Cellular therapy to treat haematological and other malignancies: progress and pitfalls

The recent Food and Drug Administration (FDA) approval of a cellular therapy to treat castration resistant prostate cancer has reinforced the potential of cellular therapy to consolidate current pharmacological approaches to treating cancer. The emergence of the cell manufacturing facility to facilitate clinical translation of these new methodologies allows greater access to these novel therapies. Here we review different strategies currently being explored to treat haematological malignancies with a focus on adoptive allogeneic or autologous transfer of antigen specific T cells, NK cells or dendritic cells. These approaches all aim to generate immunological responses against overexpressed tissue antigens, mismatched minor histocompatability antigens or tumour associated antigens. Current successes and limitations of these different approaches will be discussed with an emphasis on challenges encountered in generating long term engraftment, antigen selection and implementation as well as therapeutic immune monitoring of clinical responses, with examples from recent clinical trials.     

Immunotherapy of cancer through targeting of minor histocompatibility antigens

Minor histocompatibility antigens are allogeneic targets of T-cell mediated graft-versus-tumour effects following allogeneic stem cell transplantation. Recent research has identified several minor histocompatibility antigens as tumour proteins and has also disclosed their unique properties in both the induction and the effector phase of graft-versus-tumour effects. Targeting tumour-specific minor histocompatibility antigens by adoptive immunotherapy will battle against tumour tolerance and evoke allo-immune responses, thereby enhancing graft-versus-tumour effects against leukaemia and solid tumours. Recently acquired knowledge of the role of donor immunisation status, new techniques in the generation of minor histocompatibility antigen-specific cytotoxic T lymphocytes in vitro, and innovative principles in vaccination will help to design clinical trials that exploit minor histocompatibility antigens in the immunotherapy of cancer.     

Generation of anti-tumour effector T cells from naïve T cells by stimulation with dendritic/tumour fusion cells

Tumour-draining lymph node T cells are an excellent source of effector T cells that can be used in adoptive tumour immunotherapy because they have already been sensitized to tumour-associated antigens in vivo. However, such tumour-specific immune cells are not readily obtained from the host due to poor immunogenicity of tumours and reduced host immune responses. One obstacle in implementation of adoptive immunotherapy has been insufficient sensitization and expansion of tumour-specific effector cells. In this study, we aim to improve adoptive immunotherapy by generating anti-tumour effector T cells from naïve T lymphocytes. We attempted to achieve this by harnessing the advantages of dendritic cell (DC)-based anti-cancer vaccine strategies. Electrofusion was routinely employed to produce fusion cells with 30–40% efficiency by using the poorly immunogenic murine B16/F10 cell line, D5 cells, and DC generated from bone marrow cells. CD62L-positive T cells from spleens of naïve mice and the fusion cells were cocultured with a low concentration of IL-2. After 9 days of culture, the antigen-specific T cells were identified with an upregulation of CD25 and CD69 expression and a downregulation of CD62L expression. These cells secreted IFN-γ upon stimulation with irradiated tumour cells. Moreover, when transferred into mice with 3-day established pulmonary metastases, these cells with coadministration of IL-2 exhibited anti-tumour efficacy. In contrast, naïve T cells cocultured with a mixture of unfused DC and irradiated tumour cells did not exhibit anti-tumour efficacy. Our strategy provides the basis for a new approach in adoptive T cell immunotherapy for cancer.     

T cell receptor binding affinity governs the functional profile of cancer-specific CD8+ T cells

