In situ thermal ablation augments antitumor efficacy of adoptive T cell therapy

Abstract

Purpose: The aim of this study is to investigate whether radiofrequency ablation (RFA) improves the efficacy of adoptive T cell immunotherapy in preclinical mouse cancer models.

Method: Mice implanted subcutaneously (sc) with syngeneic colon adenocarcinoma or melanoma were treated with sub-curative in situ RFA (90?°C, 1?min). Trafficking of T cells to lymph nodes (LN) or tumors was quantified by homing assays and intravital microscopy (IVM) after sham procedure or RFA. Expression of trafficking molecules (CCL21 and intercellular adhesion molecule-1 [ICAM-1]) on high endothelial venules (HEV) in LN and tumor vessels was evaluated by immunofluorescence microscopy. Tumor-bearing mice were pretreated with RFA to investigate the therapeutic benefit when combined with adoptive transfer of in vitro-activated tumor-specific CD8⁺ T cells.

Results: RFA increased trafficking of naïve CD8⁺ T cells to tumor-draining LN (TdLN). A corresponding increase in expression of ICAM-1 and CCL21 was detected on HEV in TdLN but not in contralateral (c)LN. IVM revealed that RFA substantially enhanced secondary firm arrest of lymphocytes selectively in HEV in TdLN. Furthermore, strong induction of ICAM-1 in tumor vessels was associated with significantly augmented trafficking of adoptively transferred in vitro-activated CD8⁺ T cells to tumors after RFA. Finally, preconditioning tumors with RFA augmented CD8⁺ T cell-mediated apoptosis of tumor targets and delayed growth of established tumors when combined with adoptive T cell transfer immunotherapy.

Conclusions: These studies suggest that in addition to its role as a palliative therapeutic modality, RFA may have clinical potential as an immune-adjuvant therapy by augmenting the efficacy of adoptive T cell therapy.     

Requirements for ICAM-1 immunogene therapy of lymphoma

Intercellular cell adhesion molecule-1 (ICAM-1) is a cell-surface glycoprotein capable of eliciting bidirectional signals that activate signalling pathways in leukocytes, endothelial, and smooth muscle cells. Gene transfer of xenogeneic ICAM-1 into EL-4 lymphomas causes complete tumor rejection; however, it is unknown whether the mechanism responsible involves the "foreignness" of the ICAM-1 transgene, bidirectional signalling events, ICAM-1-receptor interaction, or a combination of the latter. To begin to address this question, we constructed four different therapeutic expression vectors encoding full-length ICAM-1, and forms in which the N-terminal ligand-binding domains and cytoplasmic tail had been deleted. Mouse EL-4 tumors (0.5 cm in diameter), which actively suppress the immune response, were significantly inhibited in their growth following injection of expression plasmids encoding either full-length xenogenic (human) ICAM-1, or a functional cytoplasmic domain-deficient form that retains ligand-binding activity. Efficacy of ICAM-1-mediated antitumor immunity was significantly augmented by administration of the antivascular drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA), which suppressed blood supply to the tumor, leading to enhanced leukocyte infiltration, and complete tumor eradication in a gene dosage and CD8(+) T cell and NK cell-dependent fashion. Generation of potent cytotoxic T cell (CTL)-mediated antitumor immunity was reflected by ICAM-1-facilitated apoptosis of tumor cells in situ. In contrast, nonfunctional ICAM-1 lacking the N-terminal ligand-binding Ig domain failed to generate antitumor immunity, even in the presence of DMXAA. These studies demonstrate that ICAM-1-stimulated antitumor immunity can overcome tumor-mediated immunosuppression, particularly when employed in combination with an attack on the tumor vasculature. The ligand-binding domain of ICAM-1 is essential for generating antitumor immunity, whereas the cytoplasmic domain and bidirectional activation of tumor signalling pathways are not essential.     

调节性T细胞与恶性肿瘤的过继免疫治疗

过继免疫治疗利用自身免疫细胞进行体外扩增培养,然后回输到体内,它通过增强机体免疫力来对抗肿瘤,对肿瘤细胞具有靶向杀伤性作用,是最有可能彻底清除手术或放化疗后残存肿瘤细胞的治疗手段.但是,调节性T细胞(Regulatory T cells,Tregs)在多种恶性肿瘤患者外周血及微环境中普遍存在着比例上调的情况,这不仅会导致机体固有免疫的负性平衡,还会影响过继免疫治疗的疗效,使其不能发挥最大的抗肿瘤作用.通过对Tregs日渐深入的了解,人们已经研究出多种抑制及消除Tregs的治疗策略.     

