭示肿瘤免疫治疗的耐受机制。方法:利用流式细胞术检测TIM-3在荷瘤小鼠中肿瘤浸润淋巴细胞各个亚群的表达分布;检测肿瘤浸润淋巴细胞中TIM-3+各亚群的活化、增殖水平以及免疫卡控点分子以及效应分子的表达情况;同时利用Mitotracker Deep Red试剂盒检测肿瘤中肿瘤浸润淋巴细胞亚群中的线粒体总体数量;利用Seahorse实验检测肿瘤浸润淋巴细胞亚群的耗氧效率及糖酵解水平;利用细胞毒杀伤实验检测肿瘤浸润淋巴细胞亚群的特异性杀伤作用;利用TIM-3与PD-1单克隆抗体联合治疗荷瘤小鼠,观察小鼠生长曲线,总体生存期或肺转移结节数量;利用流式细胞术检测抗体治疗后24小时或96小时的荷瘤小鼠肿瘤浸润淋巴细胞的群体分布变化及群体表型变化;根据荷瘤小鼠肿瘤浸润淋巴细胞群体表型变化提示,设计改进的肿瘤免疫治疗方案,利用TIM-3,PD-1和LAG-3单克隆抗体同时治疗荷瘤小鼠,观察小鼠生长曲线;利用流式细胞术检测三联组合抗体治疗后24小时荷瘤小鼠肿瘤浸润淋巴细胞群体的分布及表型变化;利用细胞毒杀伤实验检测抗体治疗后肿瘤浸润淋巴细胞的特异性杀伤功能。结果:我们发现TIM-3高表达于肿瘤浸润CD4+,CD8+细胞,Treg细胞,)型树突状细胞及I型肿瘤相关巨噬细胞。特别是在Treg上,TIM-3表达比例约80%。TIM-3+的CD4+及CD8+T细胞高表达CD44、OX40等T细胞活化分子及细胞增殖相关分子Ki67,低表达CD62L,IL7R等幼稚T细胞分子。研究进一步发现T细胞效应分子IFN-γ与颗粒酶B主要表达在TIM-3+T细胞群体。尽管普遍认为肿瘤中的PD-1+TIM-3+T细胞是耗竭T细胞,但从几个指标如线粒体总量,糖酵解水平来看,PD-1+TIM-3+较PD-1-TIM-3-T细胞群体是活化更高的效应细胞,并且具有更高水平的特异性杀伤作用。相比PD-1单克隆抗体单独治疗,TIM-3与PD-1单克隆抗体联合治疗能够显著抑制小鼠肠癌MC38的生长;TIM-3与PD-1单克隆抗体联合治疗能够显著延长卵巢癌荷瘤小鼠的生存期,而PD-1单克隆抗体单独治疗并不能延长荷瘤小鼠生存期。并且,联合治疗组中的无荷瘤小鼠再次接种肿瘤后,小鼠受免疫保护不再成瘤。在小鼠乳腺癌4T1模型中,TIM-3与PD-1单克隆抗体单独或联合治疗都不影响肿瘤生长速度。PD-1抗体单独治疗不能抑制肿瘤的肺转移,但是TIM-3与PD-1单克隆抗体联合治疗能够显著减少4T1肿瘤的肺部转移结节数量。通过流式细胞术分析,我们发现TIM-3与PD-1单克隆抗体联合治疗24或96小时后能够显著增强CD8+与CD4+T细胞的增殖与效应功能,包括颗粒酶A/B或IFNγ的表达比例增加。同时发现,TIM-3抗体与PD-1抗体联合治疗24或96小时后,另一个免疫抑制受体分子LAG-3显著上调。使用TIM-3,PD-1以及LAG-3单克隆抗体联合治疗后发现,三者的联合治疗能够进一步改善TIM-3与PD-1单克隆抗体联合治疗的效果,并且治疗效果的改善与CD8+T细胞表达的颗粒酶B的比例上调有关。细胞毒杀伤实验进一步验证了 TIM-3,PD-1以及LAG-3单克隆抗体联合治疗显著改善了 CD8+T细胞特异性细胞毒杀伤作用。结论:TIM-3是肿瘤中T细胞的一个活化标志,TIM-3+肿瘤浸润T细胞是效应性T细胞。在MC38小鼠肿瘤模型中,TIM-3+PD-1+群体不是耗竭T细胞群体,而是高度活化的效应性T细胞。TIM-3单克隆抗体能够联合PD-1单克隆抗体在多种肿瘤模型中显著改善PD-1抗体的免疫治疗效果,克服一些肿瘤对PD-1单克隆抗体的耐药性。肿瘤浸润T细胞的高度活化导致上调TIM-3,PD-1,LAG-3以及GITR等免疫检查点分子,这些分子相互交叉调控表达以控制肿瘤微环境中活化的T细胞功能。TIM-3,PD-1以及LAG-3单克隆抗体三者联合治疗能够进一步改善TIM-3与PD-1抗体的联合治疗效果,增强肿瘤浸润淋巴细胞的细胞毒作用。第二部分 人源化TIM-3单克隆抗体的功能作用研究背景:TIM-3被认为是抗肿瘤免疫应答的负性调控分子。TIM-3的以下几个特性使其成为改进现有免疫疗法的理想靶标。首先,TIM-3特异性高表达于肿瘤浸润T细胞,可通过靶向肿瘤浸润T细胞进行更精确的治疗,能够降低非特异性毒性。其次,TIM-3下游的信号转导与CTLA-4和PD-1的信号转导不同,使其能够与CTLA-4或PD-1抗体联合使用。第三,一些临床前数据显示,TIM-3抗体与PD-1抗体联合作用具有比PD-1抗体单独作用更好的效果。因此,单独靶向TIM-3以及联合PD-1或CTLA-4单克隆抗体的肿瘤免疫治疗具有巨大潜力。第一部分的数据显示在肿瘤微环境中TIM-3表达于大部分Treg,CD4+以及CD8+T细胞。我们也证明了在多种小鼠肿瘤模型包括结直肠癌,卵巢癌以及乳腺癌模型中,PD-1与TIM-3单克隆抗体联合治疗克服了单独使用PD-1抗体的耐药性,并且改善了 PD-1抗体的治疗疗效。TIM-3与PD-1单克隆抗体联合治疗能够显著增强CD4+与CD8+T细胞的增殖和效应功能。这些数据都说明TIM-3作为新型的肿瘤免疫治疗靶点,在临床上具有很大的潜力。亟需开发针对人TIM-3的抗体,改善现有肿瘤免疫治疗的困境。目的:开发靶向人TIM-3的单克隆抗体;筛选亲和性高并能够阻断TIM-3与其配体PtdSer结合的单克隆抗体;研究人源化抗体在体外对T细胞的功能作用;研究抗体在人源化小鼠模型中的抗肿瘤功能。方法:构建稳定表达TIM-3的细胞株,并利用流式细胞术筛选亲和性较高的杂交瘤克隆株;体外UV辐照诱导小鼠脾脏细胞凋亡,流式细胞术验证磷酰酯丝氨酸暴露于凋亡细胞表面,并且验证磷酰酯丝氨酸作为配体与TIM-3的结合能力;利用流式细胞术检测TIM-3抗体阻断TIM-3与其配体磷酰酯丝氨酸结合的能力;体外诱导T细胞表达TIM-3,并且在TIM-3抗体存在的情况下,体外重新再刺激高表达TIM-3的T细胞,ELISA法检测TIM-3抗体对T细胞分泌IFN-γ的作用;利用人源化小鼠模型,使用TIM-3单克隆抗体或PD-1单克隆抗体,或TIM-3与PD-1单克隆抗体联合或IgG对照对人HT29结直肠癌治疗,检测TIM-3单克隆抗体在人源化体系中对肿瘤治疗的功效。结果:本项研究构建了过表达人以及猴TIM-3 IgV结构域的质粒,并通过稳转筛选,流式分选得到稳定表达细胞株。利用稳定表达TIM-3 IgV结构域的细胞株,筛选出能够结合人以及猴TIM-3 IgV结构域的杂交瘤克隆株,并筛选出结合能力较强的克隆株。筛选出的克隆株经过人源化改造后,并在工业生产表达体系中表达、纯化出单克隆抗体。