~较高病死率的恶性肿瘤严重威胁人类健康，亟待开发更加有效的治疗手段。近年来，肿瘤免疫疗法异军突起，针对CD19的嵌合抗原受体(CAR)-T细胞在以B细胞淋巴瘤为代表的血液系统肿瘤治疗方面取得了重大突破。然而，该疗法在实体瘤治疗中的尝试结果却不尽如人意。究其原因，主要与下列三个因素相关：首先，实体瘤抗原异质性突出，不同肿瘤缺乏一个像CD19一样的共同特异性靶点；其次，实体瘤存在一个免疫抑制性肿瘤微环境，输入体内的CAR-T细胞在这个微环境中的活性受到抑制或被耗竭；最后，相对处于血液系统中的B淋巴瘤细胞而言，处于组织中的实体瘤细胞受CAR-T等免疫细胞浸润和接触的难度较大。
我们实验室前期的研究结果表明，γδT细胞受体(TCR)能以与抗体类似的方式直接识别应激诱导的完整肿瘤相关性蛋白抗原，无主要组织相容性复合体（MHC）限制性，且γδTCR的互补决定区(CDR)3δ是抗原结合与识别的关键部位。与抗体不同的是，肿瘤特异性CDR3δ广谱结合和识别多种肿瘤相关抗原。因此，与传统CAR-T细胞所使用的scFv结构相比，单链γδTCRV/V结构的抗原识别广谱性在靶向实体瘤异质性抗原时具有明显的优势。白细胞介素7（IL-7）在维持T细胞生存、促进T细胞增殖、刺激活化的T细胞从效应型向记忆型转化以及延长记忆型T细胞的存活时间等多方面发挥重要作用，而且临床应用副作用少，患者耐受性好。CC类趋化因子配体19（CC-chemokine Ligand 19, CCL19）能够与多种免疫细胞和多种肿瘤组织表达的CC类趋化因子受体7（CCR7）结合，具有增强效应T细胞在实体瘤内浸润和招募更多免疫细胞进入肿瘤组织的作用。
基于上述考虑，本研究综合利用γδTCRV/V、IL-7和CCL19在实体瘤异质性抗原识别、免疫抑制性肿瘤微环境内T细胞的活性维持和免疫细胞在实体瘤组织中浸润等方面的优势，在传统CAR-T细胞的基础上，构建针对实体瘤的/CAR-IL-7-CCL19-T细胞，并在体内外检测其抗肿瘤效应，以期为临床实体瘤的CAR-T细胞治疗提供一种新型策略。
首先，本研究构建和制备CAR-IL-7-CCL19-T细胞。我们先构建了CAR-IL-7-CCL19慢病毒载体。通过同源重组的方法，将本实验室前期鉴定的两种具有肿瘤结合特异性的CDR3序列：OT3(V2)和GTM(V1)，分别插入到完整的V9/V2和V4/V1单链结构中，并替换原有第三代CAR分子(scFv-CD8a-CD28-CD137-CD3Zeta)的胞外单链抗体部分(scFv)，构建9/2(OT3)CAR和4/1(GTM)CAR；通过PCR克隆完整的人IL-7和CCL19的cDNA序列，并通过P2A序列将其依次串联在CAR分子的下游；在CCL19分子的下游通过IRES序列串联红色荧光报告基因mCherry分子，用来检测感染效率。上述两种慢病毒载体pLVX-2(OT3)CAR-IL7-CCL19-IRES-mCherry (简称:OT37X19)和pLVX-1(GTM)CAR-IL7-CCL19-IRES-mCherry (简称:GTM7X19) 经菌液PCR、酶切和测序鉴定，确认构建成功。接着，我们进行了慢病毒包装和CAR、IL-7及CCL19表达鉴定及体外细胞毒活性测定。通过质粒转染293T细胞可获得滴度达108的慢病毒；用病毒感染T细胞的MOI值为10，平均感染效率为15.6%；CAR-IL-7-CCL19-T细胞培养至4天时平均扩增7.0倍；ELISA结果显示细胞培养上清中IL-7的浓度为398pg/mL，CCL19为4757pg/mL；LDH法检测细胞毒活性结果显示，OT37X19-T细胞和GTM7X19-T细胞分别对9种和8种实体瘤细胞系具有显著性杀伤活性。综上所述，慢病毒感染T细胞所制备的CAR-IL-7-CCL19-T细胞在体外能够分泌IL-7和CCL19，并且能够通过其表达的CAR识别多种不同组织来源的实体瘤细胞系，并对其发挥显著的细胞毒作用。
进一步，我们分选了CD8+T细胞进行基因修饰，构建了CAR-IL-7-CCL19-CD8+T细胞，并比较其与病毒修饰T细胞所得到的CAR-IL-7-CCL19-T细胞及天然T细胞的细胞毒活性差异。实验结果表明， OT37X19-CD8+T细胞对HepG2的杀伤效率显著高于OT37X19-T细胞(p<0.0001)；GTM7X19-CD8+T细胞对BGC803和LoVo的杀伤效率显著高于GTM7X19-T (p<0.0001)；GTM7X19-CD8+T细胞对A549和LoVo的杀伤效率显著高于天然T细胞(p<0.05)。上述结果表明，在感染效率没有显著差异的条件下，慢病毒修饰CD8+T细胞得到的CAR-IL-7-CCL19-CD8+T细胞对某些肿瘤的细胞毒活性优于修饰T所得到的CAR-IL-7-CCL19-T细胞，并且对某些肿瘤的细胞毒活性优于天然γδT细胞。进一步，我们对CAR-IL-7-CCL19-T细胞敏感的肿瘤细胞表面分子进行了分析。