TCR transgenes and transgene cassettes for TCR gene therapy: status in 2008

The genetic introduction of T cell receptor genes into T cells has been developed over the past decade as a strategy to induce defined antigen-specific T cell immunity. With the potential value of TCR gene therapy well-established in murine models and the feasibility of infusion of TCR-modified autologous T cells shown in a first phase I trial, the next key step will be to transform TCR gene transfer from an experimental technique into a robust clinical strategy. In this review, we discuss the different properties of the TCR transgene and transgene cassette that can strongly affect both the efficacy and the safety of TCR gene transfer. This paper is a focussed research review based on a presentation given at the sixth annual meeting of the Association for Immunotherapy of Cancer (CIMT), held in Mainz, Germany,15–16 May 2008.     

Virus-specific T cells for therapy--approaches, problems, solutions

Adoptive T cell therapy is the transfer of T cells to a patient in order to combat disease. This procedure is mainly being used but not limited to the treatment of viral infections and malignancies including virus-associated tumors. Depending on the clinical context, the T cell donor may be the same patient or another donor, usually a healthy person. Recent research is centered on the use of antigen-specific T cells, but T cells of uncharacterized specificity can be successfully used in some clinical conditions where target antigens are not known. Depending on underlying scientific hypotheses and preferred technologies, the therapeutic T cells may be anything from monoclonal to highly polyclonal; they may be specific for one epitope, several epitopes from one antigen, or various antigens; they may have been selected during the preparation process for their specificity, their functional capacity, their survival and proliferation in vitro, or the expression of surface markers associated with desirable functional properties. In this minireview, we give a brief overview on selected approaches, problems and solutions in adoptive T cell therapy. We focus on an area where T cell therapy has been particularly successful but is still calling for improvement: herpesviral disease in patients after transplantation.     

Tumor reactivity of immune T cells in short-term culture

 The adoptive transfer of immune T cells is capable of mediating the regression of established neoplasms in a variety of animal tumor models. The antitumor activity is invariably proportional to the number of cells transferred, thus methods to expand immune cell number while maintaining therapeutic efficacy have been extensively investigated. Here we demonstrate that a short-term culture of immune T cells can amplify the T cell number and enhance the therapeutic reactivity against established pulmonary tumor, while maintaining immunological specificity. In contrast, the therapeutic reactivity of immune T cells against established subcutaneous tumor is diminished by short-term culture. While cultured immune T cells are not cytotoxic in a 4-h Cr-release assay, they do specifically secrete interferon γ upon stimulation with tumor cells. T cells cultured after a single exposure to tumor are even more active against pulmonary tumor than T cells cultured from mice immunized repeatedly. This culture system can rapidly induce T cell proliferation and differentiation into mature effector cells, and the resulting cells demonstrate an enhanced ability to treat visceral metastases, but a decreased ability to treat subcutaneous tumor. Thus T cells cultured after a single exposure to tumor represent an ideal population of cells for use in human adoptive immunotherapy trials. Received: 18 July 1996 / Accepted: 27 September 1996     

Dendritic cell-based cancer immunotherapy targeting MUC-1

Vaccination therapy using dendritic cells (DC) as antigen presenting cells (APC) has shown significant promise in laboratory and animal studies as a potential treatment for malignant diseases. Pulsing of autologous DCs with tumor-associated antigens (TAA) is a method often used for antigen delivery and choice of suitable antigens plays an important role in designing an effective vaccine. We identified two HLA-A2 binding novel 9-mer peptides of the TAA MUC1, which is overexpressed on various hematological and epithelial malignancies. Cytotoxic T cells generated after pulsing DC with these peptides were able to induce lysis of tumor cells expressing MUC1 in an antigen-specific and HLA-restricted fashion. Within two clinical studies, we demonstrated that vaccination of patients with advanced cancer using DCs pulsed with MUC1 derived peptides is well tolerated without serious side effects and can induce immunological responses. Of 20 patients with metastatic renal cell carcinoma, 6 patients showed regression of metastases with 3 objective responses (1 CR, 2 PR). Furthermore, we found that in patients responding to treatment T cell responses for antigens not used for treatment occurred suggesting that antigen spreading in vivo might be a possible mechanism of mediating antitumor effects. These results demonstrate that immunotherapy in patients with advanced malignancies using autologous DCs pulsed with MUC1 derived peptides can induce immunological and clinical responses. However, further clinical studies are needed to identify the most potent treatment regimen that can consistently mediate an antitumor immune response in vivo. This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2004 (PIVAC 4)”, held in Freudenstadt-Lauterbad, Black Forest, Germany, on 22–25 September 2004.     