Protocol for gene transduction and expansion of human T lymphocytes for clinical immunogene therapy of cancer

In preparation of a clinical phase I/II study in renal cell carcinoma (RCC) patients, we developed a clinically applicable protocol that meets good clinical practice (GCP) criteria regarding the gene transduction and expansion of primary human T lymphocytes. We previously designed a transgene that encodes a single chain (sc) FvG250 antibody chimeric receptor (ch-Rec), specific for a RCC tumor-associated antigen (TAA), and that genetically programs human T lymphocytes with RCC immune specificity. Here we describe the conditions for activation, gene transduction, and proliferation for primary human T lymphocytes to yield: (a) optimal functional expression of the transgene; (b) ch-Rec-mediated cytokine production, and (c) cytolysis of G250-TAA(POS) RCC by the T-lymphocyte transductants. Moreover, these parameters were tested at clinical scale, i.e., yielding up to 5-10 x 10(9) T-cell transductants, defined as the treatment dose according to our clinical protocol. The following parameters were, for the first time, tested in an interactive way: (1) media compositions for production of virus by the stable PG13 packaging cell; (2) T-lymphocyte activation conditions and reagents (anti-CD3 mAb; anti-CD3+anti-CD28 mAbs; and PHA); (3) kinetics of T-lymphocyte activation prior to gene transduction; (4) (i) T-lymphocyte density, and (ii) volume of virus-containing supernatant per surface unit during gene transduction; and (5) medium composition for T-lymphocyte maintenance (i) in-between gene transduction cycles, and (ii) during in vitro T-lymphocyte expansion. Critical to gene transduction of human T lymphocytes at clinical scale appeared to be the use of the fibronectin fragment CH-296 (Retronectin) as well as Lifecell) X-fold cell culture bags. In order to comply with GCP requirements, we used: (a) bovine serum-free human T-lymphocyte transduction system, i.e., media supplemented with autologous patients' plasma, and (b) a closed cell culture system for all lymphocyte processing. This clinical protocol routinely yields 30-65% scFvG250 ch-Rec(POS) T lymphocytes in both healthy donors and RCC patients.     

Enhanced antitumor effect of combination intravesical mitomycin C and bacillus Calmette-Guerin therapy in an orthotopic bladder cancer model

Intravesical immunotherapy with bacillus Calmette-Guerin (BCG) is currently the most successful adjuvant agent for the treatment and/or prophylaxis of non-muscle-invasive bladder cancer (NMIBC). However, NMIBCs recur in 60-70% of cases and 30% of these recurrent tumors present with a higher grade and more invasive properties. Patients that do not respond to intravesical BCG therapy are considered to be a challenge for urologists. Thus, novel conservative possibilities should be explored. To test the efficacy of a novel therapeutic approach, we examined the antitumor effect of combination therapy by intravesical administration of mitomycin C (MMC) plus BCG, infusing the two drugs simultaneously, in an orthotopic bladder cancer model. Intravesical BCG and MMC administration showed a dose-dependent survival (n=8 per group). The combination of MMC and BCG provided a significant survival advantage compared to the BCG-alone (p=0.035) and MMC-alone groups (p=0.040) (n=8 per group). The group with combined MMC/BCG exhibited a survival period similar to that achieved with an amount eight times higher that of BCG (n=10 per group). Ki-67 labeling index of cancer cells, showing tumor proliferation, was significantly lower in the combined group compared to the BCG-alone (p<0.05), MMC-alone (p<0.01) and control groups (p<0.01). No difference was detected between the combined group and the BCG-alone group with regard to CD3, T-cell infiltration and CD68 macrophage activity. The combined MMC/BCG treatment decreased the tumor appearance rate, improved the survival period and reduced the cellular proliferation rate in tumors compared to the BCG-alone treatment. The results suggest that the combined intravesical MMC/BCG treatment induced an enhanced antitumor effect against bladder tumors. The combined MMC/BCG treatment also showed a survival period similar to that achieved using a dose eight times higher of BCG-alone.     

