Evaluation of basic procedures for adoptive immunotherapy for gastric cancer

Peripheral blood lymphocytes (PBL) of gastric cancer patients in advanced stages showed lymphokine activated killer (LAK) activities comparable to those of healthy donors, suggesting potential applicability of LAK cells induced from PBL stimulated with recombinant interleukin-2 (rIL-2) in adoptive immunotherapy (AIT) for gastric cancer. In order to generate a large number of LAK cells from PBL, lymphocytes were cultured with both rIL-2 and phytohemagglutinin (PHA). In this culture, the numbers of cells increased to a greater extent than those in culture with rIL-2 alone but cytotoxic activity did not augment, thus suggesting that this procedure would not afford sufficient clinical effects. On the other hand, a large number of LAK cells with high anti-tumor activities were efficiently induced from spleen cells of the patients by culture of rIL-2; hence clinical usefulness of these LAK cells is anticipated. In regional lymph node lymphocytes (RLNL) cultured with rIL-2, the cytotoxic activities were lower than in those induced in PBL, and a characteristic increase of CD8 + CD11 + suppressor T cells was observed after incubation with rIL-2. Nevertheless, an increase of CD4 + 4B4⁺ helper inducer T cells was also observed in RLNL after the culture with rIL-2. Furthermore, high cytotoxic activities were induced in RLNL in some cases in which metastasis to the regional lymph nodes was not detected. When gastric cancer patients were pretreated with biological response modifiers (BRM), especially with Lentinan, LAK cells from PBL showed higher NK and LAK activities as compared with those of patients without BRM pretreatment.This work was partly supported by a grant from Hokkoku Cancer Research Foundation.     

Cancer immunotherapy takes a multi-faceted approach to kick the immune system into gear

Cancer accounts for about every fourth death in the United States, with approximately 1,500 people dying each day as a result of this disease. Despite some progress in the last decades, these numbers alone undoubtedly demonstrate the urgent need for new and more efficient treatments. Immunotherapy aims to activate an efficient immune response against tumors or even prevent cancers from occurring in the first place. It is a growing field currently flourishing with several successful trials, some of which have led to the recent approval of new anti-cancer drugs by the Food and Drug Administration (FDA). This review addresses the manifold strategies that immunotherapy has taken in the past and discusses the most recent achievements in the field.     

Bone metastasis as a prognostic factor in breast cancer patients with liver metastasis given OK-432-combined adoptive immunotherapy via the hepatic artery

The outcome in 31 patients with liver metastases from breast cancer given OK-432-combined adoptive immunotherapy via the hepatic artery was analyzed. Patients received intraarterial OK-432, a streptococcal preparation, followed by the transfer of autologous lymphocytes cultured with autologous tumor extract and interleukin-2 for 9–13 days. Liver lesions were evaluable in 11 of the 12 patients with bone metastasis (group A) and in 16 of the 19 patients without bone metastasis (group B). Complete response (CR) in the liver was attained in 8 patients in group A, but in only 1 in group B (p < 0.01). In group A, radiological features of all metastatic foci of bone improved after CR in the liver. Moreover, the median survival time (MST) of group A (20 months) was longer (p=0.06) than that of group B patients with extra-hepatic metastasis (n=12; MST=6 months), while group B patients with liver metastasis alone (n=7) showed a MST similar to that of group A. Thus, loco-regional immunotherapy via the hepatic artery was found to be useful in controlling both liver and bone metastasis from breast cancer. Moreover, in breast cancer patients with liver metastasis, bone metastasis appears to be a prognostic factor associated with good response to this immunotherapy.Abbreviations MST median survival time - CR complete response - PR partial response - MDP metyl-diphosphonate     

肝癌免疫治疗的研究进展

肝细胞肝癌是全球发病率和死亡率最高的恶性肿瘤之一,发病率和死亡率呈逐年上升趋势。我国是肝癌大国,每年肝癌的死亡病例数位居全球第一。免疫治疗是继手术、化疗和放疗之后新兴的癌症治疗手段,其通过解除肿瘤微环境对免疫细胞的抑制作用并激活机体免疫功能,实现控制和杀伤肿瘤细胞。常用的免疫治疗的方法有免疫检查点治疗、过继免疫治疗和肿瘤疫苗治疗等。与传统治疗手段相比,免疫治疗因具有增强机体免疫功能、延缓肿瘤进展、延长患者生存时间等优点,逐渐成为基础和临床研究的热点。文中就免疫治疗在肝癌领域的研究进展作一综述。     