利用过表达TIM-3 IgV结构域的稳转细胞株,验证了工业表达体系生产的人源化抗体能够结合TIM-3 IgV结构域。同时,在体外诱导凋亡细胞,并验证了磷脂酰丝氨酸在凋亡细胞上的暴露。研究发现,筛选的抗体能够有效的阻断TIM-3融合蛋白与其配体磷脂酰丝氨酸的结合。在抗体的功能研究中发现,体外刺激活化T细胞可诱导表达TIM-3,TIM-3+CD4+T细胞比例可达48%以上,TIM-3+CD8+T细胞比例达77%以上。当在TIM-3单克隆抗体存在的条件下重新刺激这些T细胞时,筛选的TIM-3单克隆抗体candidate 1能够显著增加T细胞分泌IFN-γ的量。在TIM-3抗体candidate 1体内功能研究中,利用NSG人源化小鼠模型,回输人PBMC后接种人结直肠癌细胞株HT29。研究发现,PD-1抗体Nivolumab能够轻微减缓肿瘤生长速度。人TIM-3抗体candidate 1与Nivolumab联合治疗与Nivolumab抗体单独治疗组相比能够显著抑制肿瘤的生长速度。结论:本项研究成功筛选出与人及猴TIM-3亲和性高,并能够有效阻断TIM-3与其配体磷脂酰丝氨酸结合的单克隆抗体。此外,对其中亲和性最高的单克隆抗体ca ndidate 1进行了功能鉴定,证明了 candidate 1能够在体外增强T细胞的效应功能,并能够在体内与Nivolumab协同发挥抗肿瘤功能作用。第三部分 TIM-3在调节性T细胞上的功能作用研究背景:调节性T细胞(Treg)在机体感染和损伤期间起到维持免疫耐受,预防自身免疫及自身炎症疾病等作用。Treg是细胞免疫中至关重要的成分,是维持机体免疫应答平衡的关键因素。Foxp3缺陷型小鼠以及Foxp3等位基因缺陷的人会产生严重的全身性器官特异性自身免疫疾病和淋巴组织增生疾病。Treg在抑制抗肿瘤免疫应答与移植耐受中也发挥至关重要的作用。Treg细胞存在于正常的组织中,但是比例较低。在不同的组织中,Treg细胞表达不同的趋化因子受体与粘附分子。但是Treg细胞是如何在组织中富集并发挥功能作用的机制尚不明确。在肿瘤中,Treg细胞可占CD4+T细胞的30%-50%。研究显示肿瘤浸润Treg细胞上调多种免疫调控受体,包括CTLA-4,ICOS,TIM-3以及GITR等,以及抑制性细胞因子白介素-10(IL-10),转化生长因子β(TGFβ)等。本团队之前报道了在非小细胞肺癌患者中TIM-3在肿瘤浸润Treg细胞中高表达。最近研究进一步报道显示TIM-3+Treg 比 TIM-3-Treg抑制功能更强。除了肿瘤组织以外,TIM-3也被证实在其他一些组织中表达。这些研究提示TIM-3可能参与调控Treg细胞在组织中发挥的作用。在第一部分的研究中,我们同样证明了在肿瘤中Treg细胞特异性上调TIM-3的表达。然而,TIM-3在肿瘤Treg细胞中具体发挥何种作用以及作用机制尚不明确。目的:为了进一步阐明TIM-3在肿瘤组织Treg细胞中的作用和分子机制,利用Affymetrix人全基因组表达芯片研究人组织中TIM-3+Treg与TIM-3-Treg的基因表达差异,进一步理解TIM-3在Treg中表达的意义;构建Treg细胞条件型敲除TIM-3小鼠;进一步检测TIM-3在Treg细胞中缺失后对肿瘤生长的影响,并研究TIM-3对Treg功能的调控机制。方法:从人头颈鳞状细胞癌组织中分选出TIM-3+Treg细胞与TIM-3-Treg细胞,并使用Affymetrix Human Transcriptome Array 2.0基因芯片进行基因表达差异分析,系统地研究两种Treg细胞亚群的基因表达差异;利用CRISPR技术构建Treg细胞条件型敲除TIM-3小鼠,并检测确认TIM-3在Treg中特异性敲除;接种免疫原性较高的肠癌MC38肿瘤细胞与免疫原性较低的黑色素瘤B16肿瘤细胞,观察TIM-3在Treg细胞中敲除后对肿瘤生长的影响;流式分析肿瘤微环境中肿瘤浸润淋巴细胞的分布及表型特征的变化;体外分选肿瘤组织中的Treg细胞,并建立微型Treg抑制实验检测TIM-3敲除后对Treg抑制功能的影响。结果:通过流式细胞术分选出人头颈鳞状细胞癌组织中的TIM-3+Treg细胞与TIM-3-Treg 细胞,利用 Affymetrix Human Transcriptome Array 2.0 基因芯片进行基因表达分析,结果发现TIM-3+Treg细胞与TIM-3-Treg细胞有大量差异表达的基因,并且TIM-3+Treg细胞高表达效应T细胞标志的分子,TIM-3-Treg细胞高表达幼稚T细胞标志分子。我们进一步构建了 TIM-3+/-与TIM-3f/+小鼠,并通过与Foxp3YPF-Cre小鼠杂交后最终获得TIM-3+/-Foxp3Cre与TIM-3f/-Foxp3Cre基因型小鼠用于研究TIM-3在Treg细胞上的功能。通过流式细胞术检测肿瘤组织中Treg上TIM-3的表达,确认了TIM-3f/-Foxp3Cre小鼠在Treg细胞中特异性敲除TIM-3。研究发现免疫原性较强的MC38肿瘤在TIM-3f/-Foxp3Cre小鼠中的生长速度比TIM-3+/-Foxp3Cre对照小鼠显著迟缓,但B16小鼠肿瘤的生长并没有受到TIM-3在Treg细胞中缺失的影响。通过流式细胞术分析肿瘤微环境中的淋巴细胞分布及表型,我们发现TIM-3在Treg细胞中缺失后,肿瘤浸润免疫细胞增加,Th1和CD8+T细胞介导的免疫应答增强。此外,通过体外建立微型Treg抑制实验,发现来自TIM-3f/-Foxp3Cre荷瘤小鼠肿瘤组织中的Treg细胞抑制应答细胞增殖能力降低。这些结果说明TIM-3对于肿瘤组织Treg的免疫抑制功能是必需的。结论:人的癌症组织样本基因芯片表达显示TIM-3+Treg细胞是效应Treg细胞。条件型敲除TIM-3后,Treg抑制功能降低。在肿瘤模型中,TIM-3在Treg细胞中的缺失导致肿瘤免疫抑制的降低,免疫原性较高的肿瘤细胞生长得到控制。TIM-3在Treg细胞中的缺失造成肿瘤浸润淋巴细胞增多,Th1和CD8+T细胞介导的免疫应答增强。因此,TIM-3对Treg的抑制功能非常重要,特异性靶向Treg中的TIM-3能够极大程度提高肿瘤免疫治疗的疗效。更多还原