Combination of active specific immunotherapy or adoptive antibody or lymphocyte immunotherapy with chemotherapy in the treatment of cancer

Successful treatment of cancer patients with a combination of monoclonal antibodies (mAb) and chemotherapeutic drugs has spawned various other forms of additional combination therapies, including vaccines or adoptive lymphocyte transfer combined with chemotherapeutics. These therapies were effective against established tumors in animal models and showed promising results in initial clinical trials in cancer patients, awaiting testing in larger randomized controlled studies. Although combination between immunotherapy and chemotherapy has long been viewed as incompatible as chemotherapy, especially in high doses meant to increase anti-tumor efficacy, has induced immunosuppression, various mechanisms may explain the reported synergistic effects of the two types of therapies. Thus direct effects of chemotherapy on tumor or host environment, such as induction of tumor cell death, elimination of regulatory T cells, and/or enhancement of tumor cell sensitivity to lysis by CTL may account for enhancement of immunotherapy by chemotherapy. Furthermore, induction of lymphopenia by chemotherapy has increased the efficacy of adoptive lymphocyte transfer in cancer patients. On the other hand, immunotherapy may directly modulate the tumor’s sensitivity to chemotherapy. Thus, anti-tumor mAb can increase the sensitivity of tumor cells to chemotherapeutic drugs and patients treated first with immunotherapy followed by chemotherapy showed higher clinical response rates than patients that had received chemotherapy alone. In conclusion, combination of active specific immunotherapy or adoptive mAb or lymphocyte immunotherapy with chemotherapy has great potential for the treatment of cancer patients which needs to be confirmed in larger controlled and randomized Phase III trials.     

CAR-T Cell Therapy for Breast Cancer: From Basic Research to Clinical Application

Breast cancer rises as the most commonly diagnosed cancer in 2020. Among women, breast cancer ranks first in both cancer incidence rate and mortality. Treatment resistance developed from the current clinical therapies limits the efficacy of therapeutic outcomes, thus new treatment approaches are urgently needed. Chimeric antigen receptor (CAR) T cell therapy is a type of immunotherapy developed from adoptive T cell transfer, which typically uses patients'' own immune cells to combat cancer. CAR-T cells are armed with specific antibodies to recognize antigens in self-tumor cells thus eliciting cytotoxic effects. In recent years, CAR-T cell therapy has achieved remarkable successes in treating hematologic malignancies; however, the therapeutic effects in solid tumors are not up to expectations including breast cancer. This review aims to discuss the development of CAR-T cell therapy in breast cancer from preclinical studies to ongoing clinical trials. Specifically, we summarize tumor-associated antigens in breast cancer, ongoing clinical trials, obstacles interfering with the therapeutic effects of CAR-T cell therapy, and discuss potential strategies to improve treatment efficacy. Overall, we hope our review provides a landscape view of recent progress for CAR-T cell therapy in breast cancer and ignites interest for further research directions.     

IL-12 enhances efficacy and shortens enrichment time in cytokine-induced killer cell immunotherapy

Cytokine-induced killer (CIK) cells are T cell derived ex vivo expanded cells with both NK and T cell properties. They exhibit potent anti-tumor efficacy against various malignancies in preclinical models and have proven safe and effective in clinical studies. We combined CIK cell adoptive immunotherapy with IL-12 cytokine immunotherapy in an immunocompetent preclinical breast cancer model. Combining CIK cells with IL-12 increased anti-tumor efficacy in vivo compared to either therapy alone. Combination led to full tumor remission and long-term protection in 75% of animals. IL-12 treatment sharply increased the anti-tumor efficacy of short-term cultured CIK cells that exhibited no therapeutic effect alone. Bioluminescence imaging based in vitro cytotoxicity and in vivo homing assays revealed that short-term cultured CIK cells exhibit full cytotoxicity in vitro, but display different tumor homing properties than fully expanded CIK cells in vivo. Our data suggest that short-term cultured CIK cells can be “educated” in vivo, producing fully expanded CIK cells upon IL-12 administration with anti-tumor efficacy in a mouse model. Our findings demonstrate the potential to improve current CIK cell-based immunotherapy by increasing efficacy and shortening ex vivo expansion time. This holds promise for a highly efficacious cancer therapy utilizing synergistic effects of cytokine and cellular immunotherapy.     

