Annelies Jorritsma

Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy

The transfer of T cell receptor (TCR) genes can be used to induce immune reactivity toward defined antigens to which endogenous T cells are insufficiently reactive. This approach, which is called TCR gene therapy, is being developed to target tumors and pathogens, and its clinical testing has commenced in patients with cancer. In this study we show that lethal cytokine-driven autoimmune pathology can occur in mouse models of TCR gene therapy under conditions that closely mimic the clinical setting. We show that the pairing of introduced and endogenous TCR chains in TCR gene-modified T cells leads to the formation of self-reactive TCRs that are responsible for the observed autoimmunity. Furthermore, we demonstrate that adjustments in the design of gene therapy vectors and target T cell populations can be used to reduce the risk of TCR gene therapy–induced autoimmune pathology.

A rapid and potent DNA vaccination strategy defined by in vivo monitoring of antigen expression.

Induction of immunity after DNA vaccination is generally considered a slow process. Here we show that DNA delivery to the skin results in a highly transient pulse of antigen expression. Based on this information, we developed a new rapid and potent intradermal DNA vaccination method. By short-interval intradermal DNA delivery, robust T-cell responses, of a magnitude sufficient to reject established subcutaneous tumors, are generated within 12 d. Moreover, this vaccination strategy confers protecting humoral immunity against influenza A infection within 2 weeks after the start of vaccination. The strength and speed of this newly developed strategy will be beneficial in situations in which immunity is required in the shortest possible time.

Prospects and limitations of T cell receptor gene therapy

Adoptive transfer of antigen-specific T cells is an attractive means to provide cancer patients with immune cells of a desired specificity and the efficacy of such adoptive transfers has been demonstrated in several clinical trials. Because the T cell receptor is the single specificity-determining molecule in T cell function, adoptive transfer of TCR genes into patient T cells may be used as an alternative approach for the transfer of tumor-specific T cell immunity. On theoretical grounds, TCR gene therapy has two substantial advantages over conventional cellular transfer. First, it circumvents the demanding process of in vitro generation of large numbers of specific immune cells. Second, it allows the use of a set of particularly effective TCR genes in large patient groups. Conversely, TCR gene therapy may be associated with a number of specific problems that are not confronted during classical cellular therapy. Here we review our current understanding of the potential and possible problems of TCR gene therapy, as based on in vitro experiments, mouse model systems and phase I clinical trials. Furthermore, we discuss the prospects of widespread clinical application of this gene therapy approach for the treatment of human cancer.

T-Cell Receptor Gene Therapy of Established Tumors in a Murine Melanoma Model

Adoptive cell transfer therapy using tumor-infiltrating lymphocytes for patients with metastatic melanoma has demonstrated significant objective response rates. One major limitation of these current therapies is the frequent inability to isolate tumor-reactive lymphocytes for treatment. Genetic engineering of peripheral blood lymphocytes with retroviral vectors encoding tumor antigen-specific T-cell receptors (TCRs) bypasses this restriction. To evaluate the efficacy of TCR gene therapy, a murine treatment model was developed. A retroviral vector was constructed encoding the pmel-1 TCR genes targeting the B16 melanoma antigen, gp100. Transduction of C57BL/6 lymphocytes resulted in efficient pmel-1 TCR expression. Lymphocytes transduced with this retrovirus specifically recognized gp100-pulsed target cells as measured by interferon-γ secretion assays. Upon transfer into B16 tumor-bearing mice, the genetically engineered lymphocytes significantly slowed tumor development. The effectiveness of tumor treatment was directly correlated with the number of TCR-engineered T cells administered. These results demonstrated that TCR gene therapy targeting a native tumor antigen significantly delayed the growth of established tumors. When C57BL/6 lymphocytes were added to antigen-reactive pmel-1 T cells, a reduction in the ability of pmel-1 T cell to treat B16 melanomas was seen, suggesting that untransduced cells may be deleterious to TCR gene therapy. This model may be a powerful tool for evaluating future TCR gene transfer-based strategies.

