Characterization of spontaneous tumor antigen-reactive T cell responses in melanoma patients and treatment of human melanoma with optimized T cell receptor transgenic T cells in a xenotransplantation model

One of the leading human malignancies is cancer, a disease characterized by mostly uncontrolled growth of abnormal cells in the body, often forming metastases. It´s interaction with the immune system, that can influence the disease progression, has repeatedly been reported and gained increasing importance over the last decades. However, most of the current immunotherapeutic approaches for cancer treatment are limited, partly due to the immune controlling capacities of tumors. The development of novel experimental approaches and therapeutic strategies, that support and complement the tumor-directed pre-existing abilities of the immune system, will be critical for the success of future cancer immunotherapies. Therefore, and because of the general need of novel treatment strategies, the presented thesis focused on four different approaches to optimize cancer immune therapeutic interventions. Functionally competent tumor-specific memory T cells have been reported to efficiently control tumor growth and be required for a durable prevention of tumor recurrence and metastasis formation. Therefore, the first project dealed with spontaneously induced tumor antigen-reactive memory T cell responses in the peripheral blood (PB) of melanoma and dysplastic nevi patients. Thereby, using a short-term IFNgamma ELISPOT assay, high frequencies of pre-existing T cell responses were detected in both cohorts ex vivo. As antigen-presenting cells, I have used autologous dendritic cells, generated from monocytes which were pulsed with 13 different 50 amino acid long synthetic melanoma polypeptides, derived from immunodominant regions of tumor-associated antigens (TAAs). Thereby, the nature of the identified T cell responses revealed to be polyvalent and HLA-unrestricted. Moreover, the designed polypeptide sequences, that covered MHC class I- and II-restricted epitopes, are optimal for presentation on a wide range of HLA types and therefore, were recognized by CD4+ and CD8+ T cells. I have shown that the long peptide sequence of the NA17-A antigen induce high frequencies of NA17-A-reactive memory T cells, but not NA17-A-specific regulatory T cell (Treg) responses. Thus, particular importance for future vaccination approaches might therefore have to be considered. Furthermore, the demonstration that the TAA-reactive T cell frequencies increased after resection of the non-metastasized tumors or dysplastic nevi indicates that the presence of the lesions controls pre-existing T cell responses. Additionally, these investigations also showed antigen-specific Treg in the PB and revealed that, in some patients, Treg have an immunosuppressive potential. In general, however, an essential role of Treg in controlling spontaneous T cell responses in non-metastasized melanoma and dysplastic nevi patients could not be detected. Based on these findings, besides Treg, the tumor- or lesion-mediated TAA-reactive memory T cell suppression might also be facilitated through other potential effectors of immunosuppression, including combinations of soluble and cellular mediators. Summarized, the investigated polypeptides efficiently elucidated pre-existing T cell responses in melanoma and dysplastic nevi patients and thus indicate that those polyvalent responses could be therapeutically reactivated in consequence of an appropriate peptide vaccination treatment. For the second optimization approach a human melanoma murine xenotransplantation model has been established. Thereby, the in vivo efficiency and biologic relevance of human T cells, which were transduced with functionally expressed, in vitro optimized, T cell receptor (TCR) constructs, specific for the antigens MDM2 or gp100 were investigated. For the first time in vivo, two novel strategies that aimed to avoid hybrid mispaired TCR chain formation and thereby potential autoimmune disease-causing “off-target” reactions were tested. These approaches comprised the molecular design of the TCR alpha/beta interphase and a single chain TCR framework. Thereby, optimized MDM2 as well as gp100 transgenic T cells revealed the ability to significantly control human melanoma tumor growth. Moreover, the engineered T cells persisted in vivo and controlled in the same animals even a secondary tumor growth in a memory T cell-like manner. I could also demonstrate that a combinational treatment of adoptive T cell therapy using TCR gene-modified human T cells with local low dose tumor irradiation (2 Gy), led to an improved T cell targeting into the tumor and additionally further increased the tumor growth control. Consequently, in order to improve adoptive cancer immunotherapeutic approaches the in vivo experiments confirmed the feasibility and efficiency of the applied TCR design. In the third project presented in this thesis, a novel tumor cell-mediated immunosuppressive mechanism, facilitated by the cell adhesion molecules CEACAM6 and L1CAM, was addressed. Thereby, these molecules were detected in various tumor cell lines and using short-term IFNgamma ELISPOT and 51Cr release cytotoxicity assays it could be shown that their specific blockade resulted in increased, functional effector T cell responses. Therefore, in order to modulate the strength of T cell responses against malignant edited tumor cells that escaped immune recognition, CEACAM6 and L1CAM may consequently be attractive targets for novel immunotherapeutic interventions. Finally, to increase the capacity of oncolytic virotherapies, in a fourth project, I optimized the viral transport of the Newcastle disease virus (NDV) to human tumor cells. In this approach, the loading of human T cells with NDV was specified for the first time in an in vitro co-culture system. Moreover, in a “hitchhiking”-like mechanism T cells have shown to be capable to transfer cell surface-attached NDV to MCF-7 tumor cells. In addition, monolayers of tumor cells were efficiently destroyed through oncolytic NDV-modified activated T cells. Based on this combinational therapy, it can be proposed that effector T cells can facilitate as potent carrier vehicles to transport NDV to tumor sites, resulting in focused tumor cell destruction. In summary, the findings of the four different approaches, presented in this thesis, indicate novel perspectives for the optimization of future immunotherapeutic interventions in order to elicit and support protective T cell responses against clinically apparent malignant tumors

