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.     

Role of memory T cell subsets for adoptive immunotherapy

Adoptive transfer of primary (unmodified) or genetically engineered antigen-specific T cells has demonstrated astonishing clinical results in the treatment of infections and some malignancies. Besides the definition of optimal targets and antigen receptors, the differentiation status of transferred T cells is emerging as a crucial parameter for generating cell products with optimal efficacy and safety profiles.Long-living memory T cells subdivide into phenotypically as well as functionally different subsets (e.g. central memory, effector memory, tissue-resident memory T cells). This diversification process is crucial for effective immune protection, with probably distinct dependencies on the presence of individual subsets dependent on the disease to which the immune response is directed as well as its organ location.Adoptive T cell therapy intends to therapeutically transfer defined T cell immunity into patients. Efficacy of this approach often requires long-term maintenance of transferred cells, which depends on the presence and persistence of memory T cells. However, engraftment and survival of highly differentiated memory T cell subsets upon adoptive transfer is still difficult to achieve. Therefore, the recent observation that a distinct subset of weakly differentiated memory T cells shows all characteristics of adult tissue stem cells and can reconstitute all types of effector and memory T cell subsets, became highly relevant. We here review our current understanding of memory subset formation and T cell subset purification, and its implications for adoptive immunotherapy.     

T cell avidity and tumor recognition: implications and therapeutic strategies

In the last two decades, great advances have been made studying the immune response to human tumors. The identification of protein antigens from cancer cells and better techniques for eliciting antigen specific T cell responses in vitro and in vivo have led to improved understanding of tumor recognition by T cells. Yet, much remains to be learned about the intricate details of T cell – tumor cell interactions. Though the strength of interaction between T cell and target is thought to be a key factor influencing the T cell response, investigations of T cell avidity, T cell receptor (TCR) affinity for peptide-MHC complex, and the recognition of peptide on antigen presenting targets or tumor cells reveal complex relationships. Coincident with these investigations, therapeutic strategies have been developed to enhance tumor recognition using antigens with altered peptide structures and T cells modified by the introduction of new antigen binding receptor molecules. The profound effects of these strategies on T cell – tumor interactions and the clinical implications of these effects are of interest to both scientists and clinicians. In recent years, the focus of much of our work has been the avidity and effector characteristics of tumor reactive T cells. Here we review concepts and current results in the field, and the implications of therapeutic strategies using altered antigens and altered effector T cells.     

Type 1 CD8^＋ T Cells are Superior to Type 2 CD8^＋ T Cells in Tumor Immunotherapy due to Their Efficient Cytotoxicity, Prolonged Survival and Type 1 Immune Modulation

CD8^＋ cytotoxic T （Tc） cells play a crucial role in host immune responses to cancer, and in this context, adoptive CD8^＋ Tc cell therapy has been studied in numerous animal tumor models. Its antitumor efficacy is, to a large extent, determined by the ability of Tc cells to survive and infiltrate tumors. In clinical trials, such in vitro-activated T cells often die within hours to days, and this greatly limits their therapeutic efficacy. CD8^＋ Tc cells fall into two subpopulations based upon their differential cytokine secretion. In this study, we in vitro generated that ovalbumin （OVA）-pulsed dendritic cell （DCovA）-activated CD8^＋ type 1 Tc （Tcl） cells secreting IFN-T, and CD8^＋ type 2 Tc （Tc2） cells secreting IL-4, IL-5 and IL-10, which were derived from OVA-specific T cell receptor （TCR） transgenic OT I mice. We then systemically investigated the in vitro and in vivo effector function and survival of Tcl and Tc2 cells, and then assessed their survival kinetics after adoptively transferred into C57BL/6 mice, respectively. We demonstrated that, when compared to CD8^＋ Tc2, Tcl cells were significantly more effective in perforin-mediated cytotoxicity to tumor cells, had a significantly higher capacity for in vivo survival after the adoptive T cell transfer, and had a significantly stronger therapeutic effect on eradication of well-established tumors expressing OVA in animal models. In addition, CD8^＋ Tcl and Tc2 cells skewed the phenotype of CD4^＋ T cells toward Thl and Th2 type, respectively. Therefore, the information regarding the differential effector function, survival and immune modulation of CD8^＋ Tcl and Tc2 cells may provide useful information when preparing in vitro DC-activated CD8^＋ T cells for adoptive T cell therapy of cancer.     