【Abstract】 The First Part:The Expression of TIM-3 in Tumor Microenvironment and the Anti-Tumor Mechanisms of Targeting TIM-3Background:Recently,tumor immunotherapy has achieved remarkable clinical results.Although the immune checkpoint blockade strategy for cytotoxic T lymphocyte antigen 4(CTLA-4)and programmed death 1(PD-1)has shown great success,the majority of cancer patients have not yet benefit from this new therapy.In addition to PD-1 and CTLA-4,a variety of immunomodulatory molecules are expressed in tumor infiltrating lymphocytes(TIL).T cell immunoglobulin mucin domain 3(TIM-3)has shown to mediate immune tolerance in mouse models of infection,alloimmunity,autoimmunity,and tumor immunity.Previous studies found that TIM-3 is highly expressed on regulatory T cells(Tregs)in patients with non-small cell lung cancer(NSCLC),and that TIM-3 expression on CD4+T cells is associated with poor clinical outcome in cancer patients.Therefore,targeting TIM-3 is a new strategy to further improve current immunotherapy.Although a large number of experimental data showed that TIM-3 has immunosuppressive function in vivo,the specific mechanism is still unclear.In order to effectively target TIM-3 for tumor immunotherapy,further in-depth research of mechanisms is needed.These studies will provide ideas for the rational design of new combination therapies for other immune checkpoint blockades.Objective:To study the expression of TIM-3 in the tumor microenvironment and its significance;the therapeutic effect and mechanisms of TIM-3 monoclonal antibody combined with PD-1 monoclonal antibody in various mouse tumor models;the mechanism of tolerance in immunotherapyMethods:Flow cytometry was performed to determine the expression of TIM-3 in tumor infiltrating immune cells in tumor-bearing mice.The activation,proliferation,and effector molecules of TIM-3+subpopulations in tumor infiltrating lymphocytes were determined.The total number of mitochondria mass in TIM-3+or TIM-3" subpopulations of tumor infiltrating lymphocyte was determined using the Mitotracker Deep Red kit.The cytoxicity of TIM-3+or TIM-3-CD8+subsets were examined by cytolytic assay.Treat tumor-bearing mice with TIM-3 monoclonal antibody in combination with PD-1 monoclonal antibody and the growth curve or the overall survival or the number of lung metastatic nodules were observed.The population distribution and phenotypic changes of tumor infiltrating lymphocytes were detected by flow cytometry at 24 hours or 96 hours after antibodies treatments.According to the phenotypic changes of tumor-infiltrating lymphocytes in tumor-bearing mice,an improved tumor immunotherapy protocol was designed.The TIM-3.PD-1 and LAG-3 monoclonal antibodies were designed to treat tumor-bearing mice simultaneously,and the growth curves of mice were observed.Flow cytometry and cytolytic assay were performed to detect the distribution and phenotypic changes of tumor infiltrating lymphocyte in tumor-bearing mice after