Artificial antigen-presenting cells: artificial solutions for real diseases

Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo, is a promising strategy to treat a variety of life-threatening diseases. Unfortunately, current approaches for generating sufficient numbers of antigen-specific T cells lack the ability to serve as reproducible and economically viable methods. This has spurred the development of both cell- and non-cell-based artificial antigen-presenting cells to alleviate problems associated with peptide-loaded dendritic cells in current approaches to adoptive immunotherapy. Here, we review new strategies for the ex vivo generation of antigen-specific T cells and their clinical application. These new approaches have the potential to spearhead a new era of successful adoptive immunotherapy for cancer and infectious diseases.     

Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB

The ex vivo priming and expansion of human cytotoxic T lymphocytes (CTLs) has potential for use in immunotherapy applications for cancer and infectious diseases. To overcome the difficulty in obtaining sufficient numbers of CTLs, we have developed artificial antigen-presenting cells (aAPCs) expressing ligands for the T-cell receptor (TCR) and the CD28 and 4-1BB co-stimulatory surface molecules. These aAPCs reproducibly activate and rapidly expand polyclonal or antigen-specific CD8(+) T cells. The starting repertoire of CD8+ T cells was preserved during culture. Furthermore, apoptosis of cultured CD8(+) T cells was diminished by this approach. This approach may have important therapeutic implications for adoptive immunotherapy.     

Targeted immunotherapy of cancer with CAR T cells: achievements and challenges

The adoptive transfer of chimeric antigen receptor (CAR)-expressing T cells is a relatively new but promising approach in the field of cancer immunotherapy. This therapeutic strategy is based on the genetic reprogramming of T cells with an artificial immune receptor that redirects them against targets on malignant cells and enables their destruction by exerting T cell effector functions. There has been an explosion of interest in the use of CAR T cells as an immunotherapy for cancer. In the pre-clinical setting, there has been a considerable focus upon optimizing the structural and signaling potency of the CAR while advances in bio-processing technology now mean that the clinical testing of these gene-modified T cells has become a reality. This review will summarize the concept of CAR-based immunotherapy and recent clinical trial activity and will further discuss some of the likely future challenges facing CAR-modified T cell therapies.     

Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells

Adoptive immunotherapy holds promise as a treatment for cancer and infectious diseases, but its development has been impeded by the lack of reproducible methods for generating therapeutic numbers of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs). As a result, there are only limited reports of expansion of antigen-specific CTLs to the levels required for clinical therapy. To address this issue, artificial antigen-presenting cells (aAPCs) were made by coupling a soluble human leukocyte antigen-immunoglobulin fusion protein (HLA-Ig) and CD28-specific antibody to beads. HLA-Ig-based aAPCs were used to induce and expand CTLs specific for cytomegalovirus (CMV) or melanoma. aAPC-induced cultures showed robust antigen-specific CTL expansion over successive rounds of stimulation, resulting in the generation of clinically relevant antigen-specific CTLs that recognized endogenous antigen-major histocompatibility complex complexes presented on melanoma cells. These studies show the value of HLA-Ig-based aAPCs for reproducible expansion of disease-specific CTLs for clinical approaches to adoptive immunotherapy.     

T lymphocyte therapy of cancer

The rationale for the use of T lymphocytes to fight cancer is the immunogenicity of tumor cells. T cells are capable to recognize and finally to kill tumor cells. Adoptive cell transfer therapies provide the opportunity to overcome tolerogenic mechanisms by enabling the selection and activation of highly reactive T cell subpopulations and by manipulation of the host environment into which the T cells are introduced. The aim of this article is to review the possibilities, limitations and recent clinical experience with this novel anticancer treatment, namely with adoptive immunotherapy using antigen-specific T cells.     

CTLA-4 Modulates the Differentiation of Inducible Foxp3+ Treg Cells but IL-10 Mediates Their Function in Experimental Autoimmune Encephalomyelitis

In vitro induced Foxp3⁺ T regulatory (iTreg) cells form a novel and promising target for therapeutic tolerance induction. However, the potential of these cells as a target for the treatment of various immune diseases, as well as the factors involved in their development and function, remain debated. Here, we demonstrate in a myelin basic protein (MBP)-specific murine model of CNS autoimmune disease that adoptive transfer of antigen-specific iTreg cells ameliorates disease progression. Moreover, we show that the co-stimulatory molecule CTLA-4 mediates in vitro differentiation of iTreg cells. Finally, we demonstrate that the secreted, immunosuppressive cytokine IL-10 controls the ability of antigen-specific iTreg cells to suppress autoimmune disease. Overall, we conclude that antigen-specific iTreg cells, which depend on various immune regulatory molecules for their differentiation and function, represent a major target for effective immunotherapy of autoimmune disease.     

