Understanding the response to immunotherapy in humans

Whether the efforts of the last decade aimed at the development of vaccines against tumor-specific antigens encountered success or failure is a matter of expectations. On the bright side, we could optimistically observe that anti-cancer-vaccines stand as an outstanding example of the successful implementation of modern biotechnology tools for the development of biologically sound therapeutics. In particular, vaccines against melanoma (the prototype model of tumor immunology in humans) can reproducibly induce cytotoxic T cell (CTL) responses exquisitely specific for cancer cells. This achievement trespasses the specificity of any other anti-cancer therapy. The skeptics, on the other end, might point out that immunization only rarely leads to cancer regression, labeling, therefore, this approach is ineffective. In our opinion this judgment stems from the naïve expectation that CTL induction is sufficient for an effective immune response. Here we propose that more needs to be understood about the mechanisms required for the induction of a therapeutically relevant immune response in humans. In particular, we will discuss the variables related to cancer heterogeneity, the weight of individual patients polymorphism(s), the role of the T cell activation and differentiation and, finally, the complex relationship between immune and cancer cells within the tumor microenvironment.     

Harnessing TH9 cells in cancer immunotherapy

CD4 T cell effector subsets not only profoundly affect cancer progression, but recent evidence also underscores their critical contribution to the anticancer efficacy of immune checkpoint inhibitors. In 2012, the two seminal studies suggested the superior antimelanoma activity of T_H9 cells over other T cell subsets upon adoptive T cell transfer. While these findings provided great impetus to investigate further the unique functions of T_H9 cells and explore their relevance in cancer immunotherapy, the following questions still remain outstanding: are T_H9 cell anticancer functions restricted to melanoma? What are the factors favouring T_H9 cell effector functions? What is the contribution of T_H9 cells to cancer immunotherapy treatments? Can T_H9 cells be identified in humans and, if so, what is their clinical relevance? By reviewing the studies addressing these questions, we will discuss how T_H9 cells could be therapeutically harnessed for cancer immunotherapy strategies.     

NK cell-based immunotherapy for cancer

Natural killer (NK) cells can induce an antigen-independent immune response against malignant cells. A growing number of scientific reports and clinical studies have shown promising anti-tumor effects when using NK cell-based immunotherapy. Currently, various approaches are being used to enhance the number and function of NK cells. One approach uses cytokines to selectively boost both the number as well as the efficacy of anti-tumor functions of NK cells. Another emerging approach focuses on checkpoint inhibitors targeting the NK cell receptor. Furthermore, bi-specific and tri-specific engagers have been developed to enhance the specific immune response by cross-linking specific tumor antigens to effector cells. In addition, NK cell adoptive transfer therapies have shown promising prospects. Among the various sources of adoptive transfer NK cells, allogeneic haploidentical NK cells that have undergone short- or long-term activation or expansion have also demonstrated effective anti-tumor effects with a low rate of rejection and side effects. CAR-NKs, derived from a new type of genetic modification, show enhanced NK cell cytotoxicity, specificity, and targeting. These NK cell-based therapies have exhibited promising results in clinical trials with malignant tumors. In this review, the current progress on NK cell-based therapeutic approaches, NK cell manufacturing techniques and tumor therapy outcomes are discussed.     

Frequent occurrence of high affinity T cells against MELOE‐1 makes this antigen an attractive target for melanoma immunotherapy

We recently showed that the infusion of tumor infiltrating lymphocytes specific for the MELOE‐1 antigen was associated with a prolonged relapse‐free survival for HLA‐A2⁺ melanoma patients who received tumor infiltrating lymphocytes therapy. Here, we characterized the MELOE‐1/A2‐specific T‐cell repertoire in healthy donors and melanoma patients to further support an immunotherapy targeting this epitope. Using tetramer enrichment followed by multicolor staining, we found that MELOE‐1‐specific T cells were present in the blood of healthy donors and patients at similar frequencies (around 1 in 1×10⁵ CD8⁺ cells). These cells mainly displayed a naïve phenotype in 4/6 healthy donors and 3/6 patients, whereas high proportions of memory cells were observed in the remaining individuals of both groups. There was a recurrent usage of the Vα12.1 chain for 17/18 MELOE‐1‐specific T‐cell clones derived from healthy donors or patients, associated with diverse Vβ chains and V(D)J junctional sequences. All clones derived from melanoma patients (9/9) were reactive against the MELOE‐1_36–44 peptide and against HLA‐A2⁺ melanoma cell lines. This study documents the existence of a large TCR repertoire specific for the MELOE‐1/A2 epitope and its capacity to give rise to antitumor CTL that supports the development of immunotherapies targeting this epitope.     