Genetic approaches for antigen-selective cell therapy

Attempts to improve the efficacy of adoptive T-cell therapies have led to the development of innovative strategies that combine the high specificity of antibody molecules with the efficient trafficking properties and effector functions of immune cells. These antigen-selective cell therapies are designed to convert therapeutically important native antigens expressed on the cell surface (tumor associated antigens, viral envelope proteins) into recruitment points of effector functions, and address the goal of major histocompatibility complex- and exogenous cytokine-independent activation of mature effector T-cells. The most promising and best characterized antigen-selective strategy is based on the genetic manipulation of the recognition specificity of T-cells by grafting the recognition specificity of a monoclonal antibody onto a lymphocyte triggering molecule (TCR-associated polypeptides, Fc epsilon RI-gamma chain). Upon encountering specific antigen, cells harboring chimeric immune receptors (CIRs) are able to undergo specific stimulation and kill antigen bearing cells in both in vitro and in vivo model systems. Initial studies have focused on terminally differentiated effector cell-based protocols. However, recent data indicate that progenitor cell-based therapies allow the permanent generation of stable populations of CIR-expressing cells of multiple lineages, leading to long-term persistent systemic immunity. Emerging gene therapy strategies are based on the use of biespecic antibody fragments. The advantages of these biespecic antibody-mediated immune recruitment (BIR) approaches (trans-recruitment and multieffector activation) could complement conventional CIR-based immunotherapies. Although further scientific progress is required regarding the selection of the ideal effector cell/s and the definition of the optimal targeting and recruitment systems, clinical trials recently initiated in patients with advanced cancer and human immunodeficiency virus infection should help us to determine the real efficacy of these approaches. The relevance of these and other emerging concepts to cell-mediated immunotherapy is discussed.     

Adoptive precursor cell therapy to enhance immune reconstitution after hematopoietic stem cell transplantation

Strategies to enhance post-transplant immune reconstitution without aggravating graft-vs-host disease (GVHD) can improve the outcome of allogeneic hematopoietic stem cell transplantation. Recent preclinical studies demonstrated that the use of T cell depleted allografts supplemented with committed progenitor cells (vs stem cells only) allows enhanced immune reconstitution of specific hematopoietic lineages including myeloid, B, T, and natural killer lineages in the absence of GVHD. This novel adoptive therapy resulted in significantly improved resistance to microbial pathogens and could, in some cases, even mediate tumor immunity. Clinical protocols using adoptive transfer of committed hematopoietic progenitor cells are currently being evaluated.     

Clinical course and immune response of a renal cell carcinoma patient to adoptive transfer of autologous cytotoxic T lymphocytes

The cytotoxic T lymphocyte (CTL) is a promising candidate for an effector cell in adoptive immunotherapy for renal cell carcinoma (RCC). Here we report the clinical course and in vivo immune responses of a RCC patient with bulky retroperitoneal lymph node (RPLN) metastases who received adoptive autologous CTL therapy. A 56-year-old woman diagnosed with RCC with multiple RPLN metastases underwent unilateral nephrectomy. Autologous RCC cells were primary-cultured from surgical specimens. Before addition of peripheral blood mononuclear cells (PBMC) for CTL induction, subconfluent RCC cells were irradiated with 50 Gy. The PBMCs were then cultured on RCC cells in the induction medium supplemented with four kinds of interleukins. The induced CTLs showed the potent killing activity against autologous RCC cells in a typical MHC-class I-restricted manner. The patient received three courses of CTL therapy with a total of 10.2 x 10(9) cells, and the RPLN mass decreased markedly in size after the second course. Eosinophilia and enhanced CTL inducibility from peripheral blood were observed after CTL administrations. The patient was progression free without further treatment; however, she developed rapidly progressive glomerulonephritis more than 1 year after the last treatment. The patient died of newly developed metastases 27 months after the start of CTL therapy. At autopsy, viable RCC cells were found in multiple metastatic sites. However, only diffuse fibrous tissue was observed in the responding RPLN mass. Apparent histological divergence was observed between primary and metastatic sites.     

Enhancing adoptive T cell immunotherapy with microRNA therapeutics

Adoptive T cell-based immunotherapies can mediate complete and durable regressions in patients with advanced cancer, but current response rates remain inadequate. Maneuvers to improve the fitness and antitumor efficacy of transferred T cells have been under extensive exploration in the field. Small non-coding microRNAs have emerged as critical modulators of immune system homeostasis and T cell immunity. Here, we summarize recent advances in our understanding of the role of microRNAs in regulating T cell activation, differentiation, and function. We also discuss how microRNA therapeutics could be employed to fine-tune T cell receptor signaling and enhance T cell persistence and effector functions, paving the way for the next generation of adoptive immunotherapies.     