Restoration of CD3+CD56+ cell level improves skin lesions in severe psoriasis: A pilot clinical study of adoptive immunotherapy for patients with psoriasis using autologous cytokine-induced killer cells

Psoriasis is a chronic inflammatory skin disorder mediated by the cells and molecules of both the innate and adaptive immune systems. Autologous cytokine-induced killer (CIK) cell infusion is considered an effective and safe cancer treatment and is licensed for this use in China. Accumulated evidence indicating that CD3⁺CD56⁺ cells are significantly decreased in psoriatic patients prompted us to investigate if the restoration of CD3⁺CD56⁺ cells may be beneficial for psoriatic patients. We designed a clinical trial for psoriasis treatment that involved CIK cell infusion because CIK cells include a large amount of CD3⁺CD56⁺ T cells (NCT01894373 at www.clinicaltrials.gov). Six patients with severe psoriasis were initially enrolled, and four of them exhibited markedly lower levels of CD3⁺CD56⁺ cells in their peripheral blood (PB) relative to healthy donors. CIK cell infusion-associated toxicity was not observed in any infusion. The percentage of CD3⁺CD56⁺ cells in the PB markedly increased and the psoriasis area and severity index (PASI) synchronously decreased in four patients with lower CD3⁺CD56⁺ cell contents, and two of them obtained a more than 4-month PASI75 after completing a four-cycle treatment. However, a decrease in the CD3⁺CD56⁺ cells was observed concomitantly with disease recurrence after short-term amelioration. In contrast, no obvious improvement was observed in the two patients with nearly normal CD3⁺CD56⁺ cells in the PB before treatment. These observations suggest that the normalization of the CD3⁺CD56⁺ cell level may improve the skin lesions of severe psoriasis and warrant further clinical trials for severe psoriasis using repeated CIK adoptive immunotherapy.     

IgE immunotherapy: A novel concept with promise for the treatment of cancer

The importance of antibodies in activating immune responses against tumors is now better appreciated with the emergence of checkpoint blockade antibodies and with engineered antibody Fc domains featuring enhanced capacity to focus potent effector cells against cancer cells. Antibodies designed with Fc regions of the IgE class can confer natural, potent, long-lived immune surveillance in tissues through tenacious engagement of high-affinity cognate Fc receptors on distinct, often tumor-resident immune effector cells, and through ability to activate these cells under tumor-induced Th2-biased conditions. Here, we review the properties that make IgE a contributor to the allergic response and a critical player in the protection against parasites, which also support IgE as a novel anti-cancer modality. We discuss IgE-based active and passive immunotherapeutic approaches in disparate in vitro and in vivo model systems, collectively suggesting the potential of IgE immunotherapies in oncology. Translation toward clinical application is now in progress.     

基于单克隆抗体的肿瘤免疫疗法研究进展

一百多年前,"魔术子弹"学说首次提出了具有靶向特异性的抗体可以用来治疗疾病。此后,随着单克隆抗体制备技术的成熟,以及癌症血清疗法的发展,靶向肿瘤抗原的治疗性抗体开始进入临床,至今已有20余种抗体药物用于癌症的治疗。近两年,以免疫检查点蛋白拮抗剂、双特异性抗体、抗体药物偶联药物等为代表的新一代抗体药物,不断在治疗恶性肿瘤上取得突破性进展。本文回顾了抗肿瘤抗体的发展历程,总结了新一代抗体药物的作用机制与构建策略,以及主要临床副作用。并对基于抗体的肿瘤免疫疗法未来发展趋势进行了展望。     

Successful adoptive immunotherapy with OK432-inducible activated natural killer cells in tumor-bearing mice