A case of drug-induced interstitial pneumonia caused by S-1 and CPT-11 combination therapy for advanced colon cancer

The patient was a 77-year-old woman admitted for nausea and abdominal pain. Computed tomography (CT) revealed advanced ascending colon cancer with liver metastasis. After operation, we started combination chemotherapy of S-1 and irinotecan (CPT-11); S-1(80 mg/m2) administered orally for consecutive days followed by 14 days rest.CPT -11 (100 mg/m2) was given as a 2-hour infusion on day 1 and 15. The patient complained of high fever and subsequent dyspnea with severe hypoxemia after the first course of combination chemotherapy of S-1 and CPT-11.CT scan showed diffuse interstitial lesions with ground glass opacity on both lungs. Steroid pulse therapy with oxygen therapy remarkably improved her symptoms, and abnormal findings on CT scan also resolved. Drug lymphocyte stimulation test was positive against S-1 and negative against CPT-11. These findings were consistent with S-1-induced lung injury. Drug -induced pneumonia needs to be considered in the differential diagnosis when patients treated with S-1 and CPT-11 combination therapy present high fever and dyspnea.     

Induction of permanent regression of Friend virus (FV) leukemia by adoptive transfer of T helper and not T cytotoxic cells.

Friend virus (FV) induces a progressive erythroleukemia that can be made to permanently regress by the transfer of in vitro cultured virus-specific T cells (CTL/RFB) without any other adjunctive treatment. To determine the role of T cells in regression, CTL/RFB were enriched for specific T-cell subsets by treatment with monoclonal anti-Lyt2.2 or anti-L3T4 antibody and complement (C'). Pre-treatment of CTL/RFB with anti-Lyt2 antibody and C' did not affect permanent regression incidence, while CTL/RFB depleted of L3T4+ cells induced temporary regressions with all mice recurring. The number of splenic Lyt2+ (CD8+ equivalent) cells was constant irrespective of the leukemic status of the animals. However, the number of L3T4+ cells (CD4+ equivalent) in leukemic mice was three-fold lower than that of normal mice with regressed mice demonstrating a 30% increase in the number of L3T4+ cells compared to normals. Spleen cells from leukemic animals were also unable to produce IL-2 in response to mitogen stimulation. These results indicate that L3T4+ cells are involved in regression of erythroleukemia.     

Long-lived antitumor CD8+ lymphocytes for adoptive therapy generated using an artificial antigen-presenting cell.

PURPOSE: Antitumor lymphocytes can be generated ex vivo unencumbered by immunoregulation found in vivo. Adoptive transfer of these cells is a promising therapeutic modality that could establish long-term antitumor immunity. However, the widespread use of adoptive therapy has been hampered by the difficulty of consistently generating potent antitumor lymphocytes in a timely manner for every patient. To overcome this, we sought to establish a clinical grade culture system that can reproducibly generate antigen-specific cytotoxic T lymphocytes (CTL). EXPERIMENTAL DESIGN: We created an off-the-shelf, standardized, and renewable artificial antigen-presenting cell (aAPC) line that coexpresses HLA class I, CD54, CD58, CD80, and the dendritic cell maturation marker CD83. We tested the ability of aAPC to generate tumor antigen-specific CTL under optimal culture conditions. The number, phenotype, effector function, and in vitro longevity of generated CTL were determined. RESULTS: Stimulation of CD8(+) T cells with peptide-pulsed aAPC generated large numbers of functional CTL that recognized a variety of tumor antigens. These CTLs, which possess a phenotype consistent with in vivo persistence, survived ex vivo for prolonged periods of time. Clinical grade aAPC(33), produced under current Good Manufacturing Practices guidelines, generated sufficient numbers of CTL within a short period of time. These CTL specifically lysed a variety of melanoma tumor lines naturally expressing a target melanoma antigen. Furthermore, antitumor CTL were easily generated in all melanoma patients examined. CONCLUSIONS: With clinical grade aAPC(33) in hand, we are now poised for clinical translation of ex vivo generated antitumor CTL for adoptive cell transfer.     

Adjuvant therapy of colon cancer.

Colon cancer is an important cause of cancer-related mortality. A series of clinical trials of adjuvant systemic therapy have been performed in attempt to establish means to improve outcome in this disease. By the early 1990s, a role for 5-fluorouracil (5-FU)-based chemotherapy in stage III colon cancer had been firmly established. The precise role for chemotherapy in stage II disease remains under investigation. Progress continues toward optimizing the schedule and duration of systemic therapy, allowing for maximal efficacy with a minimum of toxicity. It appears that approximately 6 months of 5-FU and leucovorin are as effective as more prolonged regimens. Levamisole does not appear to add to the benefit of 5-FU and leucovorin. Several newer agents such as the oral fluorinated pyrimidines, irinotecan (CPT-11) and oxaliplatin have demonstrated activity in metastatic colon cancer and hold promise as potentially effective drugs to be tested in the adjuvant setting.     