Therapeutic efficacy of tumor-targeted IL2 in LTα−/− mice depends on conditioned T cells

An effective immunological eradication of tumors by the adaptive immune system depends on T cell priming, expansion of specific T cells and their effector function. It has been shown that either step may be impaired in the tumor-bearing host, and several strategies have been used to improve antitumor immune responses. In this regard, tumor-targeted IL2 therapy leads to the destruction of established melanoma metastases in fully immune competent mice as previously demonstrated. This effect has been attributed, but never directly confirmed, to the boost of antigen-experienced T cells. To this end, we demonstrate the absence of any antitumor effect of targeted IL2 in mice characterized by an impaired priming of T cell responses. Notably, in these animals tumor-targeted IL2 therapy induced tumor regression only after adoptive transfer of tumor-conditioned splenocytes. A detailed analysis revealed that T cells present within the transferred splenocytes were actively participating in the immune response as these were clonally expanded after targeted IL2 therapy. In summary, we demonstrate here that in LTα^−/− mice lacking sufficient numbers of tumor-specific T cells only the passive transfer of such cells prior to therapy restores the efficacy of tumor-targeted IL2 therapy. Thus, the antitumor effect of tumor-targeted IL2 is indeed based on the boost of pre-existing T cell responses.     

Myxoma virus combined with rapamycin treatment enhances adoptive T cell therapy for murine melanoma brain tumors

Adoptive transfer of tumor-specific T cells has shown some success for treating metastatic melanoma. We evaluated a novel strategy to improve adoptive therapy by administering both T cells and oncolytic myxoma virus to mice with syngeneic B16.SIY melanoma brain tumors. Adoptive transfer of activated CD8⁺ 2C T cells that recognize SIY peptide doubled survival time, but SIY-negative tumors recurred. Myxoma virus killed B16.SIY cells in vitro, and intratumoral injection of virus led to selective and transient infection of the tumor. Virus treatment recruited innate immune cells to the tumor and induced IFNβ production in the brain, resulting in limited oncolytic effects in vivo. To counter this, we evaluated the safety and efficacy of co-administering 2C T cells, myxoma virus, and either rapamycin or neutralizing antibodies against IFNβ. Mice that received either triple combination therapy survived significantly longer with no apparent side effects, but eventually relapsed. Importantly, rapamycin treatment did not impair T cell-mediated tumor destruction, supporting the feasibility of combining adoptive immunotherapy and rapamycin-enhanced virotherapy. Myxoma virus may be a useful vector for transient delivery of therapeutic genes to a tumor to enhance T cell responses.     

胃癌的过继性免疫治疗研究进展

胃癌是目前世界上发病率及致死率较高的恶性肿瘤之一,在东亚地区尤其显著。针对胃癌的治疗手段仍是传统的手术联合化疗、放疗为主,尽管靶向药物治疗提供了新的选择,但其对晚期胃癌的疗效仍然有限。胃癌的免疫治疗作为独特的治疗手段,在近十多年发展较为活跃,特别是过继性免疫治疗手段不断有创新。过继性免疫治疗主要依赖回输具有抗肿瘤活性的细胞,目前回输的细胞由具有非特异性抗肿瘤作用向具有特异性抗肿瘤作用演变,特别是嵌合性抗原T细胞治疗的出现,为进展期胃癌患者提供了有一种潜在的选择。本文对胃癌过继性免疫治疗中采用的不同免疫活性细胞的作用机制、临床应用等进行总结,并针对其不足提出利用基因工程技术增强治疗靶向性、降低免疫逃逸的研究方向。     

Phase I/II study of adoptive transfer of γδ T cells in combination with zoledronic acid and IL-2 to patients with advanced renal cell carcinoma