Human cell-based artificial antigen-presenting cells for cancer immunotherapy

Adoptive T-cell therapy, where anti-tumor T cells are first prepared in vitro, is attractive since it facilitates the delivery of essential signals to selected subsets of anti-tumor T cells without unfavorable immunoregulatory issues that exist in tumor-bearing hosts. Recent clinical trials have demonstrated that anti-tumor adoptive T-cell therapy, i.e. infusion of tumor-specific T cells, can induce clinically relevant and sustained responses in patients with advanced cancer. The goal of adoptive cell therapy is to establish anti-tumor immunologic memory, which can result in life-long rejection of tumor cells in patients. To achieve this goal, during the process of in vitro expansion, T-cell grafts used in adoptive T-cell therapy must be appropriately educated and equipped with the capacity to accomplish multiple, essential tasks. Adoptively transferred T cells must be endowed, prior to infusion, with the ability to efficiently engraft, expand, persist, and traffic to tumor in vivo. As a strategy to consistently generate T-cell grafts with these capabilities, artificial antigen-presenting cells have been developed to deliver the proper signals necessary to T cells to enable optimal adoptive cell therapy.     

Nanoparticles for generating antigen-specific T cells for immunotherapy

T cell therapy shows promise as an immunotherapy in both immunostimulatory and immunosuppressive applications. However, the forms of T cell-based therapy that are currently in the clinic, such as adoptive cell transfer and vaccines, are limited by cost, time-to-treatment, and patient variability. Nanoparticles offer a modular, universal platform to improve the efficacy of various T cell therapies as nanoparticle properties can be easily modified for enhanced cell targeting, organ targeting, and cell internalization. Nanoparticles can enhance or even replace endogenous cells during each step of generating an antigen-specific T cell response – from antigen presentation and T cell activation to T cell maintenance. In this review, we discuss the unique applications of nanoparticles for antigen-specific T cell therapy, focusing on nanoparticles as vaccines (to activate endogenous antigen presenting cells (APCs)), as artificial Antigen Presenting Cells (aAPCs, to directly activate T cells), and as drug delivery vehicles (to support activated T cells).     

An improved method for the generation of human lymphokine activated killer cells

In an effort to make interleukin-2/lymphokine-activated killer cell (IL-2/LAK) therapy safer for cancer patients, we examined the efficacy of using Fenwal PL732 bags as tissue culture flasks. These bags can be sterilly connected using tubing kits thus reducing the risk of contamination to the cells. Peripheral blood mononuclear cells were obtained from normal donors or cancer patients undergoing IL-2/LAK cell therapy. Following Ficoll-Hypaque purification, these cells were incubated in the presence of IL-2 in either PL732 plastic bags or standard tissue culture flasks. Our results showed that LAK cells could be generated from either normal donors or cancer patients in the bags as well as in the flasks. Comparisons were made of the LAK cell populations obtained from the two sources and showed that each was similar in terms of morphology as determined by Wright stain differentials. The populations of cells were also similar in regard to cell surface phenotype as determined by flow cytometric analysis. In addition, recoveries from either tissue culture vessel as well as cell viability of the LAK cells were comparable. Finally, the LAK cells obtained from both sources were assessed for cytolytic activity against the tumor cell lines K562 and Daudi. These results showed that the cytolytic activity of the LAK cells against these target cells was the same whether the cells were obtained from the flasks or the bags.     