treatment with triple-combined antibodies.Results:We found that TIM-3 is highly expressed in tumor infiltrating CD4+,CD8+,Treg,type 1 dendritic cells and type 1 tumor associated macrophages.Especially on Treg,the percentage of TIM-3+subset is about 80%.TIM-3+subpopulations in CD4+and CD8+T cells highly express activation molecules CD44,OX40 and cell proliferation-related molecule Ki67,but express low levels of naive molecules such as CD62L,IL7R.In addition,the TIM-3+conventional CD4+and CD8+TILs express higer levels of IFN-y and granzyme B when compared to TIM-3-TIL.Although PD-1+TIM-3+TILs have been described as exhausted T cells,PD-1+TIM-3+TILs have slightly higher number of mitochondrial mass compared to PD-1-TIM-3-TILs.Seahorse assay showed that PD-1+TIM-3+TILs have similar level of oxygen comsuption rate but higer extracellular acidification rate compared to PD-1-TIM-3-TILs,indicating PD-1+TIM-3+TILs are more activated.Cytolytic assay also showed that PD-1+TIM-3+TILs are more potent effector T cells compared to PD-1-TIM-3-subset.Compared with PD-1 monoclonal antibody treatment alone,TIM-3 mAb combined with PD-1 mAb significantly inhibited the tumor growth in mouse intestinal cancer MC38 model.TIM-3 mAb combined with PD-1 mAb could significantly prolong the overall survival of tumor bearing mice in ID8 model,but the treatment with PD-1 mAb alone did not have any effect.In the mouse breast cancer model 4T1,neither treatment of PD-1 mAbs or TIM-3 mAbs alone or their combination did affect tumor growth rate.Treatment with PD-1 mAb alone did not reduce the lung metastasis in this model.In contrast,TIM-3 mAb combined with PD-1 mAb could significantly reduce the number of lung metastatic nodules.With flow cytometry analysis,we found that TIM-3 mAb combined with PD-1 mAb significantly enhanced the proliferation and effector function of CD4+and CD8+T cells at 24 hours or 96 hours.At the same time,we found that another immunoinhibitory receptor molecule,LAG-3,was significantly up-regulated 24 or 96 hours after TIM-3 and PD-1 mAbs combination therapy.Treatment with the combination of TIM-3.PD-1 and LAG-3 mAbs could further improve the efficacy of immunotherapy and the improved therapeutic effect was due to the increase of the cytolytic function and the percentage of granzyme B expressed by CD8+T cells.ConclusionTIM-3 is an activation marker of tumor infiltrating T cells.TIM-3 is a hallmark of activated T cells in our syngeneic transplant tumor model.The TIM-3+PD-1+ population is not an exhausted but a functional T cell population.The TIM-3 mAb can be combined with the PD-1 mAb to significantly improve the immunotherapeutic efficacy of the PD-1 mAb alone in multiple tumor models,overcoming the resistance to PD-1 mAb therapy in some tumors.TIM-3,PD-1,LAG-3,GITR