Generating natural killer cells for adoptive transfer: expanding horizons

Natural killer (NK) cells are unique innate lymphoid cells that have therapeutic potential in adoptive cell transfer-based cancer immunotherapy that has been established across a range of early-phase clinical trials. NK cells for use in adoptive transfer therapies are obtained from various sources, including primary NK cells from peripheral blood or apheresis products (autologous or allogeneic) and umbilical cord blood. NK cells have also been generated from CD34+ hematopoietic progenitors, induced pluripotent stem cells, embryonic stem cells and malignant cell lines. Apheresis-derived NK cell products are often administered after brief cytokine-based ex vivo activation, ideally aiming for in vivo expansion and proliferation. NK cells from other sources or from smaller volumes of blood require a longer period of expansion prior to therapeutic use. Although ex vivo NK cell expansion introduces a concern for senescence and exhaustion, there is also an opportunity to achieve higher NK cell doses, modulate NK cell activation characteristics and apply genetic engineering approaches, ultimately generating potent effector cells from small volumes of readily available starting materials. Herein the authors review the field of clinical-grade NK cell expansion, explore the desirable features of an idealized NK cell expansion approach and focus on techniques used in recently published clinical trials.     

Potency assays for therapeutic live whole cell cancer vaccines.

Therapeutic cancer vaccines are under development with the goal of enhancing the body's immune response to cancer cells sufficient to arrest cancer cell growth. Among the various approaches being used are those based on whole tumor cells. Developing a suitable measure of the potency of such vaccines presents a significant challenge because neither cellular associated markers nor in vivo biological responses that are correlated with efficacy have been identified; nevertheless, manufacturers and regulatory agencies will need to develop methods to evaluate these products. At this moment, the challenge for manufacturers who are developing whole cell vaccines is to demonstrate batch-to-batch consistency for the vaccine used in clinical studies and to show that comparable vaccine batches have the same capacity to achieve an acceptable level of biological activity that may be related to efficacy. This is particularly challenging in that animal models to test that activity do not exist and direct serological or immunological correlates of clinical protection are not available because protection has not yet been established in clinical trials. In the absence of well-defined biological markers and tests for manufacturing consistency, manufacturers and regulators will need to rely heavily on a highly reproducible manufacturing process--the consistency of the process therefore becomes critical. In developing regulatory approaches to whole cell cancer vaccines, the experience from the field of infectious disease vaccines should be examined for general guidance. A framework that draws heavily on the field of infectious disease vaccines is presented and suggests that at this point in the development of this new class of products, it is reasonable to develop data on quantitative antigen expression as a measure of potency with the expectation that when clinical efficacy has been established it will confirm the appropriateness of this approach. But because this will not be known until the end of a pivotal trial, a bioassay should be considered and run in parallel. Several examples of bioassays are presented along with their advantages and disadvantages. The final selection of a potency assay for use in lot release of a commercializable therapeutic whole cell vaccine ultimately will depend on the totality of the data available at the time of approval by regulatory agencies. Based on information currently available, it is likely that quantitative antigen expression or a bioassay could be used to measure potency. If both are determined to be acceptable, the use of quantitative antigen expression could be considered for routine lot release, while the bioassay could be reserved for use as one of the elements in establishing comparability when manufacturing changes are being considered after approval.     

Recent advances in stem cells therapy: A focus on cancer,Parkinson’s and Alzheimer’s

Stem cells serve as potential therapeutics due to their high proliferative capacity, low immunogenic reactivity and their differentiating capabilities. Several pre-clinical and early-stage clinical studies are carried out to treat genetic diseases, cancers and neurodegenerative disorders with promising preliminary results. However, there are still many challenges that scientists are trying to overcome such as the unclear expression profile of stem cells in vivo, the homing of stem cells to the site of injury and their potential immune-reactivity. Prospective research lies in gene editing of autologous stem cells in vitro and safe injection of these modified cells back into patients. Here, we review the clinical trials executed using stem cell therapy in an attempt to cure challenging diseases like cancer, Parkinson’s and Alzheimer’s diseases.     