Challenges in T cell receptor gene therapy

The function of T lymphocytes as orchestrators and effectors of the adaptive immune response is directed by the specificity of their T cell receptors (TCRs). By transferring into T cells the genes encoding antigen-specific receptors, the functional activity of large populations of T cells can be redirected against defined targets including virally infected or cancer cells. The potential of therapeutic T cells to traffic to sites of disease, to expand and to persist after a single treatment remains a major advantage over the currently available immunotherapies that use monoclonal antibodies. Here we review recent progress in the field of TCR gene therapy, outlining challenges to its successful implementation and the strategies being used to overcome them. We detail strategies used in the optimization of affinity and surface expression of the introduced TCR, the choice of T cell subpopulations for gene transfer, and the promotion of persistence of gene-modified T cells in vivo. We review the safety concerns surrounding the use of gene-modified T cells in patients, discussing emerging solutions to these problems, and describe the increasingly positive results from the use of gene-modified T cells in recent clinical trials of adoptive cellular immunotherapy. The increasing sophistication of measures to ensure the safety of engineered T cells is accompanied by an increasing number of clinical trials: these will be essential to guide the effective translation of cellular immunotherapy from the laboratory to the bedside.     

Protective capacity of virus-specific T cell receptor-transduced CD8 T cells in vivo

The transfer of T cell receptor (TCR) genes by viral vectors represents a promising technique to generate antigen-specific T cells for adoptive immunotherapy. TCR-transduced T cells specific for infectious pathogens have been described, but their protective function in vivo has not yet been examined. Here, we demonstrate that CD8 T cells transduced with the P14 TCR specific for the gp33 epitope of lymphocytic choriomeningitis virus exhibit protective activities in both viral and bacterial infection models in mice.     

Expression of MEL-14 antigen is not an absolute requirement for dissemination to lymph nodes after adoptive transfer of murine T lymphocyte clones

The inability of established antigen-specific murine T lymphocyte clones to recirculate well in vivo has been attributed to loss of the surface glycoprotein gp90MEL-14, which is important for specific adherence to post-capillary high endothelial venules in peripheral lymph nodes (LN). Defective recirculation of clones may contribute to inefficient adoptive immunotherapy when compared with fresh immune spleen or LN populations. To optimize models of adoptive immunotherapy, we sought to improve recirculation of Thy-1.2+, L3T4+ clones by inducing reexpression of MEL-14 antigen (gp90MEL-14). Clones were analyzed after treatment with differentiating agents, incubation in the presence or absence of recombinant interleukin 2 (rIL 2), coincubation in vitro with nonirradiated Thy-1.1 LN or thymus cells, or adoptive transfer into Thy-1.1 hosts. We were unable to demonstrate induction of gp90MEL-14 in any case. However, although clones remained MEL-14 negative, they were able to disseminate widely after subcutaneous adoptive transfer in the presence of clone-specific antigen and rIL 2 into Thy-1.1 mice pretreated with cyclophosphamide. Withdrawal of exogenous rIL 2 was associated with rapid disappearance of clones from all sites. We conclude that murine T cell clones undergo a step in terminal differentiation that precludes surface expression of gp90MEL-14 and that these clones would be unlikely to provide a source of long-lived recirculating memory T lymphocytes. However, under appropriate circumstances it is possible for antigen-specific clones to disseminate widely among host LN and mediate short-term immune responses.     

Directed differentiation of induced pluripotent stem cells towards T lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies (1). CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines (2-7). However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies. TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases (8-10). However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic (11-13), HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture (14-16). Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs. Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.     