肺癌患者Ⅰ类和Ⅱ类辅助T细胞的变化

目的：研究肺癌患者血浆及外周单个核细胞（ＰＢＭＣ）诱生ＩＬ－２、ＩＬ－４水平及患者Ⅰ类和Ⅱ类辅助Ｔ细胞（Ｔｈ１和Ｔｈ２）细胞功能。方法：用细胞因子诱生及ＥＬＩＳＡ技术,测定３０例肺癌术前患者、１２例术后患者血清及外周血单个核细胞诱生的ＩＬ－２、ＩＬ－４水平,以ＩＬ－２水平反映Ｔｈ１细胞功能,以ＩＬ－４水平代表Ｔｈ２细胞功能。结果：肺癌患者血清及ＰＢＭＣ诱生产生ＩＬ２水平减低,而ＩＬ－４水平升高（Ｐ     

58例进展期胃癌内介入治疗的临床分析与疗效评价

目的：观察进展期胃癌内介入治疗方案的疗效和毒副反应。方法：对58例经临床确诊的进展期胃癌患者施行内介入治疗。根据病情向患者瘤体供血动脉内灌注不同剂量的5FU、CF、PDD、MMC、ADM，加超液化碘油10ml-20ml后，同时行明胶海绵颗粒靶动脉栓塞，每间隔4—6周重复注药1次，每位患者连续治疗2次后进行总体疗效评价。结果：58例进展期胃癌患者行内介入治疗后，临床症状平均缓解率为90．20％，局部肿块完全缓解（CR）3例（5．17％），部分缓解（PR）47例（81．03％），总有效人数50例，总体有效率为86．21％。出现发热41例（70．69％），体温最高达39．8℃，上腹疼痛35例（60．34％），上述症状均为化疗后导致的瘤体局部坏死所引起，经对症处理后，1周左右均可自行缓解；58例施行内介入治疗患者中，Ⅰ度胃肠反应40例（68．97％），Ⅱ度12例（20．69％），Ⅲ度5例（8．62％）；I度骨髓抑制38例（65．51％），Ⅱ度10例（7．24％），无Ⅲ度、Ⅳ度骨髓抑制病例发生。结论：内介入方法治疗进展期胃癌，疗效肯定，毒副反应轻微，具有临床推广应用价值。     

Inhibition of HER1 signaling pathway enhances antitumor effect of endocrine therapy in breast cancer

Epidermal growth factor receptor (EGFR)/HER1 is expressed at high levels in at least 20% of breast cancers. This high expression correlates with a poor prognosis in patients with breast cancer. Experimental and clinical findings suggest that aberrant activation of tyrosine receptor kinases, such as HER1 pathway, play a causal role in the development of antiestrogen resistance in breast cancer. Recent preclinical and clinical evidence shows that inhibition of growth factor signaling pathways suppresses the growth of malignant cells without serious toxicities. To test the hypothesis that inhibition of the HER1 signaling pathway enhances the antitumor effect of endocrine therapy, a promising signal transduction inhibitor (STI) of HER1 tyrosine kinase, gefitinib, and an estrogen receptor (ER) antagonist, fulvestrant, were administered to human breast cancer cells. Our experimental results have revealed that gefitinib additively enhances the antitumor effect of fulvestrant in estrogen receptor (ER)-positive breast cancer cells under estrogen-supplemented conditions. An additive increase in the protein expression level of a cyclin-dependent kinase inhibitor, p21 may play a key role of this additive cytostatic effect. The rationale and future perspectives of the combined use of STIs with endocrine therapy in breast cancer are discussed.     

A short-term dietary supplementation of high doses of vitamin E increases T helper 1 cytokine production in patients with advanced colorectal cancer.

PURPOSE: Patients with advanced cancer exhibit multifaceted defects in their immune capacity, which are likely to contribute to an increased susceptibility to infections and disease progression and to constitute a barrier to immunotherapeutic interventions. A chronic inflammatory condition associated with increased oxidative stress has been suggested as one of the responsible mechanisms behind the tumor-induced immune suppression. We, therefore, speculated that supplementation with the antioxidant vitamin E could enhance the immune functions in patients with advanced cancer. EXPERIMENTAL DESIGN: This hypothesis was here tested in twelve patients with colorectal cancer (Dukes' C and D) who, prior to intervention with chemo- or radiotherapy, received a daily dose of 750 mg of vitamin E during a period of 2 weeks. RESULTS: Short-term supplementation with high doses of dietary vitamin E leads to increased CD4:CD8 ratios and to enhanced capacity by their T cells to produce the T helper 1 cytokines interleukin 2 and IFN-gamma. In 10 of 12 patients, an increase of 10% or more (average, 22%) in the number of T cells producing interleukin 2 was seen after 2 weeks of vitamin E supplementation, as compared with peripheral blood monocyte samples taken before treatment (P = 0.02). Interestingly, there seemed to be a more pronounced stimulatory effect by vitamin E on na?ve (CD45RA(+)) T helper cells as compared with T cells with a memory/activated phenotype. CONCLUSIONS: Dietary vitamin E may be used to improve the immune functions in patients with advanced cancer, as a supplement to more specific immune interventions.     