细胞治疗的典范：嵌合抗原受体T细胞疗法

嵌合抗原受体T(CAR-T)细胞疗法是一种利用合成受体特异性靶向抗原的过继性细胞疗法(ACT),目前在血液肿瘤的治疗中有极大的临床应用价值。虽然美国食品药品监督管理局(FDA)已经批准两款CAR-T药物上市,但CAR-T疗法在治疗过程中仍然存在一些副作用,如细胞因子释放综合征(CRS)、神经毒性、B细胞功能缺失等。同时,CAR-T疗法在实体瘤治疗中的效果甚微,主要原因是缺乏特异性靶点以及肿瘤微环境对CAR-T细胞功能的抑制等。文中将从CAR的结构设计、临床应用、合成生物学对新型CAR的优化来阐述应用CAR-T细胞疗法治疗肿瘤所面临的挑战及广阔前景。     

Monocyte and interferon based therapy for the treatment of ovarian cancer

Cytokines and cells of the innate immune system have been shown to be critical regulators in the elimination, equilibrium and escape of malignant cells. Despite in vitro and in vivo evidence, components of the innate immune system have shown limited efficacy in the treatment of ovarian cancer. Intraperitoneal immunotherapies are a promising field that has not yet been fully explored in ovarian cancer. Cytokine immunotherapy using interferon alpha (IFN-α) and interferon gamma (IFN-γ) has predominantly been used intraperitoneally in ovarian cancer, with promising results. Early studies also showed that autologous monocytes infused into the peritoneum have anti-tumor properties. Combination therapies have been shown to be more effective in treating cancer than mono-therapies. Based on these observations the combination of cell therapy with cytokine therapy may provide a unique strategy for the treatment of chemotherapy resistant solid cancers.     

Biomechanics and mechanobiology in functional tissue engineering

The field of tissue engineering continues to expand and mature, and several products are now in clinical use, with numerous other preclinical and clinical studies underway. However, specific challenges still remain in the repair or regeneration of tissues that serve a predominantly biomechanical function. Furthermore, it is now clear that mechanobiological interactions between cells and scaffolds can critically influence cell behavior, even in tissues and organs that do not serve an overt biomechanical role. Over the past decade, the field of “functional tissue engineering” has grown as a subfield of tissue engineering to address the challenges and questions on the role of biomechanics and mechanobiology in tissue engineering. Originally posed as a set of principles and guidelines for engineering of load-bearing tissues, functional tissue engineering has grown to encompass several related areas that have proven to have important implications for tissue repair and regeneration. These topics include measurement and modeling of the in vivo biomechanical environment; quantitative analysis of the mechanical properties of native tissues, scaffolds, and repair tissues; development of rationale criteria for the design and assessment of engineered tissues; investigation of the effects biomechanical factors on native and repair tissues, in vivo and in vitro; and development and application of computational models of tissue growth and remodeling. Here we further expand this paradigm and provide examples of the numerous advances in the field over the past decade. Consideration of these principles in the design process will hopefully improve the safety, efficacy, and overall success of engineered tissue replacements.     

Bioreactor design for clinical-grade expansion of stem cells

The many clinical trials currently in progress will likely lead to the widespread use of stem cell-based therapies for an extensive variety of diseases, either in autologous or allogeneic settings. With the current pace of progress, in a few years' time, the field of stem cell-based therapy should be able to respond to the market demand for safe, robust and clinically efficient stem cell-based therapeutics. Due to the limited number of stem cells that can be obtained from a single donor, one of the major challenges on the roadmap for regulatory approval of such medicinal products is the expansion of stem cells using Good Manufacturing Practices (GMP)-compliant culture systems. In fact, manufacturing costs, which include production and quality control procedures, may be the main hurdle for developing cost-effective stem cell therapies. Bioreactors provide a viable alternative to the traditional static culture systems in that bioreactors provide the required scalability, incorporate monitoring and control tools, and possess the operational flexibility to be adapted to the differing requirements imposed by various clinical applications. Bioreactor systems face a number of issues when incorporated into stem cell expansion protocols, both during development at the research level and when bioreactors are used in on-going clinical trials. This review provides an overview of the issues that must be confronted during the development of GMP-compliant bioreactors systems used to support the various clinical applications employing stem cells.     

Technological progress and challenges towards cGMP manufacturing of human pluripotent stem cells based therapeutic products for allogeneic and autologous cell therapies

Recent technological advances in the generation, characterization, and bioprocessing of human pluripotent stem cells (hPSCs) have created new hope for their use as a source for production of cell-based therapeutic products. To date, a few clinical trials that have used therapeutic cells derived from hESCs have been approved by the Food and Drug Administration (FDA), but numerous new hPSC-based cell therapy products are under various stages of development in cell therapy-specialized companies and their future market is estimated to be very promising. However, the multitude of critical challenges regarding different aspects of hPSC-based therapeutic product manufacturing and their therapies have made progress for the introduction of new products and clinical applications very slow. These challenges include scientific, technological, clinical, policy, and financial aspects. The technological aspects of manufacturing hPSC-based therapeutic products for allogeneic and autologous cell therapies according to good manufacturing practice (cGMP) quality requirements is one of the most important challenging and emerging topics in the development of new hPSCs for clinical use. In this review, we describe main critical challenges and highlight a series of technological advances in all aspects of hPSC-based therapeutic product manufacturing including clinical grade cell line development, large-scale banking, upstream processing, downstream processing, and quality assessment of final cell therapeutic products that have brought hPSCs closer to clinical application and commercial cGMP manufacturing.     