Control of spontaneous ovarian tumors by CD8+ T cells through NKG2D-targeted delivery of antigenic peptide

Background

There is an urgent need to develop targeted therapies for the control of advanced stage ovarian cancer because it is the most deadly gynecologic cancer. Antigen-specific immunotherapy is a promising approach because of the potential of the immune system to specifically target tumors without the toxicity associated with traditional chemoradiation. However, one of the major limitations for antigen-specific cancer immunotherapy is the pre-existing immune tolerance against endogenous targeted tumor antigens that frequently evolves during carcinogenesis. Here, we described the creation of a therapeutic agent comprised of a tumor-homing module fused to a functional domain capable of selectively rendering tumor cells sensitive to foreign antigen-specific CD8+ T cell-mediated immune attack, thereby circumventing many aspects of immune tolerance. The tumor-homing module, NKG2D, specifically binds to NKG2D ligand that is commonly overexpressed in ovarian tumors. The functional domain is comprised of the Fc portion of IgG2a protein and foreign immunogenic CD8⁺ T cell epitope flanked by furin cleavage sites (R), which can be recognized and cleaved by furin that is highly expressed in the tumor microenvironment.

Results

We show that this therapeutic chimeric protein specifically loaded antigenic epitope onto the surface of NKG2D ligand-expressing ovarian tumor cells, rendering ovarian tumors susceptible to antigen-specific CTL-mediated killing in vitro. Furthermore, we show that intraperitoneal administration of our therapeutic chimeric protein followed by adoptive transfer of antigen-specific CD8+ T cells generates potent antitumor effects and significant accumulation of antigen-specific CD8+ T cells in the tumor loci.

Conclusions

Our findings have promise for bypassing immune tolerance to enhance cancer immunotherapy.

     

Loss of Fas (CD95/APO-1) expression by antigen-specific cytotoxic T cells is reversed by inhibiting DNA methylation

Elimination of clonally expanded peripheral CD8 T cells was thought to involve apoptosis induction mediated principally by TNF, but recently Fas (CD95/APO-1) has been shown to play a role in certain responses. Here we study Fas expression and sensitivity to its ligation on murine CD8 cells specific for the CW3 antigen expressed by transfected P815 cells. Fas was progressively downregulated after successive in vitro restimulations of antigen-specific CD8 cells, until clones became Fas negative and totally resistant to the effects of recombinant Fas ligand. In contrast, Fas expression by in vivo restimulated antigen-specific cells did not diminish. Loss of Fas expression in vitro was not totally irreversible, since it could be reinduced by inhibition of DNA methylation. Understanding how Fas may be differentially regulated in vivo and in vitro is an important issue for the optimal manipulation of T cells for adoptive immunotherapy protocols.     

IL-2 during in vitro priming promotes subsequent engraftment and successful adoptive tumor immunotherapy by persistent memory phenotypic CD8(+) T cells

Adoptive T cell tumor immunotherapy potentially consists of two protective components by the transferred effector cells, the immediate immune response and the subsequent development of memory T cells. The extent by which adoptively transferred CD8(+) CTL are destined to become memory T cells is ambiguous as most studies focus on the acute effects on tumor shortly following adoptive transfer. In this study we show that a substantial fraction of the input CTL develop into memory cells that reject a s.c. tumor challenge. The use of exogenous IL-2 or a combination of IL-2 and IL-4, but not solely IL-4, during the ex vivo culture for the CTL inoculation was necessary for efficient development of CD8(+) memory T cells. Thus, an important component of adoptive immunotherapy using CTL is the production of CD8(+) Ag-specific memory cells which is primarily favored by IL-2 receptor signaling during ex vivo generation of the effector CTL.     

Evaluating the Cellular Targets of Anti-4-1BB Agonist Antibody during Immunotherapy of a Pre-Established Tumor in Mice

Background

Manipulation of the immune system represents a promising avenue for cancer therapy. Rational advances in immunotherapy of cancer will require an understanding of the precise correlates of protection. Agonistic antibodies against the tumor necrosis factor receptor family member 4-1BB are emerging as a promising tool in cancer therapy, with evidence that these antibodies expand both T cells as well as innate immune cells. Depletion studies have suggested that several cell types can play a role in these immunotherapeutic regimens, but do not reveal which cells must directly receive the 4-1BB signals for effective therapy.

Methodology/Principal Findings

We show that re-activated memory T cells are superior to resting memory T cells in control of an 8-day pre-established E.G7 tumor in mice. We find that ex vivo activation of the memory T cells allows the activated effectors to continue to divide and enter the tumor, regardless of antigen-specificity; however, only antigen-specific reactivated memory T cells show any efficacy in tumor control. When agonistic anti-4-1BB antibody is combined with this optimized adoptive T cell therapy, 80% of mice survive and are fully protected from tumor rechallenge. Using 4-1BB-deficient mice and mixed bone marrow chimeras, we find that it is sufficient to have 4-1BB only on the endogenous host αβ T cells or only on the transferred T cells for the effects of anti-4-1BB to be realized. Conversely, although multiple immune cell types express 4-1BB and both T cells and APC expand during anti-4-1BB therapy, 4-1BB on cells other than αβ T cells is neither necessary nor sufficient for the effect of anti-4-1BB in this adoptive immunotherapy model.