流式细胞术检测结果显示，肿瘤细胞系SKOV3、OVCAR-8、A549和HepG2细胞表面都有TCR识别性配体ULBP5的高水平表达，而SKOV3 和HepG2细胞还高表达另一TCR识别性配体MSH2。我们分析了抗原识别结合与细胞毒活性的关系。结果显示，对CAR-IL-7-CCL19-T细胞杀伤敏感的肿瘤细胞系都与CDR3肽OT3和GTM有不同程度的结合，并且5种肿瘤细胞HepG2、OVCAR-8、A549、BGC803和HR8348与GTM的结合强弱与其对GTM7X19-T细胞的杀伤敏感程度高低呈正相关(p=0.0120, r=0.9553)。上述结果表明，CAR-IL-7-CCL19-T细胞主要依靠肿瘤特异性V/V结合和识别实体瘤相关抗原，高表达已知γδTCR应激配体的实体瘤细胞对CAR-IL-7-CCL19-T细胞的细胞毒活性敏感。
最后，我们选择了体外细胞毒活性实验中对OT37X19-T细胞和GTM7X19-T细胞的杀伤活性都敏感的卵巢癌细胞系OVCAR-8进行体内抑瘤实验。结果显示，与Vector-T阴性对照组相比， OT37X19-T细胞和GTM7X19-T细胞治疗组小鼠体内的瘤体细胞平均荧光信号强度在首次治疗开始后的第12天都显著性减小（**：p=0.0039, ***：p=0.0002）。但是从首次治疗开始后的第18天开始，OT37X19-T细胞和GTM7X19-T细胞两个治疗组的小鼠出现了严重不良反应并陆续死亡。病理检查结果显示，发生不良反应小鼠的肝脏和肺脏等脏器出现了不同程度的炎症和水肿；冰冻组织切片苏木素-伊红（HE）染色显示，发生不良反应小鼠的肝脏和肺脏中浸润了大量淋巴细胞。这些结果表明，OT37X19-T细胞和GTM7X19-T细胞在体内都能够有效抑制卵巢癌细胞系OVCAR-8的生长，但是可能发生了急性移植物抗宿主反应（GVHD），或是存在这些细胞在脏器中非特异性聚集和/或剂量不当等问题。为此，我们已经在此次体内实验的基础上制订了新的体内给药方案（包括调整剂量和用药次数），以期在新的体内抑瘤实验中对此次实验所出现的严重不良反应现象予以充分澄清，并对OT37X19-T细胞和GTM7X19-T细胞体内抑制卵巢癌细胞系OVCAR-8的有效性和安全性剂量范围进行详细界定。
综上所述，利用γδTCR对肿瘤相关抗原识别的广谱特征，又结合IL-7和CCL19在T细胞生存、归巢和趋化等方面所具备的有利于CAR-T细胞发挥肿瘤免疫治疗功能的特点，本研究首次构建了以单链V/V直接识别实体瘤相关抗原，并自分泌IL-7和CCL19的CAR-IL-7-CCL19-T细胞。体外实验结果显示，该细胞具备较广泛的实体瘤杀伤活性，并且对某些高表达γδTCR配体的实体瘤细胞系的杀伤活性优于天然T细胞。体内抑瘤实验表明，CAR-IL-7-CCL19-T细胞能够有效抑制卵巢癌细胞系OVCAR-8的生长。本研究成果综合考虑γδT细胞的特性和细胞因子、趋化因子的功能，为实体瘤的CAR-T细胞治疗提供了一种新型策略。
 

~Malignant tumors with high mortality are a serious threat to human health, and more effective treatment methods should be developed urgently. In recent years, tumor immunotherapy has gained new prominence, and chimeric antigen receptor (CAR)-T cells targeting CD19 have made a major breakthrough in the treatment of blood system tumors represented by B-cell lymphoma. However, attempts in treating solid tumors have been less than satisfactory. The reasons are related to the following three main factors: First, antigen heterogeneity is prominent in solid tumor, different tumors lack a common specific target like CD19; Secondly, the activity of CAR-T cells was inhibited or depleted in immunosuppressive tumor microenvironment of solid tumor. Finally, unlike B lymphoma cells in blood flowing system, solid tumors build many barriers and blockades for CAR-T cells to infiltrate into tumor tissues and contact tumor cells.
