K562工程细胞联合IL-2扩增方案对人NK细胞扩增和活化的效果

目的：评价新建立的K562工程细胞联合IL-2扩增方案对人NK细胞扩增和活化的效果。 方法：采集健康志愿者和肿瘤患者的外周血PBMC并分离NK细胞,采用前期构建的K562工程细胞(将IL-15、4-1BBL和IL-18在白血病K562细胞上进行跨膜表达获取）联合IL-2培养方案对NK细胞进行扩增和活化,以流式细胞术检测NK细胞的扩增效果和NK细胞表面受体表达水平,CCK-8法检测扩增后NK细胞对肿瘤细胞的杀伤活性和ADCC活性,CCK-8法检测在培养方案扩增末期加入TKD多肽对NK细胞的活化效果。结果：对于健康志愿者的NK细胞,新建立扩增培养方案可使NK细胞在PBMC中的比例提高至（93±3）%;使NK细胞中活化性受体NKG2D、CD94、NKp30、NKp44和NKp46的比例分别提高60%、40%、20%、40%和63%,而抑制性受体的表达变化不大;扩增后NK细胞对白血病细胞K562、肺癌细胞A549、肝癌细胞SMMU-7721和乳腺癌细胞MCF-7的杀伤活性分别提高了19%、29%、26%和28%,其ADCC活性从（33±5.6）%上升至（65±12）%;方案中增加TKD可使NK细胞的杀伤活性从（86±4）%提高至（96±2）%。对于肿瘤患者的NK细胞,新扩增方案使其在PBMC的比例提高至（90.0±8.0）%,其对K562细胞的杀伤活性提高了17%左右。 结论： K562工程细胞联合IL-2扩增方案可高效扩增NK细胞,明显激活其杀伤活性,扩增和活化的NK细胞可满足临床治疗的需要。     

Designing antibodies for oncology

Summary The past five years have witnessed the emergence of monoclonal antibodies as important therapeutics for cancer treatment. Lower toxicity for antibodies versus small molecules, the potential for increased efficacy by conjugation to radioisotopes and cellular toxins, or the ability to exploit immune cell functions have led to clinical performances on par or superior to conventional drug therapies. This review outlines the various immunoglobulin design strategies currently available, techniques used to reduce Ig antigenicity and toxicity, and points to consider during the manufacture of antibodies for use in clinical oncology.     

Novel anti-EPHA2 antibody,DS-8895a for cancer treatment

Overexpression of EPHA2 has been observed in multiple cancers and reported to be associated with poor prognosis. Here, we produced an afucosylated humanized anti-EPHA2 monoclonal antibody (mAb), DS-8895a for cancer treatment. The antibody recognizes the extracellular juxtamembrane region of EPHA2 and therefore can bind to both full-length and truncated forms of EPHA2, which are anchored to cell membranes and recently reported to be produced by post-translational cleavage in tumors. DS-8895a exhibited markedly increased antibody dependent cellular cytotoxicity (ADCC) in vitro and also inhibited tumor growth in EPHA2-positive human breast cancer MDA-MB-231 and human gastric cancer SNU-16 xenograft mouse models. Moreover, DS-8895a in combination with cisplatin (CDDP) showed better efficacy than each of the monotherapies did in the human gastric cancer model. These results suggest that a novel antibody, DS-8895a has therapeutic potential against EPHA2-expressing tumors.     

Pharmacogenomics of EGFR-targeted therapies in non-small cell lung cancer:EGFR and beyond

Commonly observed aberrations in epidermal growth factor receptor (EGFR) signaling have led to the development of EGFR-targeted therapies for various cancers, including non–small cell lung cancer (NSCL...     