Conclusions/Significance

This study establishes αβ T cells rather than innate immune cells as the critical target in anti-4-1BB therapy of a pre-established tumor. The study also demonstrates that ex vivo activation of memory T cells prior to infusion allows antigen-specific tumor control without the need for reactivation of the memory T cells in the tumor.     

In vivo cyclophosphamide and IL-2 treatment impedes self-antigen-induced effector CD4 cell tolerization: implications for adoptive immunotherapy

The development of T cell tolerance directed toward tumor-associated Ags can limit the repertoire of functional tumor-reactive T cells, thus impairing the ability of vaccines to elicit effective antitumor immunity. Adoptive immunotherapy strategies using ex vivo expanded tumor-reactive effector T cells can bypass this problem; however, the susceptibility of effector T cells to undergoing tolerization suggests that tolerance might also negatively impact adoptive immunotherapy. Nonetheless, adoptive immunotherapy strategies can be effective, particularly those utilizing the drug cyclophosphamide (CY) and/or exogenous IL-2. In the current study, we used a TCR-transgenic mouse adoptive transfer system to assess whether CY plus IL-2 treatment rescues effector CD4 cell function in the face of tolerizing Ag (i.e., cognate parenchymal self-Ag). CY plus IL-2 treatment not only enhances proliferation and accumulation of effector CD4 cells, but also preserves the ability of these cells to express the effector cytokine IFN-gamma (and to a lesser extent TNF-alpha) in proportion to the level of parenchymal self-Ag expression. When administered individually, CY but not IL-2 can markedly impede tolerization, although their combination is the most effective. Although effector CD4 cells in CY plus IL-2-treated self-Ag-expressing mice eventually succumb to tolerization, this delay results in an increased level of in situ IFN-gamma expression in cognate Ag-expressing parenchymal tissues as well as death via a mechanism that requires direct parenchymal Ag presentation. These results suggest that one potential mechanism by which CY and IL-2 augment adoptive immunotherapy strategies to treat cancer is by impeding the tolerization of tumor-reactive effector T cells.     

In vivo functional efficacy of tumor-specific T cells expanded using HLA-Ig based artificial antigen presenting cells (aAPC)

Adoptive immunotherapy for treatment of cancers and infectious diseases is often hampered by a high degree of variability in the final T cell product and in the limited in vivo function and survival of ex vivo expanded antigen-specific cytotoxic T cells (CTL). This has stimulated interest in development of standardized artificial antigen presenting cells (aAPC) to reliably expand antigen specific CTL. However, for successful immunotherapy the aAPC ex vivo generated CTL must have anti-tumor activity in vivo. Here, we demonstrate that HLA-Ig based aAPC stimulated tumor-specific CTL from human peripheral blood T lymphocytes showed robust expansion and functional activity in a human/SCID mouse melanoma model. HLA-Ig based aAPC expanded CTL were detected in the peripheral blood up to 15 days after transfer. Non-invasive bioluminescence imaging of tumor bearing mice demonstrated antigen dependent localization of transferred CTL to the tumor site. Moreover, adoptive transfer of HLA-Ig based aAPC generated CTL inhibited the tumor growth both in prevention and treatment modes of therapy and was comparable to that achieved by dendritic cell expanded CTL. Thus, our data demonstrate potential therapeutic in vivo activity of HLA-Ig based aAPC expanded CTL to control tumor growth.     

Immunotherapy for head and neck cancer

Head and neck cancer represents a challenging disease. Despite recent treatment advances, which have improved functional outcomes, the long-term survival of head and neck cancer patients has remained unchanged for the past 25 years. One of the goals of adjuvant cancer therapy is to eradicate local regional microscopic and micrometastatic disease with minimal toxicity to surrounding normal cells. In this respect, antigen-specific immunotherapy is an attractive therapeutic approach. With the advances in molecular genetics and fundamental immunology, antigen-specific immunotherapy is being actively explored using DNA, bacterial vector, viral vector, peptide, protein, dendritic cell, and tumor-cell based vaccines. Early phase clinical trials have demonstrated the safety and feasibility of these novel therapies and the emphasis is now shifting towards the development of strategies, which can increase the potency of these vaccines. As the field of immunotherapy matures and as our understanding of the complex interaction between tumor and host develops, we get closer to realizing the potential of immunotherapy as an adjunctive method to control head and neck cancer and improve long-term survival in this patient population.     