Trispecific antibodies for cancer immunotherapy

Despite the clinical success of monoclonal and bispecific antibodies, there are still limitations in the therapeutic effect of malignant tumours, such as low response rate, treatment resistance, and so on, inspiring the exploration of trispecific antibodies (TsAbs). TsAbs further improve the safety and efficacy and has great clinical potential through three targets combination and formats optimization. This article reviews the development history and the target combination features of TsAbs. Although there are still great challenges in the clinical application of TsAbs, it is undeniable that TsAbs may be a breakthrough in the development of antibody drugs.     

Cancer,cytokines, and cytotoxic cells: Interleukin-2 in the immunotherapy of human neoplasms

Summary Modern immunotherapy of human cancer has evolved as a rapidly expanding field of clinical and experimental research. Employing the systemic application of recombinant interleukin-2 (IL-2) in humans, Rosenberg and colleagues from the National Cancer Institute reported the regression of advanced metastatic tumors in approximately 10%–30% of patients treated. The additional adoptive transfer of autologous patient-derived activated lymphocytes was performed to enhance therapeutic efficacy. While the exact mechanisms of IL-2 based immunotherapy in cancer remain unclear, it has been hypothesized that both the IL-2 activated lymphocyte and its secretory products such as interferon- or tumor-necrosis factor may contribute to the lysis of tumor cells in vivo. Accordingly, research has been directed toward enhancing both the activation state and the specificity of IL-2 induced killer cells in humans. Based on in vitro and animal data, the retransfusion of tumor-infiltrating lymphocytes has been shown to mediate the regression of metastatic neoplasms in up to 50% of patients receiving systemic IL-2. Considerable toxicity from the use of high-dose IL-2 has prompted attempts to develop low-dose regimens which allow for the outpatient treatment of patients presenting poor prognosis. While in most clinical trials involving IL-2, patient follow-up has been short, and no or only limited data have become available from controlled prospective and randomized clinical studies, IL-2 has shown some promise in patients with metastatic renal cell cancer or malignant melanoma. Novel approaches toward the improvement of clinical efficacy of IL-2 include local (e.g., intracavitary) application or combinations with other cytokines such as interferon- or cytostatic drugs.Abbreviations LAK lymphokine-activated killer - IL-2 interleukin-2 - NK natural killer - MHC major histocompatibility complex - TCR T-cell receptor - CTL cytotoxic T lymphocyte - TIL tumor-infiltrating lymphocyte - ALAK adherent lymphokine-activated, killer - IFN- interferon alpha - TNF- tumor necrosis factor alpha - DTIC dacarbazine     

Gene-based strategies for the immunotherapy of cancer

 T lymphocytes play a crucial role in the host’s immune response to cancer. Although there is ample evidence for the presence of tumor-associated antigens on a variety of tumors, they are seemingly unable to elicit an adequate antitumor immune response. Modern cancer immunotherapies are therefore designed to induce or enhance T cell reactivity against tumor antigens. Vaccines consisting of tumor cells transduced with cytokine genes in order to enhance their immunogenicity have been intensely investigated in the past decade and are currently being tested in clinical trials. With the development of novel gene transfer technologies it has now become possible to transfer cytokine genes directly into tumors in vivo. The identification of genes encoding tumor-associated antigens and their peptide products which are recognized by cytotoxic T lymphocytes in the context of major histocompatibility complex class I molecules has allowed development of DNA-based vaccines against defined tumor antigens. Recombinant viral vectors expressing model tumor antigens have shown promising results in experimental models. This has led to clinical trials with replication-defective adenoviruses encoding melanoma-associated antigens for the treatment of patients with melanoma. An attractive alternative concept is the use of plasmid DNA, which can elicit both humoral and cellular immune responses following injection into muscle or skin. New insights into the molecular biology of antigen processing and presentation have revealed the importance of dendritic cells for the induction of primary antigen-specific T cell responses. Considerable clinical interest has arisen to employ dendritic cells as a vehicle to induce tumor antigen-specific immunity. Advances in culture techniques have allowed the generation of large numbers of immunostimulatory dendritic cells in vitro from precursor populations derived from blood or bone marrow. Experimental immunotherapies which now transfer genes encoding tumor-associated antigens or cytokines directly into professional antigen-presenting cells such as dendritic cells are under evaluation in preclinical studies at many centers. Gene therapy strategies such as in vivo cytokine gene transfer directly into tumors as well as the introduction of genes encoding tumor-associated antigens into antigen-presenting cells hold considerable promise for the treatment of patients with cancer. Received: 20 January 1997 / Accepted: 17 February 1997     