Tumour-associated macrophages act as a slow-release reservoir of nano-therapeutic Pt(IV) pro-drug

Therapeutic nanoparticles (TNPs) aim to deliver drugs more safely and effectively to cancers, yet clinical results have been unpredictable owing to limited in vivo understanding. Here we use single-cell imaging of intratumoral TNP pharmacokinetics and pharmacodynamics to better comprehend their heterogeneous behaviour. Model TNPs comprising a fluorescent platinum(IV) pro-drug and a clinically tested polymer platform (PLGA-b-PEG) promote long drug circulation and alter accumulation by directing cellular uptake toward tumour-associated macrophages (TAMs). Simultaneous imaging of TNP vehicle, its drug payload and single-cell DNA damage response reveals that TAMs serve as a local drug depot that accumulates significant vehicle from which DNA-damaging Pt payload gradually releases to neighbouring tumour cells. Correspondingly, TAM depletion reduces intratumoral TNP accumulation and efficacy. Thus, nanotherapeutics co-opt TAMs for drug delivery, which has implications for TNP design and for selecting patients into trials.National Cancer Institute (U.S.) (Grant RO1-CA034992

Oncolytic Viruses for Cancer Therapy: Overcoming the Obstacles

Targeted therapy of cancer using oncolytic viruses has generated much interest over the past few years in the light of the limited efficacy and side effects of standard cancer therapeutics for advanced disease. In 2006, the world witnessed the first government-approved oncolytic virus for the treatment of head and neck cancer. It has been known for many years that viruses have the ability to replicate in and lyse cancer cells. Although encouraging results have been demonstrated in vitro and in animal models, most oncolytic viruses have failed to impress in the clinical setting. The explanation is multifactorial, determined by the complex interactions between the tumor and its microenvironment, the virus, and the host immune response. This review focuses on discussion of the obstacles that oncolytic virotherapy faces and recent advances made to overcome them, with particular reference to adenoviruses

Immunogenic Chemotherapy Sensitizes Tumors to Checkpoint Blockade Therapy

Checkpoint blockade immunotherapies can be extraordinarily effective, but might benefit only the minority of patients whose tumors are pre-infiltrated by T cells. Here, using lung adenocarcinoma mouse models, including genetic models, we show that autochthonous tumors that lacked T cell infiltration and resisted current treatment options could be successfully sensitized to host antitumor T cell immunity when appropriately selected immunogenic drugs (e.g., oxaliplatin combined with cyclophosphamide for treatment against tumors expressing oncogenic Kras and lacking Trp53) were used. The antitumor response was triggered by direct drug actions on tumor cells, relied on innate immune sensing through toll-like receptor 4 signaling, and ultimately depended on CD8 + T cell antitumor immunity. Furthermore, instigating tumor infiltration by T cells sensitized tumors to checkpoint inhibition and controlled cancer durably. These findings indicate that the proportion of cancers responding to checkpoint therapy can be feasibly and substantially expanded by combining checkpoint blockade with immunogenic drugs

T‐cell modulation by cyclophosphamide for tumour therapy

The power of T cells for cancer treatment has been demonstrated by the success of co‐inhibitory receptor blockade and adoptive T‐cell immunotherapies. These treatments are highly successful for certain cancers, but are often personalized, expensive and associated with harmful side effects. Other T‐cell‐modulating drugs may provide additional means of improving immune responses to tumours without these disadvantages. Conventional chemotherapeutic drugs are traditionally used to target cancers directly; however, it is clear that some also have significant immune‐modulating effects that can be harnessed to target tumours. Cyclophosphamide is one such drug; used at lower doses than in mainstream chemotherapy, it can perturb immune homeostasis, tipping the balance towards generation of anti‐tumour T‐cell responses and control of cancer growth. This review discusses its growing reputation as an immune‐modulator whose multiple effects synergize with the microbiota to tip the balance towards tumour immunity offering widespread benefits as a safe, and relatively inexpensive component of cancer immunotherapy

The role of myeloid cells in cancer therapies

Recent clinical trials have demonstrated the ability to durably control cancer in some patients by manipulating T lymphocytes. These immunotherapies are revolutionizing cancer treatment but benefit only a minority of patients. It is thus a crucial time for clinicians, cancer scientists and immunologists to determine the next steps in shifting cancer treatment towards better cancer control. This Review describes recent advances in our understanding of tumour-associated myeloid cells. These cells remain less studied than T lymphocytes but have attracted particular attention because their presence in tumours is often linked to altered patient survival. Also, experimental studies indicate that myeloid cells modulate key cancer-associated activities, including immune evasion, and affect virtually all types of cancer therapy. Consequently, targeting myeloid cells could overcome limitations of current treatment options

Common TLR5 mutations control cancer progression

The mechanisms regulating tumor-associated inflammation are incompletely understood. In this issue of Cancer Cell, Rutkowski and colleagues indicate that TLR5 signaling deficiency, which occurs in ∼10% of the population, changes interactions with commensal microbiota and deregulates a cascade of inflammatory events that can suppress or accelerate extraintestinal cancers

Tumor microenvironment: no effector T cells without dendritic cells

Successful antitumor immunity is thought to require T cell entry into tumors, though mechanisms regulating this process remain unclear. In this issue of Cancer Cell, Spranger et al. indicate that chemokines produced by intratumoral Batf3 dendritic cells are critical for effector T cell recruitment. The findings have implications for immunotherapy