Our previous studies have shown that T cell receptor (TCR) can directly recognize intact tumor-associated protein antigens induced by stress in a similar way to antibodies, and complementary determinant region (CDR)3 in TCR is the key site for antigen binding and recognition. Unlike antibodies, tumor-specific CDR3 can bind to and recognize a wide spectrum of tumor-related antigens. Therefore, compared with the scFv structure used in traditional CAR-T cells, the single-stranded TCRV/V structure has the advantage of effectively recognizing a wide spectrum of heterogeneous antigens in solid tumor tissue. Human IL-7 plays an important role in maintaining the survival of T cells, promoting the proliferation of T cells, stimulating the transformation of activated T cells from effector type to memory type, and prolonging the survival time of memory T cells. Moreover, it has few clinical side effects and is well tolerated by patients. Chemokine CCL19 can enhance the infiltration of effector T cells in solid tumors and chemotaxis many kinds of other immune cells into tumor tissues by binding to the receptor CCR7 expressed in a variety of immune cells and a variety of tumor tissues.
Based on the above consideration, this present study comprehensively utilized the advantage of γδTCRV/V, IL-7 and CCL19 in recognition of heterogeneous solid tumor antigens, T cells activity maintenance within tumor microenvironment and infiltration of immune cells into tumor tissues, to build a novel type of genetically engineered /CAR-IL-7-CCL19-T for solid tumor cells on the basis of the traditional CAR-T cells, and detected in vivo and in vitro its anti-tumor effect, with an aim to provide a neotype strategy for clinical CAR-T cells treatment of solid tumor.
Firstly, we constructed and prepared /CAR-IL-7-CCL19-T cell. At first, we constructed /CAR-IL-7-CCL19 lentivirus vectors. With homologous recombination technology, OT3(V2) and GTM(V1), two kinds of selected CDR3 sequences with specific tumor-binding capability were used in replacing the corresponding parts of CDR3 sequences in single chain of V9/V2 and V4/V1, and then the region of scFv of the 3rd generation CAR (scFv-CD8a-CD28-CD137-CD3Zeta) was substituted by V9/V2(OT3) or V4/V1(GTM) to generate neotype CARs. Whole cDNA sequences of human IL-7 and CCL19 were cloned by PCR and connected in tandem with P2A sequences to the downstream of CAR molecule. A red fluorescent reporter gene mCherry was connected to CCL19 with IRES to detect transfection efficacy. Two kinds of plasmids 9/2(OT3)-IL-7-CCL19(OT37X19 in brief) and 4/1(GTM)-IL-7-CCL19(GTM7X19 in brief) were constructed successfully after having been identified with such methods as enzyme digestion and sequencing.  Then we carried out lentivirus packaging and identified the expression of /CAR, IL-7 and CCL19, and ex vivo cytotoxicity experiment. Lentiviruses with a titer of 108 were obtained by transfection of 293T cells with plasmids, and the normal expressions of /CAR, IL-7 and CCL19 were confirmed by WB and ELISA. We then transfected with lentivirus. The MOI value was 10 and the average infection efficiency was 15.6%. By cell numerating, we found  cells increased by an average of 7 folds 4 days after lentivirus transfection. ELISA analysis showed that IL-7 was 398pg/mL and CCL19 was 4757pg/mL in supernatant of transfected cells. The LDH assay showed that OT37X19- cells and GTM7X19- cells had significant killing activity against 9 and 8 tumor cells respectively. The above results indicated that /CAR-IL-7-CCL19-T cells prepared by transfecting  with lentivirus can secrete IL-7 and CCL19 in vitro, and can recognize a variety of solid tumor cells derived from different tissues through their expression of CAR, and exert a significant cytotoxic effect on them.