Current immunotherapy of melanoma

The immunotherapy of patients with metastatic melanoma is currently at a crossroads. Indeed, recent results from vaccine strategies worldwide have revealed a strikingly low overall response rate in patients with stage IV melanoma. Although disappointing, we have gained valuable insight and knowledge about how vaccines interact with the host immune response and to melanoma. However, although an immunological response to therapy is often reported from various clinical trials, it does not contribute to a patient's long term survival. It has been proven time and again that an immunological response to therapy does not necessarily translate into a meaningful clinical response. This frustrating dichotomy of response continues to vex investigators, providing a glaring example of our poor understanding of the immunologic response to cancer. Thus, we remain at the crossroads of understanding and treatment. On the one hand, we have dramatically advanced the field of tumor immunology/immunotherapy over the last 20 years. On the other hand, we have made little headway in truly developing effective treatment options for patients with stage IV disease. We must realize our previous shortcomings and failures in order to learn from them and develop improved therapies. The future of immunotherapy remains a bright ray of hope for everyone, with the road to success paved with the previous hard work of thousands of clinicians and researchers everywhere. Towards this end, this review hopes to provide the reader with the current state of affairs for the immunotherapy of melanoma as well as a primer of where we might be heading in the future.     

The role of dendritic cells in neuroblastoma: Implications for immunotherapy

Neuroblastoma is a solid tumor, which is originated from some neural tissues. The immune system including the innate and adaptive immune system fights against this tumor. Dendritic cells (DCs) play an important role in this way by recognizing tumor antigens and activating specific types of T cells. These cells are derived from monocytes that are induced by inflammatory factors secreted by different cells in the tumor microenvironment (TME). There are different types of DCs, including monocyte-derived DCs (moDC), plasmacytoid DCs (pDC), conventional DCs type 1 and 2 (cDC1 and cDC2), and Langerhans cells. DCs connect the innate and the adaptive part of the immune system and have an important role in anti-tumor immunity. There are some vaccines that involve specific types of DCs, which can be used to prevent neuroblastoma. Also, we can use the combination of inflammatory factors and DCs as a substitute for chemotherapy.     

Breast cancer immunotherapy

Breast cancer is a leading cause of cancer-related deaths in women worldwide.Although tumorectomy,radiotherapy,chemotherapy and hormone replacement therapy have been used for the treatment of breastcancer,there is no effective therapy for patients with invasive and metastatic breast cancer.Immunotherapymay be proved effective in treating patients with advanced breast cancer.Breast cancer immunotherapyincludes antibody based immunotherapy,cancer vaccine immunotherapy,adoptive T cell transferimmunotherapy and T cell receptor gene transfer immunotherapy.Antibody based immunotherapy such as themonoclonal antibody against HER-2/neu (trastuzumab) is successfully used in the treatment of breast cancerpatients with over-expressed HER-2/neu,however,HER-2/neu is over-expressed only in 25-30% of breastcancer patients.Cancer vaccine immunotherapy is a promising method to treat cancer patients.Cancervaccines can be used to induce specific anti-tumor immunity in breast cancer patients,but cannot induceobjective tumor regression.Adoptive T cell transfer immunotherapy is an effective method in the treatment ofmelanoma patients.Recent advances in anti-tumor T cell generation ex vivo and limited clinical trial data havemade the feasibility of adoptive T cell transfer immunotherapy in the treatment of breast cancer patients.T cellreceptor gene transfer can redirect the specificity of T cells.Chimeric receptor,scFv(anti-HER-2/neu)/zetareceptor,was successfully used to redirect cytotoxic T lymphocyte hybridoma cells to obtain anti-HER-2/neupositive tumor cells,suggesting the feasibility of treatment of breast cancer patients with T cell receptor genetransfer immunotherapy.Clinical trials will approve that immunotherapy is an effective method to cure breastcancer disease in the near future.Cellular & Molecular Immunology.2004;1(4):247-255.     