The use of endogenous T cells for adoptive transfer

Adoptive T-cell therapy involves the ex vivo enrichment and expansion of tumor-reactive T cells for infusion. As an immune-based approach, adoptive therapy has become an increasingly attractive modality for the treatment of patients with cancer due to its potential for high specificity, non-cross resistance with conventional therapies, and promise of long-term immunoprotection. In recent years, a resurgence in discoveries underlying T-cell recognition, tumor immune evasion, and T-cell memory and differentiation coupled with the development of several enabling technologies have facilitated a renewed focus in the field of adoptive therapy and its transition to the clinical arena as a treatment modality for patients with cancer. In this review, endogenous T cells derived from peripheral blood or tumor sites will be presented as a source of effector cells for adoptive therapy and strategies to isolate, manipulate, and enhance the function of antigen-specific T cells in vitro and to augment their in vivo efficacy and persistence by host immunomodulation are presented in the context of an ever-increasing inventory of preclinical and clinically available reagents. Optimizing the combination of adoptive cellular therapy and other immune-based and conventional approaches will herald a new generation of research and clinical opportunities for cancer immunotherapy.     

Immunomodulation in the treatment of haematological malignancies

Despite the continuous advances in immunology and cancer biology, haematological malignancies are often incurable. Conventional chemotherapy and radiation are efficacious for some lymphoma and leukaemia, however relapse and progressive disease often occurs. The evidence that the immune system can play an essential role in controlling cancer progression provide a basis for the development of active therapies, such as immunization, aimed to evoke or amplify a tumour-specific immune response. However, the inability of the patient’s own immune system to mount effective responses against tumour antigens is a major limit of vaccination approaches. The adoptive transfer of effectors of the adaptive immune system is an attractive strategy to circumvent the limitations of autologous immune responses. Donor lymphocyte infusion and the transfer of monoclonal antibodies (MoAbs) have been the first forms of adoptive therapy approved for clinical use and are still fundamental components of immunotherapy of haematological malignancies. Due to the continuous characterization of tumour-specific antigen, the development of tumour-tailored therapies that exploit the specificity of antibodies and T cell receptors (TCRs) is progressing rapidly. This review highlights the current advances in the field of adoptive immunotherapy of haematological malignancies, starting by elucidating the ongoing progress in passive transfer of MoAbs. We will also discuss recent advances in the adoptive transfer with tumour-specific high avidity T cells, which can be generated ex vivo by the transfer of gene constructs encoding single chain antibodies or TCRs, thus redirecting T cell specificity to selected tumour antigens. The ability to produce gene-modified T cells of desired specificity and defined functional activity may improve in the future T cell based immunotherapy of cancer.     

Impaired cytolytic activity in peripheral blood T cells from renal cell carcinoma patients

Classic T lymphocyte cytotoxicity is mediated through the T cell receptor (TCR). Defects in TCR signal transduction and cytolytic activity have been reported in tumor infiltrating T lymphocytes. We hypothesized that impaired cytotoxicity occurs in peripheral blood T cells from renal cell carcinoma (RCC) that can be reversed by exposure to rhIL-2. Peripheral blood mononuclear cells (PBMC) from 29 RCC patients and 29 healthy volunteers were isolated and cultured in the absence or presence of 10 IU/ml rhIL-2. A redirected cytotoxicity assay that requires TCR signal transduction was used with chromium-labeled P815 target cells, effector PBMC and anti-CD3 antibody. Target cell lysis was measured in "lytic units" (LU). Mean LU from RCC patients was lower than that of healthy volunteers (105.8 LU vs. 194.6 LU, P = 0.025). Exposure to rhIL-2 increased T cell-mediated lysis in both groups. Disruption of T cell cytotoxicity in RCC patients can be overcome by exposure to rhIL-2.     

Immune anticancer response: recent advances in the treatment of renal cell carcinoma

Recent advances in the field of immunobiology have provided many opportunities for anticancer-immunotherapy. Because they express tu-mor antigen, tumor cells can be kill by T cells. Renal Cell Carcinoma (RCC) is an immunogenic tumor and metastatic RCC is presently treated by cytokines. Anticancer immunity may be achieved by different strategies: allogeneic hematopoietic cell transplantation, vaccination with peptides, vaccination with loaded dendritic cells or adoptive cellular therapy in which specific T cells are isolated and expanded in vitro and then infused to patients. In our group, we have chosen the adoptive transfer of in vitro activated T cells with autologous tumor antigen loaded dendritic cells. To determine the best strategy of anticancer-immunotherapy, we need rigorous control of the specificity and the phenotype of the cell therapy product linked with the immunological status of the patient (before and after infusion) and with the clinical response.     