We had demonstrated that the NK cell mediated cytotoxicity of murine spleen cells could be augmented byin vivo priming and subsequentin vitro challenge with a streptococcal preparation OK432, and the cell surface phenotype of induced killer cells was Thy-1⁺, asialo GM1⁺, suggesting that the activated cells were of NK lineage (OK-NK cell). We had also clarified that IL-2 played a major role in inducing the OK-NK cells via the production of IFN-. In this study, we examined the effect of adoptive transfer of OK-NK cells on syngeneic tumors in mice. Mice were implanted with SP2 myeloma cells intraperitoneally (i.p.), or C26 colon adenocarcinoma cells subcutaneously to make the models of peritonitis carcinomatosa or solid tumor, and the OK-NK cells were transferred i.p. or intratumorally, adoptively. By the adoptive transfer of OK-NK cells, 92% of mice bearing SP2-tumor had be cured. The tumor growth of C26-solid tumor was inhibited, and the survival rate of mice bearing C26-tumor was significantly increased. The intratumoral remnants of¹²⁵I-labelled OK-NK cells were 61, 27 and 8% at 4, 12 and 36h after intratumoral transfer, respectively. By multiple transfer of OK-NK cells, the antitumor effect was more effectively augmented than that of a single transfer. Results in this study suggested that OK-NK cells could be useful for the therapy of cancer patients.     

Adoptive immunotherapy of cancer: biological response modifiers and cytotoxic cell therapy

Immunotherapy has been developed for the treatment of metastatic cancers refractory to conventional therapies. Immunotherapy utilizes immune cells and/or biological response modifiers (BRMs) to induce an anti-tumor response mediated by the patient's immune system. BRMs, including lymphokines and cytokines, are used as single agents or in combination for cancer therapy. Some BRMs, particularly interleukin 2 (IL-2), can activate and expandin vitro lymphocytes with anti-tumor reactivity which will be adoptively transferred to the patient. To enhance the therapeutic effect of immunotherapy, gene therapy is currently under investigation and involves the insertion of cytokine genes in immune cells or in tumor cells. The development and future of cancer immunotherapy will be discussed in this review.     

Phage antibody display libraries: a powerful antibody discovery platform for immunotherapy

Phage display technology (PDT), a combinatorial screening approach, provides a molecular diversity tool for creating libraries of peptides/proteins and discovery of new recombinant therapeutics. Expression of proteins such as monoclonal antibodies (mAbs) on the surface of filamentous phage can permit the selection of high affinity and specificity therapeutic mAbs against virtually any target antigen. Using a number of diverse selection platforms (e.g. solid phase, solution phase, whole cell and in vivo biopannings), phage antibody libraries (PALs) from the start point provides great potential for the isolation of functional mAb fragments with diagnostic and/or therapeutic purposes. Given the pivotal role of PDT in the discovery of novel therapeutic/diagnostic mAbs, in the current review, we provide an overview on PALs and discuss their impact in the advancement of engineered mAbs.     

PD-1/PD-L1 pathway: Basic biology and role in cancer immunotherapy

Over the course of past few years, cancer immunotherapy has been accompanied with promising results. However, preliminary investigations with respect to immunotherapy concentrated mostly on targeting the immune checkpoints, nowadays, emerge as the most efficient strategy to raise beneficial antitumor immune responses. Programmed cell death protein 1 (PD-1) plays an important role in subsiding immune responses and promoting self-tolerance through suppressing the activity of T cells and promoting differentiation of regulatory T cells. PD-1 is considered as an immune checkpoint and protects against autoimmune responses through both induction of apoptosis in antigen-specific T cells and inhibiting apoptosis in regulatory T cells. Several clinical trials exerting PD-1 monoclonal antibodies as well as other immune-checkpoint blockades have had prosperous outcomes and opened new horizons in tumor immunotherapy. Nonetheless, a bulk of patients have failed to respond to these newly emerging immune-based approach and the survival rate was not satisfying. Additional strategies, especially combination therapies, has been initiated and been further promising. Attempts to identify novel and well-suited predictive biomarkers are also sensed. In this review, the promotion of cancer immunotherapy targeting PD-1 immunoinhibitory pathway is discussed.     