MD2 blockage prevents the migration and invasion of hepatocellular carcinoma cells via inhibition of the EGFR signaling pathway

BackgroundThe toll-like receptor (TLR) is an emerging signaling pathway in tumor invasion and metastasis. The activation of TLRs requires specific accessory proteins, such as the small secreted glycoprotein myeloid differentiation protein 2 (MD2), which contributes to ligand responsiveness. However, the role of MD2 in tumorigenesis and metastasis has rarely been reported. This study aimed to investigate the effects and underlying mechanisms of MD2 on the proliferation, migration, and invasion of hepatocellular carcinoma (HCC).MethodsCell counting kit 8 (CCK8), cell colony formation, wound healing, and transwell assays were conducted to determine cell viability, proliferation, migration, and invasion, respectively. Quantitative real-time PCR (qRT-PCR) was performed to assess the expression of MD2 in HCC cell lines and human normal liver cell lines as well as the silencing efficiency of MD2 blockage. Western blot and qRT-PCR assays were performed to detect the protein and mRNA expression levels of epithelial mesenchymal transformation (EMT) markers and epidermal growth factor receptor (EGFR) signaling molecules.ResultsMD2 was highly expressed in HCC tissues and cell lines. High expression of MD2 was associated with poor prognosis of HCC patients. In addition, MD2 silencing slightly inhibited the proliferation of HepG2 and HCCLM3, and significantly suppressed cell migration and invasion. Furthermore, MD2 blockage could distinctly prevent the EMT process by increasing the protein and mRNA levels of E-cadherin and Occludin, and decreasing the levels of Vimentin, N-cadherin, and Snail. Finally, the phosphorylation level of EGFR as well as its downstream molecular Src, Akt, I-κBα, and p65 were downregulated in HCC cells with MD2 silencing.ConclusionsOur findings suggest that high expression of MD2 may affect the EMT, migration, and invasion via modulation of the EGFR pathway in HCC cells.     

Neoadjuvant chemo-immunotherapy modifies CD4CD25 regulatory T cells (Treg) in non-small cell lung cancer (NSCLC) patients

Background

Regulatory T cells (Treg) are critical for cancer immune evasion; whereas natural killer (NK) cells are central for effective anti-tumor immunity including antibody-induced cellular cytotoxicity (ADCC). The predictive role of Treg levels for clinical response to chemo-immunotherapy in non-small cell lung cancer (NSCLC) as well as therapy-induced Treg changes remain to be defined.

Patients and methods

The impact of Treg on NK-mediated cetuximab-dependent cellular cytoxicity was tested in vitro. Frequency and functional activity of Treg was analyzed in 31 NSCLC stage IB-IIIA patients treated by neoadjuvant Cetuximab/Docetaxel/Cisplatin prior to surgery. Data were correlated with clinical outcome variables and Treg tumor infiltration.

Results

Treg potently inhibit NK-mediated and cetuximab-induced ADCC in vitro. In addition, a significant correlation between Treg reduction and clinical response was seen. However, the grade of tumor infiltrating Treg in resected tumors did not correlate with peripheral Treg levels. Moreover, Treg levels at diagnosis did not predict clinical response to chemo-immunotherapy.

Conclusions

The drop of Treg levels during neoadjuvant chemo-immunotherapy in NSCLC patients significantly correlates with clinical response. However, Treg at diagnosis are not linked to inferior clinical response to chemo-immunotherapy in NSCLC in vivo even though Treg efficiently inhibit ADCC in vitro.     

Increase of CIK cell efficacy by upregulating cell surface MICA and inhibition of NKG2D ligand shedding in multiple myeloma