Emerging frontiers in immuno- and gene therapy for cancer

Background aimsThe field of cell and gene therapy in oncology has moved rapidly since 2017 when the first cell and gene therapies, Kymriah followed by Yescarta, were approved by the Food and Drug Administration in the United States, followed by multiple other countries. Since those approvals, several new products have gone on to receive approval for additional indications. Meanwhile, efforts have been made to target different cancers, improve the logistics of delivery and reduce the cost associated with novel cell and gene therapies. Here, we highlight various cell and gene therapy-related technologies and advances that provide insight into how these new technologies will speed the translation of these therapies into the clinic.ConclusionsIn this review, we provide a broad overview of the current state of cell and gene therapy-based approaches for cancer treatment – discussing various effector cell types and their sources, recent advances in both CAR and non-CAR genetic modifications, and highlighting a few promising approaches for increasing in vivo efficacy and persistence of therapeutic drug products.     

Generation of cytotoxic responses in mice and human individuals against hematological malignancies using survivin-RNA-transfected dendritic cells

Survivin is a member of the inhibitors of apoptosis family and is overexpressed in many types of human cancers, making it an attractive target for T cell-based immunotherapeutic strategies. Recently, HLA-A2-binding peptides derived from the survivin protein were identified as capable of inducing specific T cell responses in cancer patients. Here we demonstrate that human survivin-specific CTLs generated from PBMC by stimulation with autologous dendritic cells transfected with survivin-RNA were cytotoxic for a range of hemopoietic malignant cell lines and primary tumor cells isolated from patients with acute myeloid leukemia. We also show that vaccination of mice with survivin-RNA-transfected dendritic cells leads to long term resistance to challenge by a survivin-expressing lymphoma, demonstrating the potential of survivin as a tumor rejection Ag. Our data provide evidence for the use of survivin as a target structure for immunotherapeutic strategies against hematological neoplasms.     

Combined blockade of TIM-3 and TIM-4 augments cancer vaccine efficacy against established melanomas

Cancer vaccines have been developed to instruct the endogenous immune responses to autologous tumors and to generate durable clinical responses. However, the therapeutic benefits of cancer vaccines remain insufficient due to the multiple immunosuppressive signals delivered by tumors. Thus, to improve the clinical efficacy of cancer immunotherapy, it is important to develop new modalities to overcome immunosuppressive tumor microenvironments and elicit effective antitumor immune responses. In this study, we show that novel monoclonal antibodies (mAbs) specifically targeting either T cell immunoglobulin mucin protein-3 (TIM-3) or T cell immunoglobulin mucin protein-4 (TIM-4) enhance the therapeutic effects of vaccination against established B16 murine melanomas. This is true for vaccination with irradiated B16 melanoma cells engineered to express the flt3 ligand gene (FVAX). More importantly, combining anti-TIM-3 and anti-TIM-4 mAbs markedly increased vaccine-induced antitumor responses against established B16 melanoma. TIM-3 blockade mainly stimulated antitumor effector activities via natural killer cell-dependent mechanisms, while CD8⁺ T cells served as the main effectors induced by anti-TIM-4 mAb. Our findings reveal that therapeutic manipulation of TIM-3 and TIM-4 may provide a novel strategy for improving the clinical efficacy of cancer immunotherapy.     

Comparison of cytotoxic T lymphocyte responses against pancreatic cancer induced by dendritic cells transfected with total tumor RNA and fusion hybrided with tumor cell

Pancreatic cancer (PC) is a deadly human malignancy. Dendritic cell (DC)-based immunotherapy with whole tumor antigens demonstrates potential efficiency in cancer treatment. Tumor RNA and tumor fusion hybrid cells are sources of whole tumor antigens for preparing DC tumor vaccines. However, the efficacy of these sources in eliciting immune responses against PC has not yet to be directly compared. In the present study, patient-derived PC cells and DCs were fused (DC–tumor hybrids) and primary cultured PC cell-derived total RNA was electroporated into autologous DCs (DC–tumor RNA). The antitumor immune responses induced by DC–tumor hybrids and DC–tumor RNA were compared directly. The results showed that both RNA and hybrid methodologies could induce tumor-specific cytotoxic T lymphocyte (CTL) responses, but pulsing DCs with total tumor RNA could induce a higher frequency of activated CTLs and T-helper cells than fusing DCs with autologous tumor cells. In addition, DC–tumor RNA triggered stronger autologous tumor cell lysis than DC–tumor hybrids. It could be concluded that DCs pulsed with whole tumor RNA are superior to those fused with tumor cells in priming anti-PC CTL responses. Electroporation with total tumor RNA may be more suitable for DC-based PC vaccination.     