Requirements for Effective Antitumor Responses of TCR Transduced T Cells

Adoptive transfer of TCR gene-modified T cells has been proposed as an attractive approach to target tumors for which it is difficult or impossible to induce strong tumor-specific T cell responses by vaccination. Whereas the feasibility of generating tumor Ag-specific T cells by gene transfer has been demonstrated, the factors that determine the in vivo effectiveness of TCR-modified T cells are largely unknown. We have analyzed the value of a number of clinically feasible strategies to enhance the antitumor potential of TCR modified T cells. These experiments reveal three factors that contribute greatly to the in vivo potency of TCR-modified T cells. First, irradiation-induced host conditioning is superior to vaccine-induced activation of genetically modified T cells. Second, increasing TCR expression through genetic optimization of TCR sequences has a profound effect on in vivo antitumor activity. Third, a high precursor frequency of TCR modified T cells within the graft is essential. Tumors that ultimately progress in animals treated with this optimized regimen for TCR-based adoptive cell transfer invariably display a reduced expression of the target Ag. This suggests TCR gene therapy can achieve a sufficiently strong selective pressure to warrant the simultaneous targeting of multiple Ags. The strategies outlined in this study should be of value to enhance the antitumor activity of TCR-modified T cells in clinical trials.

Human Telomerase Reverse Transcriptase-Transduced Human Cytotoxic T Cells Suppress the Growth of Human Melanoma in Immunodeficient Mice

Immunotherapy of melanoma by adoptive transfer of tumor-reactive T lymphocytes aims at increasing the number of activated effectors at the tumor site that can mediate tumor regression. The limited life span of human T lymphocytes, however, hampers obtaining sufficient cells for adoptive transfer therapy. We have shown previously that the life span of human T cells can be greatly extended by transduction with the human telomerase reverse transcriptase (hTERT) gene, without altering antigen specificity or effector function. We developed a murine model to evaluate the efficacy of hTERT-transduced human CTLs with antitumor reactivity to eradicate autologous tumor cells in vivo. We transplanted the human melanoma cell line melAKR or melAKR-Flu, transduced with a retrovirus encoding the influenza virus/HLA-A2 epitope, in RAG-2−/− IL-2Rγ −/− double knockout mice. Adoptive transfer of the hTERT-transduced influenza virus-specific CTL clone INFA24 or clone INFA13 inhibited the growth of melAKR-Flu tumors in vivo and not of the parental melAKR melanoma cells. Furthermore, the hTERT-transduced CTL clone INFA13 inhibited tumor growth to the same extent in vivo as the untransduced CTL clone, as determined by in vivo imaging of luciferase gene-transduced melAKR-Flu tumors, indicating that hTERT did not affect the in vivo function of CTL. These results demonstrate that hTERT-transduced human CTLs are capable of mediating antitumor activity in vivo in an antigen-specific manner. hTERT-transduced MART-1-specific CTL clones AKR4D8 and AKR103 inhibited the growth of syngeneic melAKR tumors in vivo. Strikingly, melAKR-Flu cells were equally killed by the MART-1-specific CTL clones and influenza virus-specific CTL clones in vitro, but only influenza-specific CTLs were able to mediate tumor regression in vivo. The influenza-specific CTL clones were found to produce higher levels of IFNγ on tumor cell recognition than the MART-1-specific CTL clones, which may result from the higher functional avidity of the influenza virus-specific CTL clones. Also, melAKR-Flu tumors were growing faster than melAKR tumors, which may have surpassed the relatively modest antitumor effect of the MART-1-specific CTL, as compared with the influenza virus-specific CTL. Taken together, the adoptive transfer model described here shows that hTERT-transduced T cells are functional in vivo, and allows us to evaluate the balance between functional activity of the CTL and tumor growth rate in vivo, which determines the efficacy of CTLs to eradicate tumors in adoptive transfer therapy.