The role and mechanisms of double negative regulatory T cells in the suppression of immune responses

Accumulating evidence has demonstrated that regulatory T(Treg) cells play an important role in themaintenance of immunologic self-tolerance and in down-regulating various immune responses.Thus,there hasrecently been an increasing interest in studying the biology of Treg cells as well as their potential application intreating immune diseases.Many types of Treg cell subsets have been reported in a variety of disease models.Among these subsets,αβTCR~+CD3~+CD4~-CD8~- double negative(DN) Treg cells are defined by their capability ofinhibiting immune responses via directly killing effector T cells in an antigen specific fashion.Furthermore,DNTreg cells have been shown to develop regulatory activity after encountering specific antigens,partiallymediated by the acquisition of MHC-peptide complexes from antigen presenting cells(APCs).The presentationof acquired alloantigens on DN T cells allows for the specific interaction between DN Treg cells and alloantigenreactive effector T cells.Once the DN Treg and target cells have come into contact,killing is then mediated byFas/Fas-ligand interactions,and perhaps through other unidentified pathways.Further characterization of thefunctions,molecular expression and mechanisms of activation of DN Treg cells will help in the development ofnovel therapies to induce antigen specific tolerance to self and foreign antigens.Cellular & MolecularImmunology.2004;1(5):328-335.     

A novel co-stimulatory T cell antigen co-expressed on renal cell carcinoma

The A6H mAb raised primarily against human renal cell carcinoma(RCC) has previously been shown to bind strongly to RCC, tosome degree to colon carcinoma but only marginally to a varietyof normal tissues. Immunohlstochemical analysis of RCC tissuescontaining tumor-Infiltrating lymphocytes revealed that A6Hstained both tumor cells and lymphocytes. FACS analysis of humanperipheral blood cells demonstrated that A6H mAb stained 85-90%of both CD4⁺ and CD8⁺ T cells, but not granulocytes, monocytes,NK cells or B cells. Furthermore, 85-90% of naive and memoryT helper cells were stained with A6H suggesting that the A6HmAb defines unique subsets within these T cell populations.Dual staining showed that A6H mAb bind to an antigen that isclearly distinct from other cell surface molecules on T cells,including CD28, CD29, CD26, CD44 and ICAM-2. A6H mAb bindinginduced a second signal in anti-CD3 mAb activated T cells, resultingIn cell proliferation, IL-2 receptor expression and vigorousproduction of IFN_- and TNF, and production of minor amountsof IL-2. Immunoprecipitatlon with A6H mAb indicated a molecularweight of 120-140 kDa on both T cells and RCC. We suggest thatthe A6H mAb defines a unique T cell surface antigen which isinvolved in signal transduction and is expressed on subsetsof human T cells. The co-expression of A6H on T cells and tumorcells suggests a possible function related to common propertiesof these cells.     

Making and circumventing tolerance to cancer

The interactions between cancer and immune cells are complex. Even though the mutations that cause cancer can create new antigens that are potentially “visible” to T cells, in most experimental model systems the growth of tumors is accompanied by induction of T‐cell tolerance towards the tumor. How tolerance to tumors is induced and how tolerance can be broken by immunotherapy have been a main focus in cancer immunology. Here, we discuss experimental models used in cancer immunology. We argue that, while it is obviously easy for tumors to induce tolerance, it should be as easy to circumvent tolerance by the adoptive transfer of tumor‐antigen‐reactive T cells. Effective adoptive T‐cell therapy has become feasible by methods to identify TCR against tumor‐associated (self‐) antigens with high affinity and to graft a new antigen specificity to patients' T cells by TCR gene transfer.     