Antigen-specific T cell receptor (TCR) gene transfer via patient-derived T cells is an attractive approach to cancer therapy, with the potential to circumvent immune regulatory networks. However, high-affinity tumour-specific TCR clonotypes are typically deleted from the available repertoire during thymic selection because the vast majority of targeted epitopes are derived from autologous proteins. This process places intrinsic constraints on the efficacy of T cell-based cancer vaccines and therapeutic strategies that employ naturally generated tumour-specific TCRs. In this study, we used altered peptide ligands and lentivirus-mediated transduction of affinity-enhanced TCRs selected by phage display to study the functional properties of CD8⁺ T cells specific for three different tumour-associated peptide antigens across a range of binding parameters. The key findings were: (i) TCR affinity controls T cell antigen sensitivity and polyfunctionality; (ii) supraphysiological affinity thresholds exist, above which T cell function cannot be improved; and (iii) T cells transduced with very high-affinity TCRs exhibit cross-reactivity with self-derived peptides presented by the restricting human leucocyte antigen. Optimal system-defined affinity windows above the range established for natural tumour-specific TCRs therefore allow the enhancement of T cell effector function without off-target effects. These findings have major implications for the rational design of novel TCR-based biologics underpinned by rigorous preclinical evaluation.     

New clinical advances in immunotherapy for the treatment of solid tumours

Advances in understanding the mechanisms of cancer cells for evading the immune system surveillance, including how the immune system modulates the phenotype of tumours, have allowed the development of new therapies that benefit from this complex cellular network to specifically target and destroy cancer cells. Immunotherapy researchers have mainly focused on the discovery of tumour antigens that could confer specificity to immune cells to detect and destroy cancer cells, as well as on the mechanisms leading to an improved activation of effector immune cells. The Food and Drug Administration approval in 2010 of ipilumumab for melanoma treatment and of pembrolizumab in 2014, monoclonal antibodies against T‐lymphocyte‐associated antigen 4 and programmed cell death 1, respectively, are encouraging examples of how research in this area can successfully translate into clinical use with promising results. Currently, several ongoing clinical trials are in progress testing new anti‐cancer therapies based on the enhancement of immune cell activity against tumour antigens. Here we discuss the general concepts related to immunotherapy and the recent application to the treatment of cancer with positive results that support their consideration of clinical application to patients.     

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.     

Prostate cancer: advances in immunotherapy

The absence of curative therapies for advanced or recurrent forms of prostate cancer has prompted a vigorous search for novel treatment strategies. Immunotherapy encompasses one particularly promising systemic approach to the treatment of prostate cancer. Immune-based strategies for treating prostate cancer have recently been facilitated by the identification of a number of prostate tissue/tumour antigens that can be targeted, either by antibody or T cells, to promote prostate tumour cell injury or death. These same prostate antigens can also be used for the construction of vaccines to induce prostate-specific T cell-mediated immunity. Greater insight into specific mechanisms that govern antigen-specific T cell activation has brought with it a number of innovative methods to induce and enhance T cell-mediated responses against prostate tumours. For instance, autologous dendritic cells loaded with prostate antigens have proved useful to induce prostate-specific T cell activation. Similarly, in vivo manipulations of T cell costimulatory pathway receptors can greatly facilitate tumour-specific T cell activation and potentiate T cell-mediated responses against a number of malignancies, including prostate cancer. For example, blocking T cell cytotoxic lymphocyte-associated antigen 4 (CTLA-4) receptor binding to its ligand prevents the down-regulation of T cell responses and can even potentiate T cell antitumoural immunity in mouse models of prostate cancer. Androgen ablation (AA) may induce prostate tumour/tissue-specific T cell mediated inflammation and, as such, a phase II trial is currently in progress to ascertain whether CTLA-4 blockade can enhance AA-induced treatment responses in patients with advanced prostate cancer. Nevertheless, further basic and clinical investigation is still required to establish immunotherapy as a true prostate cancer treatment option.     

Avidity optimization of a MAGE-A1-specific TCR with somatic hypermutation

A T-cell receptor (TCR) with optimal avidity to a tumor antigen can be used to redirect T cells to eradicate cancer cells via adoptive cell transfer. Cancer testis antigens (CTAs) are attractive targets because they are expressed in the testis, which is immune-privileged, and in the tumor. However, CTAs are self-antigens and natural TCRs to CTAs have low affinity/avidity due to central tolerance. We previously described a method of directed evolution of TCR avidity using somatic hypermutation. In this study, we made several improvements to this method and enhanced the avidity of the hT27 TCR, which is specific for the cancer testis antigen HLA-A2-MAGE-A1_278-286. We identified eight point mutations with varying degrees of improved avidity. Human T cells transduced with TCRs containing these mutations displayed enhanced tetramer binding, IFN-γ and IL2 production, and cytotoxicity. Most of the mutations have retained specificity, except for one mutant with extremely high avidity. We demonstrate that somatic hypermutation is capable of optimizing avidity of clinically relevant TCRs for immunotherapy.     