Case Report of a Fatal Serious Adverse Event Upon Administration of T Cells Transduced With a MART-1-specific T-cell Receptor

Here, we describe a fatal serious adverse event observed in a patient infused with autologous T-cell receptor (TCR) transduced T cells. This TCR, originally obtained from a melanoma patient, recognizes the well-described HLA-A*0201 restricted 26-35 epitope of MART-1, and was not affinity enhanced. Patient 1 with metastatic melanoma experienced a cerebral hemorrhage, epileptic seizures, and a witnessed cardiac arrest 6 days after cell infusion. Three days later, the patient died from multiple organ failure and irreversible neurologic damage. After T-cell infusion, levels of IL-6, IFN-γ, C-reactive protein (CRP), and procalcitonin increased to extreme levels, indicative of a cytokine release syndrome or T-cell-mediated inflammatory response. Infused T cells could be recovered from blood, broncho-alveolar lavage, ascites, and after autopsy from tumor sites and heart tissue. High levels of NT-proBNP indicate semi-acute heart failure. No cross reactivity of the modified T cells toward a beating cardiomyocyte culture was observed. Together, these observations suggest that high levels of inflammatory cytokines alone or in combination with semi-acute heart failure and epileptic seizure may have contributed substantially to the occurrence of the acute and lethal event. Protocol modifications to limit the risk of T-cell activation-induced toxicity are discussed.

An Inducible Caspase 9 Safety Switch Can Halt Cell Therapy-Induced Autoimmune Disease

Transfer of either allogeneic or genetically modified T cells as a therapy for malignancies can be accompanied by T cell-mediated tissue destruction. The introduction of an efficient “safety switch” can potentially be used to control the survival of adoptively transferred cell populations and as such reduce the risk of severe graft-vs-host disease. In this study, we have tested the value of an inducible caspase 9-based safety switch to halt an ongoing immune attack in a murine model for cell therapy-induced type I diabetes. The data obtained in this model indicate that self-reactive T cells expressing this conditional safety switch show unimpaired lymphopenia- and vaccine-induced proliferation and effector function in vivo, but can be specifically and rapidly eliminated upon triggering. These data provide strong support for the evaluation of this conditional safety switch in clinical trials of adoptive cell therapy.

Manufacture of gene-modified human T-cells with a memory stem/central memory phenotype

Advances in genetic engineering have made it possible to generate human T-cell products that carry desired functionalities, such as the ability to recognize cancer cells. The currently used strategies for the generation of gene-modified T-cell products lead to highly differentiated cells within the infusion product, and on the basis of data obtained in preclinical models, this is likely to impact the efficacy of these products. We set out to develop a good manufacturing practice (GMP) protocol that yields T-cell receptor (TCR) gene-modified T-cells with more favorable properties for clinical application. Here, we show the robust clinical-scale production of human peripheral blood T-cells with an early memory phenotype that express a MART-1-specific TCR. By combining selection and stimulation using anti-CD3/CD28 beads for retroviral transduction, followed by expansion in the presence of IL-7 and IL-15, production of a well-defined clinical-scale TCR gene-modified T-cell product could be achieved. A major fraction of the T-cells generated in this fashion were shown to coexpress CD62L and CD45RA, and express CD27 and CD28, indicating a central memory or memory stemlike phenotype. Furthermore, these cells produced IFNγ, TNFα, and IL-2 and displayed cytolytic activity against target cells expressing the relevant antigen. The T-cell products manufactured by this robust and validated GMP production process are now undergoing testing in a phase I/IIa clinical trial in HLA-A*02:01 MART-1-positive advanced stage melanoma patients. To our knowledge, this is the first clinical trial protocol in which the combination of IL-7 and IL-15 has been applied for the generation of gene-modified T-cell products.

Immunotherapeutic strategies: the melanoma example

T-cell-based immunotherapy can be induced by nonspecific activation, by antigen-specific immunization, or by adoptive immunotherapy. In this review, progress in these areas is discussed as based on data from clinical trials for the treatment of metastatic melanoma. Nonspecific immunotherapy has been shown to result in low, but in some cases significant, levels of objective tumor responses, and is often associated with autoimmune reactions. Antigen-specific targeting of tumors via vaccination has only resulted in low to very low levels of objective responses, and these strategies seem to have most value when the T-cell repertoire is not affected by tolerance. Finally, adoptive immunotherapy can be applied by in vitro expansion of autologous lymphocytes that have escaped tolerance or by genetic transfer of allogeneic T-cell receptors (TCRs). Autologous adoptive T-cell transfer has resulted in a very high frequency of clinical responses when combined with chemotherapy and IL-2 administration in single-center studies. Although TCR gene transfer has, until now, only resulted in a low frequency of clinical responses, it does have a broader application potential, and optimization of this strategy is likely to improve its efficacy.