and other immunoregulatory receptors expressed on T cells are cross-regulated to control hyperactivated T cells in the tumor microenvironment.The combination of TIM-3,PD-1 and LAG-3 mAbs can further improve the therapeutic effect of TIM-3 and PD-1 mAbs combination.The Second Part:Functional Study of a Humanized TIM-3 Monoclonal AntibodyBackground:TIM-3 has been considered to be a negative regulator of anti-tumor immune responses.Several features of TIM-3 make it an ideal target for next generation immunotherapy.First,the selective expression of TIM-3 on T cells in tumors makes it more precisely treated by targeting tumor infiltrating T cells,which can reduce non-specific toxicity.In addition,downstream signal of TIM-3 is quite different from that of CTLA-4 and PD-1.thus these molecules can be targeted simutaneously.Third,several preclinical studies have shown that TIM-3 and PD-1 mAbs have additive antitumor activities.Thus,specific expression and intracellular signal transduction make it great target for TIM-3 blockade therapy or in combination with current PD-1 and CTLA-4 based tumor immunotherapies.Our previous data indicated that TIM-3 is expressed on tumor infiltrating CD4+and CD8+T cells in multiple types of tumors.We have also demonstrated that anti-PD-1 and anti-TIM-3 combination therapy overcomes the resistance of PD-1 mAb alone in a variety of mouse tumor models including colorectal cancer,ovarian cancer and breast cancer models.Treatment of TIM-3 and PD-1 mAbs combination significantly enhanced the effector function of CD8+T cells.These data indicated that TIM-3 has great potential for clinical application as a new target for tumor immunotherapy.There is an urgent need to develop antibodies against human TIM-3 to improve the predicament of existing tumor immunotherapy.Objective:To develop monoclonal antibodies targeting human TIM-3;to screen for monoclonal antibodies with high affinity and which can block the binding of TIM-3 to its ligand;to study the effect of humanized antibodies on T cells in vitro;to study the anti-tumor function of antibodies in humanized systemsMethods:We constructed cell lines stably expressing human or monkey TIM-3 IgV domain.These cell lines were used to screen for hybridoma clones that express high affinity TIM-3 antibodies by Flow cytometry.We also established in vitro functional assays for TIM-3 mAbs.The first is the phosphatidylserine(PtdSer)binding assay.We induced apoptotic mouse splenocytes to expose PtdSer by UV irradiation in vitro and confirmed the exposure of the PtdSer on cell surface by flow cytometry.We then used flow cytometry to determine the blocking ability of TIM-3 antibody on the binding of TIM-3 to its ligand PtdSer.We also established a T cell functional assay for TIM-3.We stimulated T cells