A roundtable on responsible innovation with autologous stem cells in Australia,Japan and Singapore

We report on a roundtable event hosted in Singapore that sought to identify some of the ethical and regulatory challenges in translating autologous cell-based interventions, particularly those claiming to involve stem cells, into safe and effective therapies and to propose some solutions to encourage responsible innovation with these products. Challenges are identified in the three areas of cell manufacturing and processing, innovative uses of autologous cells in clinical practice and standards of evidence. Proposed solutions are discussed within a co-operative model of statutory laws and regulations that can enable product development with autologous cells and professional codes and standards that can encourage ethical conduct in clinical practice. Future research should be directed toward establishing regional networks for the development of internationally consistent standards in manufacturing and ethical codes of conduct for innovating with stem cells, and other autologous cells, and fostering ongoing exchange between jurisdictions.     

Mathematical model of a personalized neoantigen cancer vaccine and the human immune system

Cancer vaccines are an important component of the cancer immunotherapy toolkit enhancing immune response to malignant cells by activating CD4⁺ and CD8⁺ T cells. Multiple successful clinical applications of cancer vaccines have shown good safety and efficacy. Despite the notable progress, significant challenges remain in obtaining consistent immune responses across heterogeneous patient populations, as well as various cancers. We present a mechanistic mathematical model describing key interactions of a personalized neoantigen cancer vaccine with an individual patient’s immune system. Specifically, the model considers the vaccine concentration of tumor-specific antigen peptides and adjuvant, the patient’s major histocompatibility complexes I and II copy numbers, tumor size, T cells, and antigen presenting cells. We parametrized the model using patient-specific data from a clinical study in which individualized cancer vaccines were used to treat six melanoma patients. Model simulations predicted both immune responses, represented by T cell counts, to the vaccine as well as clinical outcome (determined as change of tumor size). This model, although complex, can be used to describe, simulate, and predict the behavior of the human immune system to a personalized cancer vaccine.     

Autologous dendritic cell vaccine for estrogen receptor (ER)/progestin receptor (PR) double-negative breast cancer

Purpose

A wealth of preclinical information, as well as a modest amount of clinical information, indicates that dendritic cell vaccines have therapeutic potential. The aim of this work was to assess the immune response, disease progression, and post-treatment survival of ER/PR double-negative stage II/IIIA breast cancer patients vaccinated with autologous dendritic cells pulsed with autologous tumor lysates.

Methods

Dendritic cell (DC) vaccines were generated from CD14+ precursors pulsed with autologous tumor lysates. DCs were matured with defined factors that induced surface marker and cytokine production. Individuals were immunized intradermally four times. Specific delayed type IV hypersensitivity (DTH) reaction, ex vivo cytokine production, and lymphocyte subsets were determined for the evaluation of the therapeutic efficiency. Overall survival and disease progression rates were analyzed using Kaplan–Meier curves and compared with those of contemporaneous patients who were not administered DC vaccines.

Results

There were no unanticipated or serious adverse effects. DC vaccines elicited Th1 cytokine secretion and increased NK cells, CD8+ IFN-γ+ cells but decreased the percentage of CD3+ T cells and CD3+ HLA-DR+ T cells in the peripheral blood. Approximately 58% (18/31) of patients had a DTH-positive reaction. There was no difference in overall survival between the patients with and without DC vaccine. The 3-year progression-free survival was significantly prolonged: 76.9% versus 31.0% (with vs. without DC vaccine, p?Conclusion Our findings strongly suggest that tumor lysate-pulsed DCs provide a standardized and widely applicable source of breast cancer antigens that are very effective in evoking anti-breast cancer immune responses.     