扩散性抗原决定簇特异性Th2/Tr1细胞被动免疫治疗实验性变态反应性脑脊髓炎的研究

目的 :研究用扩散性抗原决定簇特异性Th2 /Tr1细胞被动免疫治疗实验性变态反应性脑脊髓炎 (EAE)的有效性和可能性。方法 :应用髓鞘蛋白脂质蛋白 (PLP) 139 15 1诱发EAE小鼠模型 ,通过转染方式建立具有高效表达IL 10特性的抗原特异性Th2 /Tr1样T细胞系 ,在EAE小鼠发病 6d后给予静脉注射。小鼠每天称体重并进行神经功能评分。实验结束时 ,取脊髓进行PLP免疫组化染色 ,应用数字化图像分析技术进行图像分析。结果 :注射扩散性抗原决定簇MBP87 99特异性Th2 /Tr1样T细胞的治疗组小鼠的临床评分明显改善 ,并伴有复发率的下降、首次复发时间的延迟 (P <0 0 5 )。PLP免疫组化染色的图像分析结果显示 ,接受MBP87 99特异性Th2 /Tr1样T细胞治疗组小鼠脊髓内PLP像素水平为正常值的 94 37%± 0 5 2 %,而对照组小鼠为正常PLP水平的 91 94%± 0 5 0 %(P <0 0 0 1) ,与对照组比较 ,治疗组小鼠脊髓内脱髓鞘病变下降 30 %。结论 :扩散性抗原决定簇特异性Th2 /Tr1细胞被动免疫治疗可明显减少脱髓鞘病变 ,改善EAE的临床症状 ,为多发性硬化的免疫治疗提供了理论依据。     

CAR-T细胞免疫疗法的现状及展望

肿瘤细胞免疫治疗被公认为是继手术、放疗和化疗之后的第四种肿瘤治疗方法,免疫疗法嵌合型抗原受体(chimeric antigen receptor,CAR)修饰的T细胞疗法(CAR-T),属于过继性细胞免疫治疗,不仅在肿瘤治疗中取得突破性进展,在感染性疾病和自身免疫性疾病等治疗中也有良好的应用前景.现就CAR-T细胞免疫疗法的发展历史、临床应用、目前存在的问题以及最新研究成果作一综述.     

Optimizing cancer immunotherapy trials: back to basics

Attempts to raise effective immunity against cancer are benefiting from information on the nature of the immunity involved and its regulation and, perhaps, now it is time to step back and define our approach in molecular terms prior to clinical testing. Although there are immunological differences between mice and patients, results from murine studies are encouraging early 'translation' of concepts to the clinic and it is vital to take immunological principles emerging from mice into clinical vaccine design. One is the requirement to break tolerance against over-expressed self-antigens, a potentially risky procedure but necessary for several cancer targets. A study in this issue of the European Journal of Immunology attempts to do this by using xenogeneic antigens, albeit with variable outcome. The unstated goal is to activate T-cell help but this can be achieved more effectively by harnessing a predictable anti-microbial repertoire. The second issue lies in the delivery of antigen. One strategy is "prime/boost" using DNA priming and boosting with a viral vector; however, this induces blocking immunity against viral proteins, and must be used judiciously. There are other physical methods to increase immunity such as electroporation, which can itself be used in 'prime/boost' sequence. These twin problems of engagement of T-cell help and delivery of adequate antigen can now be addressed by applying immunological logic to cancer vaccines.     