Multiple myeloma, which is a monoclonal plasma cell malignancy, still remains incurable despite recent progress in our understanding of this disorder. Adoptive immunotherapy of multiple myeloma using cytokine‐induced killer cells is yielding promising results in clinical trials; however, some myeloma cells still evade immune surveillance by various unknown molecular mechanisms. This study aims at increasing the efficacy of cytokine‐induced killer cells in targeting this tumor, using selective small‐molecule inhibitors which increase and stabilize surface expression of the natural killer group 2, member D ligand, major histocompatibility complex class I polypeptide‐related sequence A (MICA) on myeloma cells. We treated 2 multiple myeloma cell lines—U266 and KMS‐12‐PE—with 3 drugs. One of these drugs (sodium butyrate) is a histone deacetylase inhibitor. Another drug which was used (matrix metalloproteinase inhibitor III) blocks ligand shedding while the third drug (phenylarsine oxide) obstructs surface ligand internalization. The effect of these drugs on cell viability was determined using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay, surface ligand expression was examined using flow cytometry, and ligand shedding was assessed using enzyme‐linked immunosorbent assay. We demonstrated that cytokine‐induced killer cells have increased cytotoxicity against multiple myeloma cells after combined drug treatment than without drug pretreatment. We also established that this increased cytotoxicity was due to potent upregulation and stabilization of surface MICA on the surface of these tumor cell lines. Our study thus highlights further therapeutic options which could be used for the treatment of multiple myeloma patients.     

EGFR inhibitors augment antitumour helper T-cell responses of HER family-specific immunotherapy

Background:

Head and neck squamous cell carcinoma (HNSCC) is a major cause of cancer-related morbidity and mortality worldwide. Epidermal growth factor receptor (EGFR)-targeted therapy is an attractive strategy alternative to conventional cancer treatments for HNSCC, but its efficacy remains controversial. T-cell-based immunotherapy has been proposed as a novel therapeutic approach to improve the clinical outcome for HNSCC. In this study, we report human epidermal receptor (HER) family epitopes that induced CD4 T-cell responses to HNSCC. The results provide support for a novel strategy to treat HNSCC by combining EGFR-targeted therapy with T-cell-based immunotherapy.

Methods:

We evaluated the capacity of predicted CD4 T-cell peptide epitopes from EGFR to induce antitumour immune responses in vitro. In addition, EGFR inhibitors were evaluated for their ability to augment tumour MHC class II expression in HNSCC cell lines and subsequently increase T-cell recognition.

Results:

Among several predicted peptide epitopes, EGFR_875–889 elicited CD4 T-cell responses that were restricted by HLA-DR4, DR15, or DR53 molecules, indicating that the peptide functions as a promiscuous T-cell epitope. The peptide-reactive T cells responded to autologous dendritic cells loaded with EGFR-expressing tumour cell lysates, indicating that these epitopes are naturally processed. In addition, the CD4 T cells were capable of directly recognising and killing HNSCC cells expressing EGFR and the appropriate HLA class II molecule. T cells reactive with the EGFR_875–889 epitope could be detected in the blood of HNSCC patients. EGFR_875–889-reactive CD4 T cells were also able to recognise several peptide analogues derived from homologous regions of EGFR family members, HER-2, HER-3 and c-MET. Finally, we examined the effects of EGFR tyrosine kinase inhibition or EGFR-blocking antibodies on CD4 T-cell tumour reactivity. Treatment of tumour cells with the EGFR inhibitors enhanced tumour recognition by EGFR_875–889-reactive T cells presumably due to the upregulation of HLA-DR expression in the HNSCC cells.

Conclusion:

We identified novel CD4 T-cell EGFR epitopes and amongst these, EGFR_875–889 functions as a promiscuous helper T-cell epitope that can elicit effective antitumour T-cell responses against tumours expressing HER family members and c-MET. These observations should facilitate the translation of T-cell-based immunotherapy into the clinic for the treatment of HNSCC and provide a rational basis for EGFR inhibition, immune-targeted combination therapy.     

Immunology and immunotherapy of neuroblastoma

Purpose

This review demonstrates the importance of immunobiology and immunotherapy research for understanding and treating neuroblastoma.