Enhanced antitumor effect of RGD fiber-modified adenovirus for gene therapy of oral cancer

Current clinical success rates of adenoviral vector (Adv)-based gene therapy of squamous cell carcinoma (SCC) of the head and neck remain unsatisfactory. A major problem with this approach is thought to be related to low Adv transduction efficiency due to weak expression of the adenovirus receptor, coxsackie-adenovirus receptor (CAR), in SCC. To improve the limited infectivity of Adv in oral SCC, we constructed mutated Adv incorporating the integrin-binding motif, RGD, in the HI loop of the fiber knob. The mutated Adv infected target cells through integrins commonly expressed in oral SCC. LacZ marker gene expression after infection with this mutated Adv (Adv-F/RGD) in oral SCC cell lines that showed reduced expression of CAR was approximately 5-10 times higher than that obtained with the parental Adv containing wild-type fiber knob (Adv-F/wt). In an in vitro study, transduction of oral cancer cell lines with Adv-F/RGD expressing human IL-2 (AxCAhIL2-F/RGD) resulted in greater production of cytokine than AxCAhIL2-F/wt infection. In an in vivo therapeutic xenograft model of oral SCC in nude mice, AxCAhIL2-F/RGD demonstrated antitumor effects superior to those of AxCAhIL2-F/wt. These data suggest that exploitation of genetically altered adenovirus vectors with integrin-binding motifs may offer significant improvements in oral SCC gene therapy.     

Oxaliplatin and irinotecan: From advanced to adjuvant therapy of colon cancer

Both irinotecan and oxaliplatin have demonstrated important clinical activity in metastatic colorectal cancer. Although they appear to have similar activity in metastatic disease, only oxaliplatin has demonstrated efficacy in the adjuvant setting. Despite a survival advantage of single agent irinotecan in the second-line metastatic setting and a survival advantage for irinotecan added to first-line metastatic treatment, irinotecan has failed to show a survival or disease-free survival benefit in the adjuvant setting, and at the present time there is no role for irinotecan in the adjuvant treatment of colon cancer. In contradistinction, the addition of oxaliplatin to 5-fluorouracil/leucovorin has improved disease-free survival in two large randomized adjuvant trials. Data for overall survival in these trials have not yet demonstrated statistical significance but are expected to with maturation. Oxaliplatin/5-fluorouracil/ leucovorin should, therefore, be regarded as a reference standard for adjuvant therapy.     

奥沙利铂联合卡培他滨或替吉奥对晚期结肠癌患者疗效对比

目的 探究奥沙利铂联合卡培他滨或替吉奥对晚期结肠癌患者的治疗效果.方法 选取晚期结肠癌患者112例,依据抽签法将患者分为卡培他滨组和替吉奥组,每组各56例;给予卡培他滨组患者奥沙利铂联合卡培他滨进行化疗,给予替吉奥组患者奥沙利铂联合替吉奥进行化疗.观察比较两组患者治疗前后的白细胞计数(WBC)、血小板计数(PLT)、miRNA(miR-21)水平及恶心呕吐、手足综合征(HFS)、口腔黏膜炎、肝功能异常发生率,并比较两组疗效.结果 治疗前两组患者的WBC、PLT及miR-21水平比较,差异无统计学意义(P﹥0.05);治疗后两组患者WBC、PLT及miR-21水平较本组治疗前均降低,组间比较卡培他滨组患者的PLT高于替吉奥组,差异有统计学意义(P﹤0.05);治疗过程中卡培他滨组患者HFS及恶心呕吐发生率高于替吉奥组,差异有统计学意义(P﹤0.05);两组患者的总有效率比较,差异无统计学意义(P﹥0.05).结论 奥沙利铂联合卡培他滨或替吉奥均可对晚期结肠癌患者起到明显治疗效果,两者均可作为结肠癌的临床治疗方案.