LiveCount Assay: concomitant measurement of cytolytic activity and phenotypic characterisation of CD8(+) T-cells by flow cytometry

Tumour immunologists strive to develop efficient tumour vaccination and adoptive transfer therapies that enlarge the pool of tumour-specific and -reactive effector T-cells in vivo. To assess the efficiency of the various strategies, ex vivo assays are needed for the longitudinal monitoring of the patient's specific immune responses providing both quantitative and qualitative data. In particular, since tumour cell cytolysis is the end goal of tumour immunotherapy, routine immune monitoring protocols need to include a read-out for the cytolytic efficiency of Ag-specific cells. We propose to combine current immune monitoring techniques in a highly sensitive and reproducible multi-parametric flow cytometry based cytotoxicity assay that has been optimised to require low numbers of Ag-specific T-cells. The possibility of re-analysing those T-cells that have undergone lytic activity is illustrated by the concomitant detection of CD107a upregulation on the surface of degranulated T-cells. To date, the LiveCount Assay provides the only possibility of assessing the ex vivo cytolytic activity of low-frequency Ag-specific cytotoxic T-lymphocytes from patient material.     

T cell receptor-transgenic primary T cells as a tool for discovery of leukaemia-associated antigens

Identification of a broad array of leukaemia-associated antigens is a crucial step towards immunotherapy of haematological malignancies. However, it is frequently hampered by the decrease of proliferative potential and functional activity of T cell clones used for screening procedures. Transfer of the genes encoding the T cell receptor (TCR) alpha and beta chains of leukaemia-specific clones into primary T cells may help to circumvent this obstacle. In this study, transfer of two minor histocompatibility antigen (minor H antigen)-specific TCRs was performed and the feasibility of the use of TCR-transgenic T cells for identification of minor H antigens through cDNA library screening was investigated. We found that TCR-transgenic cells acquired the specificity of the original clones and matched their sensitivity. Moreover, the higher scale of cytokine-production by TCR-transgenic T cells permits the detection of either small amounts of antigen-positive cells or cells expressing low amounts of an antigen. When applied in equal numbers, TCR-transgenic T cells and the original T cell clones produced similar results in the screening of a cDNA library. However, the use of increased numbers of TCR-transgenic T cells allowed detection of minute amounts of antigen, barely discernible by the T cell clone. In conclusion, TCR-transfer generates a large amount of functional antigen-specific cells suitable for screening of cDNA expression libraries for identification of cognate antigens.     

Novel immunotherapies for hematologic malignancies

The immune system is designed to discriminate between self and tumor tissue. Through genetic recombination, there is fundamentally no limit to the number of tumor antigens that immune cells can recognize. Yet, tumors use a variety of immunosuppressive mechanisms to evade immunity. Insight into how the immune system interacts with tumors is expanding rapidly and has accelerated the translation of immunotherapies into medical breakthroughs. Herein, we appraise novel strategies that exploit the patient's immune system to kill cancer. We review various forms of immune-based therapies, which have shown significant promise in patients with hematologic malignancies, including (i) conventional monoclonal therapies like rituximab; (ii) engineered monoclonal antibodies called bispecific T-cell engagers; (iii) monoclonal antibodies and pharmaceutical drugs that block inhibitory T-cell pathways (i.e. PD-1, CTLA-4, and IDO); and (iv) adoptive cell transfer therapy with T cells engineered to express chimeric antigen receptors or T-cell receptors. We also assess the idea of using these therapies in combination and conclude by suggesting multi-prong approaches to improve treatment outcomes and curative responses in patients.     