Human Vγ2 Vδ2-bearing T cells have recently received much attention in cancer immunotherapy. In this study, we conducted a phase I/II clinical trial of the adoptive transfer of γδ T cells to patients with advanced renal cell carcinoma. Eleven patients who had undergone nephrectomy and had lung metastasis were enrolled. Peripheral blood γδ T cells obtained from the patients were stimulated ex vivo with 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP), a synthetic pyrophosphomonoester antigen, and transferred in combination with zoledronic acid (Zol) and teceleukin (recombinant human interleukin-2). Expanded γδ T cells exhibited potent cytotoxic activity against tumor cells in vitro, and the proportion of peripheral blood γδ T cells among CD3⁺ cells typically peaked three to 5 days after transfer. Tumor doubling time was prolonged in all 11 patients, and the best overall responses were 1 CR, 5 SD, and 5 PD, as defined based on Response Evaluation Criteria in Solid Tumors (RECIST). Although ten patients developed adverse reactions of grade ≥3, they were likely to have been the result of the concomitant infusion of Zol and IL-2, and most symptoms swiftly reverted to normal during the course of treatment. In conclusion, this clinical trial demonstrated that our regimen for the adoptive transfer of γδ T cells in combination with Zol and IL-2 was well tolerated and that objective clinical responses could be achieved in some patients with advanced renal cell carcinoma.     

Contribution of the PD-L1/PD-1 pathway to T-cell exhaustion: an update on implications for chronic infections and tumor evasion

Recent clinical data support ideas of Programmed death receptor-ligand 1 (PD-L1; also called B7-H1, CD274) playing an important role in immune evasion of tumor cells. Expression of PD-L1 on tumors strongly correlates with the survival of cancer patients. PD-L1 on tumors interacts with the co-inhibitory molecule Programmed death receptor-1 (PD-1, CD279) on T cells mediating decreased TCR-mediated proliferation and cytokine production. In animal tumor models, blockade of PD-L1/PD-1 interactions resulted in an improved tumor control. In addition, exhausted T cells during chronic viral infections could be revived by PD-L1 blockade. Thus, targeting PD-L1/PD-1 interactions might improve the efficacy of adoptive cell therapies (ACT) of chronic infections as well as cancers. Obstacles for a general blockade of PD-L1 might be its role in mediating peripheral tolerance. This review discusses the currently available data concerning the role of PD-L1 in tumor immune evasion and envisions possibilities for implementation into ACT for cancer patients. This article is a symposium paper from the conference “Cancer Immunotherapy 2006 Meets Strategies for Immune Therapy,” held in Mainz, Germany, on 4–5 May 2006.     

Therapy of murine leukemia with cyclophosphamide and immune Lyt-2+ cells: cytolytic T cells can mediate eradication of disseminated leukemia

Several animal models have been developed in which the adoptive transfer of specifically immune syngeneic T cells has been shown to mediate the eradication of established tumors. In adoptive chemoimmunotherapy (ACIT) of disseminated FBL leukemia with cyclophosphamide and immune T cells, the major effector T cell has been shown to be a noncytolytic Lyt-1+2- T cell that mediates its therapeutic effect without the participation of CTL. Because studies in other models have suggested that CTL can mediate an anti-tumor effect, the efficacy of Lyt-2+ T cells rendered highly cytolytic before adoptive transfer in ACIT of disseminated FBL was examined. The results demonstrated that such CTL had a detectable but limited therapeutic effect in the treatment of FBL. Because this limited activity of transferred purified CTL might have reflected a requirement for helper T cells to produce IL 2 for promotion of the in vivo survival and proliferation of the CTL, the effect of administering IL 2 to tumor-bearing hosts after transfer of CTL was examined. A dose of IL 2 previously shown to induce in vivo proliferation of transferred T cells rendered CTL that were minimally effective alone curative in ACIT of FBL leukemia. Thus, either lymphokine-producing T cells or the lymphokines produced by these cells are necessary for the full expression of the in vivo therapeutic potential of CTL.     