Principal results

The first suggestions of immune system–neuroblastoma interactions came from in vitro experiments showing that lymphocytes from patients were cytotoxic for their own tumor cells and from evaluations of tumors from patients that showed infiltrations of immune system cells. With the development of monoclonal antibody (mAb) technology, a number of mAbs were generated against neuroblastoma cells lines and were used to define tumor associated antigens. Disialoganglioside (GD2) is one such antigen that is highly expressed by virtually all neuroblastoma cells and so is a useful target for both identification and treatment of tumor cells with mAbs. Preclinical research using in vitro and transplantable tumor models of neuroblastoma has demonstrated that cytotoxic T lymphocytes (CTLs) can specifically recognize and kill tumor cells as a result of vaccination or of genetic engineering that endows them with chimeric antigen receptors. However, CTL based clinical trials have not progressed beyond pilot and phase I studies. In contrast, anti-GD2 mAbs have been extensively studied and modified in pre-clinical experiments and have progressed from phase I through phase III clinical trials. Thus, the one proven beneficial immunotherapy for patients with high-risk neuroblastoma uses a chimeric anti-GD2 mAb combined with IL-2 and GM-CSF to treat patients after they have received intensive cyto-reductive chemotherapy, irradiation, and surgery. Ongoing pre-clinical and clinical research emphasizes vaccine, adoptive cell therapy, and mAb strategies. Recently it was shown that the neuroblastoma microenvironment is immunosuppressive and tumor growth promoting, and strategies to overcome this are being developed to enhance anti-tumor immunotherapy.

Conclusions

Our understanding of the immunobiology of neuroblastoma has increased immensely over the past 40 years, and clinical translation has shown that mAb based immunotherapy can contribute to improving treatment for high-risk patients. Continued immunobiology and pre-clinical therapeutic research will be translated into even more effective immunotherapeutic strategies that will be integrated with new cytotoxic drug and irradiation therapies to improve survival and quality of life for patients with high-risk neuroblastoma.     

Toll-like receptor agonists and invariant natural killer T-cells enhance antibody-dependent cell-mediated cytotoxicity (ADCC)

huHMFG-1 (AS1402) is a humanised IgG1 against MUC1, which exerts tumour cell killing through antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells. Here, we explored the capacity of invariant NKT (iNKT) cells, which are known to activate NK cells, and toll-like receptor (TLR) ligands which activate both iNKT and NK cells, to enhance huHMFG-1-ADCC. Addition of iNKT cells, as well as TLR2/6, 7, 8 and 9 agonists to PBMC improved the efficacy of huHMFG-1. These results suggest that transfer of ex vivo expanded iNKT cells or TLR agonist treatment may improve the efficacy of NK cell-mediated antibody-based tumour immunotherapies.     

Phase I Trial of a Third Generation EGFR Mutant-Selective Inhibitor (D-0316) in Patients with Advanced Non-Small Cell Lung Cancer

BackgroundD-0316 is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) developed for patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) with EGFR T790M mutation that progressed after prior treatment with the first- or second-generation EGFR-TKI.MethodsThis phase I, open-label, multicenter clinical trial evaluated daily oral D-0316 administration in dose-escalation (25 to 150 mg; 17 patients) and dose-expansion (50, 100 mg; 67 patients) cohorts for safety, tolerability, anti-tumor activity, and pharmacokinetics.ResultsD-0316 was well tolerated at daily doses of 25 to 150 mg and the maximum tolerated dose (MTD) was not reached. The most common treatment-related adverse events (AEs) were platelet count decreased, electrocardiogram QT corrected interval prolonged, anemia, rash, low white blood cell count, hypertriglyceridemia, high cholesterol, headache, pruritus, cough, and aspartate transaminase (AST) or alanine transaminase (ALT) increased. Most of AEs were grade 1 or 2. In the 50 and 100 mg group, the overall response rate (ORR) was 33.3% and 45.5%, the disease control rate (DCR) was 86.7% and 93.9%, and the median PFS was 8.3 and 9.6 months, respectively. D-0316 exposure increased in proportion to dose from 25 to 150 mg. The recommended phase II dose (RP2D) was 100 mg.ConclusionD-0316 is safe, tolerable, and effective for patients with locally advanced/metastatic NSCLC with the EGFR T790M mutation who previously received EGFR-TKI.ClinicalTrials.gov Identifier{"type":"clinical-trial","attrs":{"text":"NCT03452150","term_id":"NCT03452150"}}NCT03452150.     