The tumour suppressor gene CEACAM1 is completely but reversibly downregulated in renal cell carcinoma

CEACAM1 acts as a tumour suppressor in various epithelial tumours. On the other hand, de novo expression of CEACAM1 is strongly associated with reduced disease-free survival of melanoma and non-small cell lung carcinoma patients. Since effector functions of natural killer and T cells are inhibited by homophilic CEACAM1 interaction, immune escape could be responsible for the poor prognosis of these patients. Here, we describe CEACAM1 expression in normal kidney, renal adenomas and renal cell carcinomas (RCC) using a novel antibody generated by genetic immunization. In normal kidney, CEACAM1 was found in epithelial cells of proximal tubules and in endothelial cells. In contrast, tumour cells of 30 clear cell, three chromophobic, and two chromophilic RCCs were completely devoid of CEACAM1. Renal adenomas also lacked CEACAM1 expression. Similarly, RCC cell lines CaKi1, CaKi2, A498, and RCC26 exhibited no or low-level CEACAM1 expression. However, CEACAM1 expression was transiently induced in A498 cells upon contact with allogeneic CD8+ T cells, mediated at least in part by interferon-gamma. Furthermore, the majority of tumour-infiltrating T and NK cells expressed CEACAM1 upon stimulation. Thus, transient expression of the tumour suppressor CEACAM1 by tumour cells and subsequent homophilic interaction with CEACAM1 on tumour-infiltrating lymphocytes could represent a novel immune escape mechanism in RCC.     

T细胞受体基因转染过继性免疫治疗的研究进展

利用T细胞进行过继性免疫治疗是治疗病毒感染性疾病和肿瘤的理想方法,但是用于治疗的T细胞的特异性、亲和性和数量等限制了其应用,如何获得特异、高效、一定数量的T细胞是目前亟待解决的问题。采用TCR基因转染的方法,将特异性高亲和力TCR转移到受体的T细胞中,可以特异性杀伤受体体内的肿瘤细胞。     

Antigen-specific CD4 T cell clonal expansion and differentiation in the aged lymphoid microenvironment. I. The primary T cell response is unaffected

Aging is associated with changes in the immune system that lead to decreased immunity in the elderly. Prior studies from humans and mice have shown that aged T cells exhibit numerous defects, including decreased proliferation following in vitro stimulation, suggesting that intrinsic defects exist within aged T cells, leading to defective T cell activation and clonal expansion. In vivo, however, cellular and soluble factors in the lymphoid microenvironment influence T cell function. To investigate the effects of the aged lymphoid microenvironment on T cell function, we monitored the immune response of CD4 T cells from DO11.10 TCR transgenic mice following adoptive transfer into young and aged hosts. After immunization with specific antigen similar rates of donor DO11.10 T cell division were observed in the two host types. However, at the peak of the response, greater numbers of DO11.10 T cells were found in the aged hosts. Regardless of the age of the host, the donor DO11.10 T cell population differentiated into functional effector cells. Despite the increased CD4 T cell growth in aged hosts, similar numbers of memory DO11.10 T cells were found in young and in aged hosts. As CD4 T cell clonal expansion and differentiation is not impaired in the aged microenvironment, our data suggest that diminished T cell immunity during aging is largely due to intrinsic T cell defects, rather than to extrinsic influences associated with the aged lymphoid microenvironment.     

Considerations on clinical use of T cell immunotherapy for cancer

The recognition by effector T lymphocytes of novel antigenic targets on tumor cells is the premise of specific, targeted immunotherapy of cancer. With the molecular characterization of peptide epitopes from melanoma antigens and, more recently, broadly expressed tumor antigens, there has been considerable enthusiasm for clinical evaluation of peptide tumor vaccines. Immunologic monitoring of vaccinated patients has demonstrated an expansion of CD8+ T cells that react with the relevant peptide and, more importantly, with native tumor. In most instances, however, vaccine-induced CD8+ T cell responses alone have not been sufficiently robust or sustained to translate into a high percentage of durable clinical responses. Vaccine strategies have also utilized dendritic cells (DCs) that have been modified to present tumor antigens. The superior antigen-processing capacity and co-stimulatory function of DCs convey a powerful stimulatory signal to both CD4+ and CD8+ T cells. Several strategies are attempting to broaden the immune response beyond single antigens by introducing the entire complement of tumor antigens into DCs. Adoptive immunotherapy is a promising strategy to recover tumor-reactive precursor T cells from patients, stimulate them to induce numerical expansion, and then re-infuse them. Ex vivo manipulation of the tumor-reactive T cells also permits cytotoxic therapy to be administered to the patient without damaging the effector cells. Recently, host lymphodepletion prior to adoptive transfer of effector T cells has resulted in an extremely high and sustained frequency of effectors that has achieved therapeutic efficacy against bulky metastatic disease in a substantial fraction of treated patients.