Dendritic cell recovery post-lymphodepletion: a potential mechanism for anti-cancer adoptive T cell therapy and vaccination

Adoptive transfer of autologous tumor-reactive T cells holds promise as a cancer immunotherapy. In this approach, T cells are harvested from a tumor-bearing host, expanded in vitro and infused back to the same host. Conditioning of the recipient host with a lymphodepletion regimen of chemotherapy or radiotherapy before adoptive T cell transfer has been shown to substantially improve survival and anti-tumor responses of the transferred cells. These effects are further enhanced when the adoptive T cell transfer is followed by vaccination with tumor antigens in combination with a potent immune adjuvant. Although significant progress has been made toward an understanding of the reasons underlying the beneficial effects of lymphodepletion to T cell adoptive therapy, the precise mechanisms remain poorly understood. Recent studies, including ours, would indicate a more central role for antigen presenting cells, in particular dendritic cells. Unraveling the exact role of these important cells in mediation of the beneficial effects of lymphodepletion could provide novel pathways toward the rational design of more effective anti-cancer immunotherapy. This article focuses on how the frequency, phenotype, and functions of dendritic cells are altered during the lymphopenic and recovery phases post-induction of lymphodepletion, and how they affect the anti-tumor responses of adoptively transferred T cells.     

Adoptive immunotherapy combined with intratumoral TLR agonist delivery eradicates established melanoma in mice

Toll-like receptor (TLR) agonists can trigger broad inflammatory responses that elicit rapid innate immunity and promote the activities of lymphocytes, which can potentially enhance adoptive immunotherapy in the tumor-bearing setting. In the present study, we found that Polyinosinic:Polycytidylic Acid [Poly(I:C)] and CpG oligodeoxynucleotide 1826 [CpG], agonists for TLR 3 and 9, respectively, potently activated adoptively transferred T cells against a murine model of established melanoma. Intratumoral injection of Poly(I:C) and CpG, combined with systemic transfer of activated pmel-1 T cells, specific for gp100_25–33, led to enhanced survival and eradication of 9-day established subcutaneous B16F10 melanomas in a proportion of mice. A series of survival studies in knockout mice supported a key mechanistic pathway, whereby TLR agonists acted via host cells to enhance IFN-γ production by adoptively transferred T cells. IFN-γ, in turn, enhanced the immunogenicity of the B16F10 melanoma line, leading to increased killing by adoptively transferred T cells. Thus, this combination approach counteracted tumor escape from immunotherapy via downregulation of immunogenicity. In conclusion, TLR agonists may represent advanced adjuvants within the setting of adoptive T-cell immunotherapy of cancer and hold promise as a safe means of enhancing this approach within the clinic.     

Telomere length of transferred lymphocytes correlates with in vivo persistence and tumor regression in melanoma patients receiving cell transfer therapy

Recent studies have indicated that adoptive immunotherapy with autologous antitumor tumor-infiltrating lymphocytes (TILs) following nonmyeloablative chemotherapy mediates tumor regression in approximately 50% of treated patients with metastatic melanoma, and that tumor regression is correlated with the degree of persistence of adoptively transferred T cells in peripheral blood. These findings, which suggested that the proliferative potential of transferred T cells may play a role in clinical responses, led to the current studies in which telomere length as well as phenotypic markers expressed on the administered TILs were examined. TILs that were associated with objective clinical responses following adoptive transfer possessed a mean telomere length of 6.3 kb, whereas TILs that were not associated with significant clinical responses were significantly shorter, averaging 4.9 kb (p < 0.01). Furthermore, individual TIL-derived T cell clonotypes that persisted in vivo following adoptive cell transfer possessed telomeres that were longer than telomeres of T cell clonotypes that failed to persist (6.2 vs 4.5 kb, respectively; p < 0.001). Expression of the costimulatory molecule CD28 also appeared to be associated with long telomeres and T cell persistence. These results, indicating that the telomere length of transferred lymphocytes correlated with in vivo T cell persistence following adoptive transfer, and coupled with the previous observation that T cell persistence was associated with clinical responses in this adoptive immunotherapy trial, suggest that telomere length and the proliferative potential of the transferred T cells may play a significant role in mediating response to adoptive immunotherapy.