Safety profile and anti-tumor effects of adoptive immunotherapy using gamma-delta T cells against advanced renal cell carcinoma: a pilot study

Purpose Although various types of immunotherapy have been used to improve the prognosis of patients with advanced renal cell carcinoma (RCC), adoptive immunotherapy using gamma-delta (γδ) T cells has not yet been tried. In this study, we designed a pilot study of adoptive immunotherapy using in vitro activated γδ T cells against advanced RCC to evaluate the safety profile and possible anti-tumor effects of this study. Experimental design Patients with advanced RCC after radical nephrectomy were administered via intravenous infusion in vitro-activated autologous γδ T cells every week or every 2 weeks, 6–12 times, with 70 JRU of teceleukin. Adverse events, anti-tumor effects and immunomonitoring were assessed. The anti-tumor effects were evaluated according to tumor doubling time (DT) by computed tomography (CT) and immunomonitoring was performed by flow cytometric analysis. Results Seven advanced RCC patients were entered in this study. The most common adverse events were fever, general fatigue and elevation of hepatobiliary enzymes, but no severe adverse events were seen. Prolongation of tumor DT was seen in three out of five patients; these three patients showed an increase in the number of γδ T cells in peripheral blood and also a high response to the antigen in vitro. Conclusions The results indicated that adoptive immunotherapy using in vitro-activated autologous γδ T cells was well tolerated and induced anti-tumor effects.     

Treatment of advanced metastasized breast cancer with bone marrow-derived tumour-reactive memory T cells: a pilot clinical study

Background  Breast cancer patients frequently harbour tumour-reactive memory T cells in their bone marrow (BM) but not in the blood. After reactivation ex-vivo these cells rejected autologous breast tumours in xenotransplanted mice demonstrating therapeutic potential upon reactivation and mobilization into the blood. We conducted a clinical pilot study on metastasized breast cancer patients to investigate if ex-vivo reactivation of tumour-reactive BM memory T cells and their adoptive transfer is feasible and increases the frequencies of tumour-reactive T cells in the blood. Methods  The study protocol involved one transfusion of T cells which were reactivated in vitro with autologous dendritic cells pulsed with lysate of MCF7 breast cancer cells as source of tumour antigens. Immunomonitoring included characterization of T cell activation in vitro and of tumour-specific T cells in the blood by interferon (IFN)-γ ELISPOT assay, HLA-tetramers and antigen-induced interleukin (IL)-4 secretion. Results  Twelve patients with pre-existing tumour-reactive BM memory T cells were included into the study. In all cases, the treatment was feasible and well tolerated. Six patients (responders) showed by ELISPOT assay de-novo tumour antigen-specific, IFN-γ-secreting T cells in the blood after 7 days. In contrast, non responders showed in the blood tumour antigen-induced IL-4 responses. All responders received more than 6.5 × 10³ tumour-reactive T cells. In contrast, all non responders received lower numbers of tumour antigen-reactive T cells. This was associated with reduced activation of memory T cells in activation cultures, increased amounts of CD4⁺ CD25^high regulatory T cells in the BM and increased tumour antigen-dependent IL-10 secretion. The latter was prevented by preceding depletion of regulatory T cells suggesting that regulatory T cells in the BM can inhibit reactivation of tumour-specific T cells. Conclusion  Taken together, adoptive transfer of ex-vivo re-stimulated tumour-reactive memory T cells from BM of metastasized breast cancer patients can induce the presence of tumour antigen-reactive type-1 T cells in the peripheral blood. Florian Schuetz and Katrin Ehlert contributed equally to the study.     

T-cell receptor gene-modified cells: past promises,present methodologies and future challenges

Immunotherapy constitutes an exciting and rapidly evolving field, and the demonstration that genetically modified T-cell receptors (TCRs) can be used to produce T-lymphocyte populations of desired specificity offers new opportunities for antigen-specific T-cell therapy.Overall, TCR-modified T cells have the ability to target a wide variety of self and non–self targets through the normal biology of a T cell. Although major histocompatibility complex (MHC)–restricted and dependent on co-receptors, genetically engineered TCRs still present a number of characteristics that ensure they are an important alternative strategy to chimeric antigen receptors (CARs), and high-affinity TCRs can now be successfully engineered with the potential to enhance therapeutic efficacy while minimizing adverse events. This review will focus on the main characteristics of TCR gene-modified cells, their potential clinical application and promise to the field of adoptive cell transfer (ACT), basic manufacturing procedures and characterization protocols and overall challenges that need to be overcome so that redirection of TCR specificity may be successfully translated into clinical practice, beyond early-phase clinical trials.     