Boosting the immune response: the use of iNKT cell ligands as vaccine adjuvants

Natural killer T (NKT) cells comprise a small, but important T cell subset and are thought to bridge the innate and adaptive immune responses. The discovery of NKT cells and extensive research on their activating ligands have paved the way for modulation of these potent immunoregulatory cells in order to improve the outcome of various clinical conditions. Efforts to modulate NKT cell effector functions have ranged from therapy for influenza to antitumor immunotherapy. These approaches have also led to the use of NKTcell agonists such as ??-Galactosylceramide (??-GalCer) and its analogs as vaccine adjuvants, an approach that is aimed at boosting specific B and Tcell responses to a vaccine candidate by concomitant activation of NKT cells. In this review we will provide a comprehensive overview of the efforts made in using ??-GalCer and its analogs as vaccine adjuvants. The diverse array of vaccination strategies used, as well as the role of NKTcell activating adjuvants will be discussed, with focus on vaccines against malaria, HIV, influenza and tumor vaccines. Collectively, these studies demonstrate the efficacy of NKT cell-specific agonists as adjuvants and suggest that these compounds warrant serious consideration during the development of vaccination strategies.     

Prospects and challenges of induced pluripotent stem cells as a source of hematopoietic stem cells

Many life-threatening hematological diseases are now treated by bone marrow transplantations, i.e., infusion of hematopoietic stem cells (HSCs). HSC transplantations are a valid option for the treatment of a variety of metabolic disorders, and even for solid tumors and some refractory severe autoimmune diseases. Unfortunately, the frequency and outcome of HSC transplantations are limited by a shortage of suitable donors. Induced pluripotent stem cells (iPSCs)-somatic cells that have acquired pluripotent stem cell characteristics by the ectopic expression of pluripotency-inducing factors-have been proposed as an alternative source of HSCs. Possible applications include cells of autologous, of autologous and genetically modified, or of allogeneic origin. Here, we provide a perspective on the distinct opportunities of iPSCs and discuss the challenges that lie ahead.     

Spotlight on chimeric antigen receptor engineered T cell research and clinical trials in China

T cell mediated adoptive immune response has been characterized as the key to anti-tumor immunity. Scientists around the world including in China, have been trying to harness the power of T cells against tumors for decades. Recently, the biosynthetic chimeric antigen receptor engineered T cell(CAR-T) strategy was developed and exhibited encouraging clinical efficacy, especially in hematological malignancies. Chimeric antigen receptor research reports began in 2009 in China according to our Pub Med search results. Clinical trials have been ongoing in China since 2013 according to the trial registrations on clinicaltrials.gov.. After years of assiduous efforts, research and clinical scientists in China have made their own achievements in the CAR-T therapy field. In this review, we aim to highlight CAR-T research and clinical trials in China, to provide an informative reference for colleagues in the field.     

Establishing guidelines for CAR-T cells: challenges and considerations

T cells, genetically modified by chimeric antigen receptors (CAR-T), are endowed with specificity to a desired antigen and are cytotoxic to cells expressing the targeted antigen. CAR-T-based cancer immunotherapy is a promising therapy for curing hematological malignancy, such as acute lymphoid leukemia, and is promising for extending their efficacy to defeat solid tumors. To date, dozens of different CAR-T cells have been evaluated in clinical trials to treat tumors; this necessitates the establishment of guidelines for the production and application of CAR-T cells. However, it is challenging to standardize CAR-T cancer therapy because it involves a combination of gene therapy and cell therapy. In this review, we compare the existing guidelines for CAR-T cells and discuss the challenges and considerations for establishing guidance for CAR-T-based cancer immunotherapy.     

International Meeting “Immunotherapy of Cancer: Challenges and Needs”

The main aims of the international meeting “Immunotherapy of Cancer: Challenges and Needs” were to review the state of the art of cancer immunotherapy and to identify critical issues which deserve special attention for promoting progress of research in this field, with a particular focus on the perspectives of clinical research. Novel concepts and strategies for identifying, monitoring and predicting effective responses to cancer immunotherapy protocols were presented, focused on the use of adjuvants (CpG oligonucleotides) or cytokines (IFN-alpha) to enhance the efficacy of cancer vaccines. Moreover, the possible advantages of using different types of dendritic cells (for active immunization strategies) or T cells (for adoptive immunotherapy protocols) were debated. A consensus was achieved on the need for enhancing the efficacy of cancer vaccines or adoptive cell immunotherapy by combining these strategies with other anti-cancer treatments, including chemotherapy. Finally, initiatives for promoting clinical research by establishing a strategic cooperation in the field of cancer immunotherapy based on the active participation of all the relevant actors, including public institutions responsible of Public Health, National Cancer Institutes, industry, representatives of regulatory bodies, and patients’ organizations were proposed.