Establishment of peripheral blood mononuclear cell-derived humanized lung cancer mouse models for studying efficacy of PD-L1/PD-1 targeted immunotherapy

ABSTRACT

Animal models used to evaluate efficacies of immune checkpoint inhibitors are insufficient or inaccurate. We thus examined two xenograft models used for this purpose, with the aim of optimizing them. One method involves the use of peripheral blood mononuclear cells and cell line-derived xenografts (PBMCs-CDX model). For this model, we implanted human lung cancer cells into NOD-scid-IL2Rg?/? (NSI) mice, followed by injection of human PBMCs. The second method involves the use of hematopoietic stem and progenitor cells and CDX (HSPCs-CDX model). For this model, we first reconstituted the human immune system by transferring human CD34+ hematopoietic stem and progenitor cells (HSPCs-derived humanized model) and then transplanted human lung cancer cells. We found that the PBMCs-CDX model was more accurate in evaluating PD-L1/PD-1 targeted immunotherapies. In addition, it took only four weeks with the PBMCs-CDX model for efficacy evaluation, compared to 10–14 weeks with the HSPCs-CDX model. We then further established PBMCs-derived patient-derived xenografts (PDX) models, including an auto-PBMCs-PDX model using cancer and T cells from the same tumor, and applied them to assess the antitumor efficacies of anti-PD-L1 antibodies. We demonstrated that this PBMCs-derived PDX model was an invaluable tool to study the efficacies of PD-L1/PD-1 targeted cancer immunotherapies. Overall, we found our PBMCs-derived models to be excellent preclinical models for studying immune checkpoint inhibitors.     

The establishment and application of CD3E humanized mice in immunotherapy

In the field of cancer immunotherapy, monoclonal antibody drugs, bispecific antibodies, and antibody-conjugated drugs have become the focus of current research, and gene-edited animal models play an essential role in the entire drug development process. In this study, CD3E humanized mice were established by replacing the second to the seventh exon of the Cd3e mouse gene with the same exon of the human gene. The expression of human CD3E in CD3E humanized mice was detected by RT-PCR as well as flow cytometry, also a tumor model was established based on CD3E humanized mice, and the pharmacodynamic effects of CD3E monoclonal antibodies were evaluated. The results showed that CD3E humanized mice expressed only human CD3E, and the proportion of each lymphocyte in the thymus and spleen was not significantly changed compared with wild-type mice. CD3E monoclonal antibody could promote tumor growth after treatment, which may be related to the activation-induced cell death effect caused by this CD3E antibody. In contrast, Bispecific antibody blinatumomab inhibited tumor growth significantly. Thus, the CD3E humanized mice provided an adequate animal model for evaluating the efficacy and safety of CD3E antibody drugs.     

Augmentation of the antitumor effect of adoptive immunotherapy by in vivo sensitization of EL-4 lymphoma and pre-treatment with sizofiran

The antitumor effects of adoptive immunotherapy using LAKcells treated with sizofiran (SPG) following in vivoantigen sensitization with EL-4 lymphoma (EsLAK),comparing nonsensitized LAK cells (sLAK), were studied inmice with intraperitoneal implantation of EL-4 lymphoma.EL-4 cells treated with Mitomycin C (100 g /ml) wereintroduced by inoculation into the peritoneum of C57BL/6mice for antigen sensitization. Four days later, SPG (100g) was intramuscularly injected. Three days after SPG administration, mononuclear cells obtained from the spleen were prepared for LAK cells (EsLAK). The following resultswere obtained: 1) The survival period was significantlygreater in the sLAK and EsLAK groups than in the controlgroup. The survival period in the EsLAK group wassignificantly greater than that in the sLAK group. 2) Thenumber of EL-4 cells in the peritoneal exudate cells 11days postimplantation was lowest in the EsLAK group, andthe number of lymphocytes including LGL was largest in theEsLAK group, compared with the sLAK group and the controlgroup. 3 ) The EsLAK cells showed significantly moreenhanced cytotoxic activity against EL-4 than the sLAKcells. 4) Histopathological findingsof metastatic lesions of the liver and spleen stained by HE11 days postimplantation showed less infiltrating tumorcells and more lymphocytic infiltrations in the sLAK andEsLAK groups compared with the control group. Theseresults suggest that induction of LAK cells byadministration of SPG to lymphocytes treated by in vivosensitization with tumor antigen increasesthe efficacy of adoptive immunotherapy.     