Biomaterials as carrier,barrier and reactor for cell-based regenerative medicine

Cell therapy has achieved tremendous success in regenerative medicine in the past several decades. However, challenges such as cell loss, death and immune-rejection after transplantation still persist. Biomaterials have been designed as carriers to deliver cells to desirable region for local tissue regeneration; as barriers to protect transplanted cells from host immune attack; or as reactors to stimulate host cell recruitment, homing and differentiation. With the assistance of biomaterials, improvement in treatment efficiency has been demonstrated in numerous animal models of degenerative diseases compared with routine free cell-based therapy. Emerging clinical applications of biomaterial assisted cell therapies further highlight their great promise in regenerative therapy and even cure for complex diseases, which have been failed to realize by conventional therapeutic approaches.     

Toxicological and immunological evaluation of the MHC-non-restricted cytotoxic T cell line TALL-104

 The human MHC-non-restricted cytotoxic T cell line TALL-104 has been shown to display potent antitumor effects in several animal models with spontaneous and induced malignancies. In view of its potential future use in cancer therapy, we investigated the tolerability and target-organ toxicity of these cells in various animal species. The acute toxicity of TALL-104 cell administrations was evaluated in: (a) healthy immunocompetent mice and immunodeficient (SCID) mice bearing human tumors using multiple (up to 15) intraperitoneal (i.p.) injections, and (b) healthy dogs, tumor-bearing dogs, and healthy monkeys using multiple (up to 17) intravenous (i.v.) injections. TALL-104 cells were γ-irradiated (40 Gy) prior to administration to mice and dogs, but administered without irradiation in monkeys. Cell doses ranged from 5×10⁷/kg to 10¹⁰/kg for each injection. All regimens were well tolerated, the main clinical signs observed being transient gastrointestinal effects. Moderate and transient increases in liver transaminase levels were observed in all animal species. Discrete and transient leukocytosis with neutrophilia was also noted in dogs and monkeys after i.v injections of TALL-104 cells. Histological analysis revealed foci of hepatic necrosis with lympho-/mono-/granulocytic infiltration in immunocompetent mice injected i.p. with 5×10⁹ – 10¹⁰ cells/kg. In the same mice, the colon showed an increased number of muciparous cells and alterations in the villi structure: these alterations were completely reversed by 72 h after the last injection, while liver alterations reversed more slowly (1 week). No delayed or chronic toxicity was observed in any of the animals even when non-irradiated TALL-104 cells were administered: both immunocompetent mice and healthy dogs were found to be grossly and histopathologically normal when sacrificed (1 year and 1 month after the last TALL-104 injection respectively). TALL-104 cells did not persist in these hosts. In addition, monkeys showed no molecular signs of TALL-104-cell-induced leukemia in their blood 1 year after the last cell injection. Despite immunosuppression, most of the tumor-bearing dogs as well as the healthy dogs and monkeys developed both humoral and cellular immune responses against TALL-104 cells. The data derived from these preclinical studies suggest that administration of high doses of irradiated TALL-104 cells is well tolerated and would be unlikely to induce severe toxicity if applied in clinical trials to the treatment of patients with refractory cancer. Received: 8 September 1996 / Accepted: 28 January 1997     

Adaptive T cell immunotherapy in cancer

Impaired tumor-specific effector T cells contribute to tumor progression and unfavorable clinical outcomes. As a compensatory T cell-dependent cancer immunoediting strategy, adoptive T cell therapy(ACT) has achieved encouraging therapeutic results,and this strategy is now on the center stage of cancer treatment and research. ACT involves the ex vivo stimulation and expansion of tumor-infiltrating lymphocytes(TILs) with inherent tumor reactivity or T cells that have been genetically modified to express the cognate chimeric antigen receptor or T cell receptor(CAR/TCR), followed by the passive transfer of these cells into a lymphodepleted host. Primed T cells must provide highly efficient and long-lasting immune defense against transformed cells during ACT. Anin-depth understanding of the basic mechanisms of these living drugs can help us improve upon current strategies and design better next-generation T cell-based immunotherapies. From this perspective, we provide an overview of current developments in different ACT strategies, with a focus on frontier clinical trials that offer a proof of principle. Meanwhile, insights into the determinants of ACT are discussed, which will lead to more rational, potent and widespread applications in the future.