to induce expression of TIM-3 in vitro.We then re-stimulated the TIM-3-expressing T cells in the presence or absence of TIM-3 antibody.The cytokines were measured by ELISA.To evaluate the antitumor function of the anti-human TIM-3 monoclonal antibody in humanized systems using a humanized mouse model of human colorectal cancer,four experimental groups were used i.e.control IgG group,the anti-human TIM-3 monoclonal antibody alone group,the anti-human PD-1 monoclonal antibody alone group or the anti-human TIM-3 combined with anti-human PD-1 monoclonal antibodies group.Tumor growth was recorded,and growth curves were compared.Results:We successfully constructed cell lines that overexpress human or monkey TIM-3 IgV domains,and by several rounds of fluorescence activated cell sorting(FACS)we obtained stable cell lines.The hybridoma clones that are capable of binding human or monkey TIM-3 IgV domains were screened by stable TIM-3 IgV domain expressing cell lines,and clones with strongest binding ability were selected for further development.The variable region of genes encoding immunoglobin heavy and light chains of the selected hybridoma clones were cloned and humanized and then expressed in a commercial expression system.At the same time,we induced apoptotic cells in vitro and verified the exposure of phosphatidylserine on the surface of apoptotic cells.We found that the antibody we screened was able to effectively block the binding of the TIM-3 fusion protein to its ligand phosphatidylserine.In the functional study of antibody,we found that in vitro stimulation of T cells can induce the expression of TIM-3,the proportion of CD4+TIM-3+T cells could reach more than 48%,and the proportion of CD8+TIM-3+T cells was above 77%.When these T cells were re-stimulated,we found that our anti-human TIM-3 monoclonal antibody candidate 1 significantly increased the expression of IFN-y secreted by T cells.In the in vivo functional study of candidate 1,we used the humanized mouse model NSG to inoculate human colorectal cancer cell line HT29 after adoptively transferring of human PBMC.We found that anti-PD-1 antibody Nivolumab was able to slow down tumor growth slightly.Compared with Nivolumab treatmemt group,the combination of candidate 1 and Nivolumab significantly inhibited tumor growth.Conclusion:We have successfully screened a monoclonal antibody that has high affinity and is effective in blocking the binding of TIM-3 to its ligand phosphatidylserine.We perfomed a functional assay on the most sensitive monoclonal antibody,candidate 1,which demonstrated that candidate 1 can enhance the effector function of T cells in vitro and synergizes with Nivolumab to exert more potent anti-tumor function in vivo.The Third Part:Study of the function of TIM-3 on regulatory T