Development in vitro of human CD4+ thymocytes into functionally mature Th2 cells. Exogenous interleukin-12 is required for priming thymocytes to produce both Th1 cytokines and interleukin-10

Fresh postnatal thymocyte cell suspensions were directly cloned under limiting dilution conditions with either phytohemagglutinin or toxic shock syndrome toxin-1 (TSST-1), a bacterial superantigen. Cultures contained allogenic irradiated feeder cells and interleukin (IL)-2, in the absence or presence of exogenous IL-4, interferon (IFN)-γ or IL-12. The resulting CD4⁺ T cell clones generated under these different experimental conditions were then analyzed for their ability to produce IL-2, IL-4, IL-5, IL-10, IFN-γ and tumor necrosis factor (TNF)-β in response to stimulation with phorbol 12-myristate 13-acetate (PMA)+anti-CD3 monoclonal antibody or PMA + ionomycin. Different from T cell clones generated from peripheral blood, virtually all CD4⁺ T cell clones generated from human thymocytes produced high concentrations of IL-2, IL-4 and IL-5, but no IFN-γ, TNF-β or IL-10. Moreover, after activation, these clones expressed on their surface membrane both CD30 and CD40 ligand, but not the product of lymphocyte activation gene (LAG)-3, and provided strong helper activity for IgE synthesis by allogeneic B cells. The Th2 cytokine pattern could not be modified by the addition of IFN-γ. However, upon addition of exogenous IL-12, the resulting CD4⁺ thymocyte clones produced TNF-β, IFN-γ, and IL-10 in addition to IL-4 and IL-5. These results suggest that CD4⁺ human thymocytes have the potential to develop into cells producing the Th2 cytokines IL-4 and IL-5, whereas the ability to produce both Th1 cytokines and IL-10 is acquired only after priming with IL-12.     

Changes of serum cytokines after the long term immunotherapy with Japanese hop pollen extracts

Japanese hop (Hop J) pollen has been considered as one of the major causative pollen allergens in the autumn season. We developed a new Hop J immunotherapy extract in collaboration with Allergopharma (Reinbeck, Germany) and investigated immunologic mechanisms during 3 yr immunotherapy. Twenty patients (13 asthma with rhinitis and 7 hay fever) were enrolled from Ajou University Hospital. Sera were collected before, 1 yr, and 3 yr after the immunotherapy. Changes of serum specific IgE, IgG1, and IgG4 levels to Hop J pollen extracts and serum IL-10, IL-12, TGF-beta1 and soluble CD23 levels were monitored by ELISA. Skin reactivity and airway hyper-responsiveness to methacholine were improved during the study period. Specific IgG1 increased at 1 yr then decreased again at 3 yr, and specific IgG4 levels increased progressively (p<0.05, respectively), whereas total and specific IgE levels showed variable responses with no statistical significance. IL-10, TGF-beta1 and soluble CD23 level began to decrease during first year and then further decreased during next two years with statistical significances. (p<0.05, respectively). In conclusion, these findings suggested the favorable effect of long term immunotherapy with Hop J pollen extracts can be explained by lowered IgE affinity and generation of specific IgG4, which may be mediated by IL-10 and TGF-beta1.     

Interleukin-15 enhances cytokine induced killer (CIK) cytotoxic potential against epithelial cancer cell lines via an innate pathway

CIK cells are a subset of effector lymphocytes endowed with a non-MHC restricted anti-tumor activity making them an appealing and promising cell population for adoptive immunotherapy. CIK are usually generated ex-vivo by initial priming with Interferon-γ (IFN-γ) and monoclonal antibody against CD3 (anti-CD3), followed by culture in medium containing Interleukin-2 (IL-2). Interleukin-15 (IL-15) shares with IL-2 similar biological functions and recently it has been reported to induce CIK with increased anti-leukemic potential.The aim of the study was to compare the killing efficacy of CIK generated by IL-2 alone or IL-2 and IL-15 toward tumor targets of different origins, leukemic cells and malignant cells from epithelial solid tumors. CIK bulk cultures were examined for cell proliferation, surface phenotype and cytotoxic potential against tumor cell lines K562, HL60, HeLa and MCF-7. The results showed that IL-15 is able to induce a selective expansion of CIK cells, but it is less effective in sustaining CIK cell proliferation compared to IL-2. Conversely, our data confirm and reinforce the feature of IL-15 to induce CIK cells with a potent cytotoxic activity mostly against tumor cells from epithelial solid malignancies via NKG2D-mediated mechanism.