PHA-ICC, ADCC and NK in patients with ANLL in CR: human fibroblastic interferon fails to increase NK-active cell frequency

PHA-ICC, ADCC and NK activity of PBL were studied in ten patients with ANLL in CR and in eighteen normal controls in the presence and absence of HFIF. No statistically significant differences were recorded among the two groups with regard to basic lymphocyte functions. Although the parameters of lymphocyte function remained analogous for those tested, the analysis at the single cell level revealed that HFIF stimulation increases the number of NK active cells and target binding cells among normals, but not in leukemic patients.     

Current developments in immunotherapy in the treatment of multiple myeloma

Multiple myeloma (MM) is the second most common hematologic malignancy and represents approximately 10% of all hematological neoplasms. Standard therapy consists of induction therapy followed by high‐dose chemotherapy and autologous stem cell transplantation (ASCT) or, if ASCT cannot be performed, standard doublet, triplet, or quadruplet, novel agent–containing induction treatment until progression. Although MM is still regarded as mostly incurable by current standards, the development of several novel compounds, combination therapies, and immunotherapy approaches has raised great hopes about transforming MM into an indolent, chronic disease and possibly achieving a cure for individual patients. Several new inhibitory and immunological agents have been approved or are under intensive investigation and may lead to new therapeutic options for patients with relapsed/refractory MM, for patients ineligible for ASCT, and for patients after ASCT. Especially in the field of immunotherapy, including monoclonal antibodies, checkpoint inhibition, and chimeric antigen receptor T cells, current advances are rapid and highly promising. This review aims to summarize the newest and most promising immunotherapeutic agents for MM, their clinical efficacy, their adverse event (AE) profiles, and the ways in which these AEs can best be overcome or avoided. Cancer 2018;124:2075‐85 . © 2018 American Cancer Society.     

Mature dendritic cells enriched in regulatory molecules may control regulatory T cells and the prognosis of head and neck cancer

We previously reported that regulatory T (Treg) cells expressing CTLA-4 on the cell surface are abundant in head and neck squamous cell carcinoma (HNSCC). The role of expanded Treg cells in the tumor microenvironment of HNSCC remains unclear. In this study, we reveal that the tumor microenvironment of HNSCC is characterized by the high expression of genes related to Treg cells, dendritic cells (DCs), and interleukin (IL)-17-related molecules. Increased expression of IL17A, IL17F, or IL23A contributes to a favorable prognosis of HNSCC. In the tumor microenvironment of HNSCC, IL23A and IL12B are expressed in mature dendritic cells enriched in regulatory molecules (mregDCs). The mregDCs in HNSCC are a migratory and mature phenotype; their signature genes strongly correlate with Treg signature genes in HNSCC. We also observed that IL17A was highly expressed in Th17 cells and exhausted CD8⁺ T cells in HNSCC. These data suggest that mregDCs in HNSCC may contribute to the prognosis by balancing Treg cells and effector T cells that produce IL-17. Targeting mregDCs may be a novel strategy for developing new immune therapies against HNSCC.     

Kinetics of the early recruitment of leukocyte subsets at the sites of tumor cells in the lungs: natural killer (NK) cells rapidly attract monocytes but not lymphocytes in the surveillance of micrometastasis