Secondly, we sorted CD8+T cells and prepared /CAR-IL-7-CCL19-CD8+T through transfecting CD8+T cells with lentivirus, and compared the ex vivo cytotoxicity of /CAR-IL-7-CCL19-T, /CAR-IL-7-CCL19-CD8+T cells and natural T against different solid tumors. It was found that the killing efficacy of OT37X19-CD8+ cells on HepG2 was significantly higher than that of OT37X19-T cells (p<0.0001). The killing efficacy of GTM7X19-CD8+ cells against BGC803 and LoVo was significantly higher than that of GTM7X19-T cells (p<0.0001). The above results showed that under the condition of similar transfection efficiency, CD8+ cell had stronger cytotoxicity against some tumors than T cell after lentivirus modification, and was even better than natural γδT cells. Then we analyzed the characteristics of tumor cell lines which were sensitive to cytotoxicity of /CAR-IL-7-CCL19-T cell ex vivo. FCM detection showed that expression of ULBP5 (ligand recognized by TCR) was up-regulated on the surfaces of A549, HepG2, SKOV3 and OVCAR-8, and MSH2 (another ligand recognized by TCR) were also highly expressed on surfaces of HepG2 and SKOV3. Then we analyzed the relationship between antigen recognition and cytotoxicity. The results showed that the tumor cell lines which were sensitive to cytotoxicity of /CAR-IL-7-CCL19-T cells could all be bound by CDR3 peptide OT3 or GTM in different degrees, and the percentage of five kinds of tumor cells HepG2, OVCAR-8, A549, BGC803 and HR8348 bound by GTM was positively correlated with their sensitivity to the cytotoxicity of GTM7X19-T cells (p=0.0120, r=0.9553). The above results indicated that /CAR-IL-7-CCL19-T cells mainly rely on tumor-specific V/V structure in recognition and binding of solid tumor related antigens, and solid tumor cells with high expression of known γδTCR stress ligands are sensitive to the cytotoxicity of /CAR-IL-7-CCL19-T cells.
Finally, we selected OVCAR-8, an ovarian cancer cell line sensitive to both the cytotoxicity of OT37x19-T cells and GTM7X19-T cells ex vivo, to perform tumor inhibition experiment in vivo. The results showed that under comparison with the Vector-T negative control group, the mean fluorescence signal intensity of tumor cells in OT37X19-T cells and GTM7X19-T cells treatment group decreased significantly on the 12th day after the first treatment (**: P =0.0039, ***: P =0.0002). However, from the 18th day on post the first treatment, mice in the two treatment groups, OT37X19-T cells and GTM7X19-T cells, began to develop severe adverse reactions and died successively. Pathological examination showed that different degree of inflammation and edema had occurred in the liver and lung of the mice which had developed adverse reactions. Hematoxylin-Eosin (HE) staining of frozen tissue sections showed that the liver and lung of the mice were infiltrated with a large number of lymphocytes. These results suggest that both OT37X19-T cells and GTM7X19-T cells can effectively inhibit the growth of ovarian cancer cell line OVCAR-8 in vivo, but the mice might have developed acute GVHD, and there may be some problems of non-specific aggregation of CAR-T cells into the organs and/or improper dosage administration. Therefore, based on this experiment in vivo, we have formulated a new administration plan for the next experiment in vivo (including adjustment of dosage and frequency), with an aim to fully clarify the serious adverse reactions in this experiment in vivo, and define the dose range of effectiveness and safety in detail for OT37X19-T cells and GTM7X19-T cells in inhibiting the growth of OVCAR-8.
To sum up, by utilizing the unique nature of T cells capable of sensing a wide spectrum of tumor associated antigens, together with the special characters of IL-7 and CCL19 being favorable of T cell survival, homing and chemotaxis to benefit CAR-T to function ideally in solid tumor immunotherapy, we for the first time prepared a new type of CAR-IL-7-CCL19-T cell, which can express CAR and auto-secret human IL-7 and CCL19. Experiments ex vivo revealed that this CAR-T cell possessed cytotoxicity on a wide spectrum of solid tumors and exerted higher antitumor activity than natural T cells in killing certain solid tumors highly expressing ligands of TCR. Tumor suppressive experiment in vivo showed that CAR-IL-7-CCL19-T cell could effectively inhibit the growth of solid tumor OVCAR-8, an ovarian cancer cell line. With a comprehensive consideration to nature of γδT cell, function of cytokine and chemokine, this present study provides a new strategy for the research of solid tumor immunotherapy based on CAR-T cells.