cellsBackground:Regulatory T(Treg)cells play an important role in maintaining immune tolerance and preventing autoimmunity,autoinflammatory diseases and immunopathology during tissue infection and injury.Treg cell is a critical component in cellular immunity and is a key factor in maintaining the proper balance of immune responses.Foxp3-deficient mice and humans who bear defective Foxp3 alleles develop severe systemic and organ-specific autoimmune diseases and lymphoproliferative disorders.Treg cell also plays a crucial role in inhibiting anti-tumor immune responses and maintaining transplant tolerance.Treg cells also exist in healthy tissues,but at a lower number.In different tissues,Treg cells express different chemokine receptors and adhesion molecules.But the mechanism of how Treg cells are enriched and maitained in the tissues is not clear.In tumor tissues,Treg cells can account for 30%to 50%of CD4+T cells.Studies have shown that tumor infiltrating Treg cells upregulate a variety of immune regulatory receptors such as CTLA-4,ICOS,TIM-3,GITR,etc.,as well as the inhibitory cytokines such as interleukin-10(IL-10),transforming growth factor beta(TGFβ),etc.Previous report showed that TIM-3 is specifically upregulated on tumor-infiltrating Treg cells but not on Treg cell in peripheral blood in non-small cell lung cancer patients.Recently,there have been reports that TIM-3+Treg cells have a stronger suppression function.In the first part of my study,we also further confirmed that tumoral Treg specifically up-regulated the expression of TIM-3 in our mouse models.In addition,TIM-3 was also confirmed to be expressed in tissue Treg.However,the role of TIM-3 in Treg that mediates tumor immune tolerance is not clear.Objective:In order to further understand the molecular mechanisms underlying TIM-3+Treg cell function,we will perform expression profiling studies comparing TIM-3+Treg and TIM-3-Treg isolated from head and neck squamous cell carcinoma.We will generate Treg-specific TIM-3 deficient mice.We will then determine the effect of TIM-3 deletion specifically in Treg on tumor growth.We will further study the role of TIM-3 in the function of tumor infiltrating Treg cells.Methods:To study the molecular basis of TIM-3+Treg cells,we sorted TIM-3=Treg and TIM-3-Treg cells from human head and neck squamous cell carcinoma by FACS,and we used Affymetrix Human Transcriptome Array 2.0 Gene chip to perform global gene expression analysis to examined the differentially expressed genes between two Treg subsets.Then we generated one allele of TIM-3 floxed mice(TIM-3f/+)and one allele of TIM-3 knockout mice(TIM3+/-)by CRISPR technology.We then crossed TIM-3f/+,TIM3+/-and Foxp3YPF-Cre mice to generate TIM-3+/-Foxp3Cre and TIM-3f/-Foxp3Cre mice.We confirmed Treg-specific TIM-3 