Early host defense mechanisms play a critical role for the outcome of metastatic disease but most of the initial steps of such responses against tumor cells are still unknown. Here, the specificity and kinetics of leukocyte subsets in response to intravenous inoculation of vital dye labeled Fischer 344 rat syngeneic MADB106 tumor cells were monitored in lungs in situ by immunohistochemistry and image analysis over a time-period of 6 hr. In comparison with sham injections, tumor cell inoculation induces a dynamic sequence of rapidly increasing granulocyte (+40% at 5 min), NK and T cell (+60% at 15 min) as well as monocyte (+100% at 30 min) numbers in lung tissue. Already within the first minutes frequent colocalizations of granulocytes and NK cells with tumor targets were found in situ. Within the first hour NK cells selectively kill tumor targets, because depletion of NK cells in vivo drastically increases both the number of MADB106 cells retained in lungs and the emerging numbers of lung tumor colonies. In addition, the tumor-cell-induced increase of monocytes strictly depends on the presence of NK cells because NK-depletion completely abrogates the time specific response of monocytes. Under NK depleted conditions the tumor-induced recruitment of CD4(+) T cells is more pronounced suggesting a compensatory mechanism. In contrast, B cell numbers progressively decrease within hours after cell inoculation. These findings demonstrate that NK and T cells mediate the initial steps in the surveillance of lung metastasis. NK cells rapidly kill tumor cells and subsequently recruit monocytes in vivo.     

Host-dependent variables: The missing link to personalized medicine

Individualized medicine has the potential to tailor anticancer therapy with the best response and highest safety margin to provide better patient care. However, modern targeted therapies are still being tested through clinical trials comparing preselected patient cohorts and assessed upon behaviour of group averages. Clinically manifesting malignant disease requires identification of host- and tumour-dependent variables such as biological characteristics of the tumour and its microenvironment including immune response features, and overall capacity of the host to receive, tolerate and efficiently utilize treatment. Contemporary medical oncology including clinical trial design need to refocus from assessing group averages to individuality taking into consideration time dependent host-associated characteristics and reinventing outliers to be appreciated as naturally occurring variables collectively determining the ultimate outcome of malignant disease.     

Chimeric antigen receptor transduced T cells: Tuning up for the next generation

Chimeric antigen receptor (CAR) T cell therapy has recently achieved impressive clinical outcome in patients with CD19‐positive hematologic malignancies. Extrapolation of CAR T cell treatment to solid tumors, however, has not yet yielded similar results. This might be due to intrinsic causes, e.g. insufficient CAR T cell activation or CAR toxicity as well as extrinsic factors displaying an unfavorable tumor environment for CAR T cells by raising physical and chemical barriers. In this review, we discuss the advantages as well as major obstacles of CAR T cell therapy, particularly in the context of solid tumors, and focus on efforts and novel strategies in CAR T cell development.     

Leukemia of T-helper lymphocytes: Clinical and functional features

Leukemia of T-lymphocytes is rare. Chronic and subacute forms of T-cell leukemia have been described including Sézary cell leukemia, T-cell leukemia of Japan, and rare cases of chronic lymphocytic leukemia, prolymphocytic leukemia and hairy cell leukemia. Several studies have analyzed the functional capacity of lymphocytes from patients with T-cell leukemias. In many cases Sézary cells appear to be “helper” T-cells which cooperate with B-lymphocytes in immunoglobulin synthesis. In contrast, lymphocytes from the Japanese form of T-cell leukemia have been reported to function as suppressor cells. We describe an unusual leukemia in a 40-year old man, whose clinical course was characterized by fever, jaundice, hepatosplenomegaly, anemia and neutropenia. Lymph node enlargement and skin infiltration were notably absent. The leukemic cells were atypical mononuclear cells with a high nuclear-cytoplasmic ratio and convoluted nuclei. Immunologic studies of these cells demonstrated them to be T-lymphocytes with receptors for sheep erythrocytes. They reacted with an anti-thymocyte serum and responded to T-cell mitogens and to alloantigens. The leukemic T-cells had helper activity and could cooperate with B-lymphocytes in pokeweed mitogen (PWM) stimulated immunoglobulin synthesis. Concanavalin-A (Con-A) inducible suppressor cell activity was absent. The clinical and immune studies suggest that this case represents a previously undescribed form of T-cell leukemia.