Harnessing the CD1 restricted T cell response for leukemia adoptive immunotherapy

Disease recurrence following chemotherapy and allogeneic hematopoietic cell transplantation is the major unmet clinical need of acute leukemia. Adoptive cell therapy (ACT) with allogeneic T lymphocytes can control recurrences at the cost of inducing detrimental GVHD. Targeting T cell recognition on leukemia cells is therefore needed to overcome the problem and ensure safe and durable disease remission. In this review, we discuss adoptive cells therapy based on CD1-restricted T cells specific for tumor associated self-lipid antigens. CD1 molecules are identical in every individual and expressed essentially on mature hematopoietic cells and leukemia blasts, but not by parenchymatous cells, while lipid antigens are enriched in malignant cells and unlike to mutate upon immune-mediated selective pressure. Redirecting T cells against self-lipids presented by CD1 molecules can thus provide an appealing cell therapy strategy for acute leukemia that is patient-unrestricted and can minimize risks for GVHD, implying potential prognostic improvement for this cancer.     

Preliminary validation of an activation assay for ex vivo activated T cells utilized in cancer immunotherapy

An in vitro assay that measures the activation level of ex vivo activated (EVA) T cells currently being used in the adoptive immunotherapy of metastatic renal cell carcinoma has been developed. This assay is based on the ability of activated, but not resting. T cells to proliferate in response to the protein kinase C activator, phorbol myristate (PMA). To utilize this assay for in-process monitoring and control, we have begun an initial validation of the overall reproducibility of this assay. The proliferation of activated T cells in response to PMA, as measured by the mean cpm values of (3)H-thymidine incorporated, was demonstrated to have intra-assay coefficients of variation (cv's) for individual analysts that were typically less than 10% and rarely exceeded 20%. Activated T cells could be frozen and stored for at least 6 weeks with little or no deterioration in their ability to proliferate in response to PMA. Using these cells, inter-assay cv's that were typically less than 15% were obtained by individual analysts, and overall cv's of 10% to 25% were obtained for different samples assayed by different analysts at different times. This level of variability is very reasonable for a cellular assay. Furhter validation of this assay will address the issues of sensitivity, linearity and selectivity. To date, this assay has been used to analyze over 90 patient EVA cell samples and has revealed a broad range of proliferative responses to PMA. Taken together, these results suggest that this assay may be useful in defining the potency of the activated T cell used therapeutically.     

An efficient or methodical review of immunotherapy against breast cancer

Breast cancer (BC) is one of the most widespread malignancies in women worldwide. Breast cancer is mainly classified into a few key molecular subtypes in accordance with hormone and growth factor receptor expression, etc. In spite of numerous advances in the remedy of breast cancer, the development of metastatic disease remains an untreatable and repeated basis of cancer death for women. Preclinical and clinical studies of immunotherapy in cancer remedy have been in progress for the past quite a few decades by an effort to accelerate, augment, and modulate the immune system to spot and devastate cancer cells. Advancement of cancer immunotherapy is rapidly increasing with eminent and most interesting therapy compared to other therapy like targeted therapy, cytotoxic chemotherapy, radiation as well as surgery. Cancer immunotherapy, also known as biological therapy, which denotes the controlling and by means of the patient's own immune system to goal the cancer cells rather than using an extrinsic therapy. In that way, focusing of cancer immunotherapy developing mediators that stimulates or enhances the immune system's recognition and destroying the cancer cells. This review describes a holistic outlook and deeper understanding of the biology of immunotherapy within the system of tumor microenvironment of breast cancer that improve clinical research and constructive impact on the study conclusion.     

双特异性抗体在抗病毒方面的研究进展

随着基因工程抗体的快速发展,双特异性抗体技术也日趋成熟。双特异性抗体能够同时结合两个以上不同的抗原表位,在免疫治疗中具有独特的优势。双特异性抗体己经广泛应用于癌症治疗如黑色素瘤、霍奇金淋巴瘤以及肝癌、胃癌等,以及炎症方面的治疗如类风湿性关节炎、牛皮癣与克罗恩病等。双特异性抗体在病毒免疫治疗方面则刚刚起步。文中对双特异性抗体用于病毒免疫治疗的研究进展进行了综述,特别是在体内外效果较好的产品,为用于病毒免疫治疗的双特异性抗体药物开发与研究提供一定的参考。