T cell receptor‐based cancer immunotherapy: Emerging efficacy and pathways of resistance

Adoptive cell transfer (ACT) using chimeric antigen receptor (CAR)‐modified T cells can induce durable remissions in patients with refractory B‐lymphoid cancers. By contrast, results applying CAR‐modified T cells to solid malignancies have been comparatively modest. Alternative strategies to redirect T cell specificity and cytolytic function are therefore necessary if ACT is to serve a greater role in human cancer treatments. T cell receptors (TCRs) are antigen recognition structures physiologically expressed by all T cells that have complementary, and in some cases superior, properties to CARs. Unlike CARs, TCRs confer recognition to epitopes derived from proteins residing within any subcellular compartment, including the membrane, cytoplasm and nucleus. This enables TCRs to detect a broad universe of targets, such as neoantigens, cancer germline antigens, and viral oncoproteins. Moreover, because TCRs have evolved to efficiently detect and amplify antigenic signals, these receptors respond to epitope densities many fold smaller than required for CAR‐signaling. Herein, we summarize recent clinical data demonstrating that TCR‐based immunotherapies can mediate regression of solid malignancies, including immune‐checkpoint inhibitor refractory cancers. These trials simultaneously highlight emerging mechanisms of TCR resistance. We conclude by discussing how TCR‐based immunotherapies can achieve broader dissemination through innovations in cell manufacturing and non‐viral genome integration techniques.     

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.     

Promising approaches in cancer immunotherapy

Immunotherapy is a promising field, which enhances and harnesses the powers of the host immune system against cancer and in recent years, has become a major application of the fundamental research of cancer immunology. Cancer immunotherapy is often more targeted than non-specific therapy approaches, including radiotherapy or chemotherapy, as the immune system can be trained to remember cancer cells, highlighting a durable approach that can be maintained after the treatment completion. Immunotherapy functions by directing the immune system to attack the tumour cells via targeting tumour antigens, also enhancing the existing anti-tumour immune responses. Current strategies include non-specific immunotherapy, cancer vaccines, oncolytic virus therapy, monoclonal antibodies, immune checkpoints and T cell therapy. The combination of effective approaches can increase the immunotherapy efficacy, leading to durable anti-tumour immune responses. This review will discuss the immunotherapy approaches, particularly immune checkpoints and T cell therapy, which are the most common clinical applications in cancer immunotherapy.     

Tumor-Infiltrating Lymphocyte Therapy for Melanoma: Rationale and Issues for Further Clinical Development

Cancer immunotherapy has become an important area for the future development of cancer therapy; this includes T-cell-based therapies that involve adoptive transfer of autologous T cells derived from the tumors or peripheral blood of cancer patients, vaccines, oncolytic virus therapy, and immunomodulatory antibodies and ligands. Here, we summarize the current approaches and clinical data in the field of adoptive T-cell transfer therapy using tumor-infiltrating lymphocytes (TILs) for metastatic melanoma. We also discuss current knowledge on the mechanism of transferred TILs in mediating tumor regression and the growing need for and recent advances in the identification of predictive biomarkers to better select patients for TIL therapy. The current technical limitations of current TIL expansion methods for out-scaling are discussed as well as how these are being addressed in order to further “industrialize” this form of cell therapy. Lastly, how TIL adoptive transfer can be incorporated into the current melanoma treatment continuum, especially as combination therapy with other immunomodulators and targeted drugs, is discussed.     

Optimizing dendritic cell vaccine for immunotherapy in multiple myeloma: tumour lysates are more potent tumour antigens than idiotype protein to promote anti-tumour immunity