deletion by flow cytometry.We inoculated TIM-3f/-Foxp3Cre mice and TIM-3+/-Foxp3Cre control mice with immunogenic intestinal cancer MC38 tumor cells or melanoma B16 tumor cells which are of poor immunogenicity.We subsequently monitored tumor growth in these mice.We also analyzed the phenotypic changes of tumor infiltrating lymphocytes in tumor microenvironment by flow cytometry.We sorted Treg cells from tumor tissues ex vivo by FACS and set up Treg micro suppression assay to study the effect of TIM-3 deletion on Treg suppression function.Results:We FACS purified TIM-3+Treg and TIM-3-Treg cells from human head and neck squamous cell carcinoma.We then performed gene expression analysis by using Affymetrix Human Transcriptome Array 2.0 gene chip.TIM-3+Treg and TIM-3-Treg cells have a large number of differentially expressed genes.TIM-3+Treg cells highly express genes that are also found upregulated in the effector T cells,and TIM-3-Treg cells highly express naive T cell markers.We then generated TIM-3+/-and TIM-3f/-+mice and by breeding with Foxp3YPF-Cre mice,we finally obtained TIM-3+/-Foxp3Cre and TIM-3f/-Foxp3Cre genotypes of mice.These mice were used to study the function of TIM-3 on Treg.The expression of TIM-3 on Treg in tumor tissues was detected by flow cytornetry,and its absence on tumor Treg cells was confirmed in TIM-3f/-Foxp3Cre mice.By examining the growth of mouse tumors in TIM-3f/-Foxp3Cre mice,we confirmed that TIM-3 deficiency in Treg resulted in inhibited tumor growth in immunogenic MC38 tumor.However,TIM-3 deletion in Treg cells did not affect the progression of tumors in B16 model.By analyzing the phenotypic changes on tumor infiltrating immune cells by flow cytometry,we found that after TIM-3 was deleted in Treg,tumor infiltrating immune cells were increased.In addition,Th1 immune responses were also increased.By establishing a Treg microsuppression assay in vitro,we found that Treg cells from TIM-3f/-Foxp3Cre tumor-beraing mice had a decreased suppressive function,indicating that TIM-3 is required for the suppression function of tumoral Treg cells.Conclusion:Gene chip expression profiling of human tumoral Treg cells showed that TIM-3+Treg cells have many characteristics of effector T cells,therefore are effector Treg cells suppression function of Treg is dependent on TIM-3.In MC38 tumor model,the loss of TIM-3 expression in Treg led to a decrease in immune suppression in the tumor microenvironment and inhibited tumor growth.The loss of TIM-3 expression in Treg also resulted in an increase of the percentage of total lymphocytes in the tumor.Thus,TIM-3 is very important for the suppression function of Treg,and specific targeting of TIM-3 in Treg can greatly improve the efficacy of tumor 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