Dendritic cells (DCs) are the most potent antigen-presenting cells and are the mediators of T cell immunity. Many investigators have explored the potential of using DCs as a vaccine for tumour-derived antigens in immunotherapy of B cell malignancies, and the results have been disappointing. To search for better tumour antigens to improve the efficacy of DC-based immunotherapy in myeloma, we evaluated and compared the efficacy of the vaccination of DCs pulsed with idiotype (Id) or tumour lysate in the 5TGM1 myeloma mouse model. Our results showed that Id- or tumour lysate-pulsed DC vaccines protected mice efficiently against developing myeloma, retarded tumour growth, induced tumour regression against established tumour and protected surviving mice from tumour rechallenge. The therapeutic responses were associated with an induction of strong humoral immune responses, including anti-Id or anti-lysate antibodies, and cellular immune responses including myeloma-specific CD8⁺ cytotoxic T lymphocytes, CD4⁺ type 1 T helper cells and memory T cells in mice receiving Id- or tumour lysate-pulsed DC vaccines. In addition, our studies showed that tumour lysate-pulsed DCs were more potent vaccines than the Id-pulsed DC vaccines to promote anti-tumour immunity in the model. This information will be important for improving the strategies of DC-based immunotherapy for patients with myeloma and other B cell tumours.     

Long-lived fusions of human haematological tumour cells and B-lymphoblastoid cells induce tumour antigen-specific cytotoxic T-cell responses in vitro

Tumour-specific cytotoxic T-cells (CTL) are important anti-cancer immune effectors. Most tumour cells, however, do not stimulate effective anti-tumour immune responses, in vivo or in vitro. To enhance tumour cell immunogenicity, we fused human tumour cells from haematological malignancies with the B-lymphoblastoid cell line (LCL), HMy2, to generate a panel of long-lived, self replicating LCL/tumour hybrid cell lines. The LCL/tumour hybrid cell lines expressed HLA class I and class II molecules, CD80 and CD86, and a range of known tumour associated antigens (TAAs). In vitro stimulation of PBLs from healthy, HLA-A2+ individuals by hybrid cell lines induced tumour antigen-specific CTLs to TAAs, including survivin, MAGE-A1, NY-ESO-1 and WT-1. Individual hybrid cell lines simultaneously induced CTL to multiple TAAs. In vitro stimulation of PBL from 2 patients with acute myeloid leukaemia by autologous LCL/tumour hybrid cell lines induced CTL capable of killing the patient's own tumour cells. Our data show, for the first time, that hybrid cell lines formed by fusion of HMy2 cells and haematological tumour cells induce tumour- and tumour antigen-specific cytotoxic T-cell responses in vitro. Hybrid cell lines such as those described may represent novel reagents for use in the immunotherapy of haematological malignancies.     

Preservation and redirection of HPV16E7-specific T cell receptors for immunotherapy of cervical cancer

Human papilloma virus (HPV) type 16 infections of the genital tract are associated with the development of cervical cancer (CxCa) in women. HPV16-derived oncoproteins E6 and E7 are expressed constitutively in these lesions and might therefore be attractive candidates for T-cell-mediated adoptive immunotherapy. However, the low precursor frequency of HPV16E7-specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer. To overcome this problem, we have isolated T cell receptor (TCR) genes from four different HPV16E7-specific healthy donor and patient-derived human cytotoxic T lymphocyte (CTL) clones. We examined whether genetic engineering of peripheral blood-derived CD8+ T cells in order to express HPV16E711-20-specific TCRs is feasible for adoptive transfer purposes. Reporter cells (Jurkat/MA) carrying a transgenic TCR were shown to bind relevant but not irrelevant tetramers. Moreover, these TCR-transgenic Jurkat/MA cells showed reactivity towards relevant target cells, indicating proper functional activity of the TCRs isolated from already available T cell clones. We next introduced an HPV16E711-20-specific TCR into blood-derived, CD8+ recipient T cells. Transgenic CTL clones stained positive for tetramers presenting the relevant HPV16E711-20 epitope and biological activity of the TCR in transduced CTL was confirmed by lytic activity and by interferon (IFN)-gamma secretion upon antigen-specific stimulation. Importantly, we show recognition of the endogenously processed and HLA-A2 presented HPV16E711-20 CTL epitope by A9-TCR-transgenic T cells. Collectively, our data indicate that HPV16E7 TCR gene transfer is feasible as an alternative strategy to generate human HPV16E7-specific T cells for the treatment of patients suffering from cervical cancer and other HPV16-induced malignancies.