Decoding the tumor microenvironment : a B-cell and Macrophage perspective

The immune system plays a role in many different functions in our body, and disease is no exception. Cancer is one of the biggest challenges of the twenty first century and vast efforts have been made to understand this disease. Since the 1900s, we have evidence based that the immune system participates in cancer progression, but it was only some decades ago that we started to investigate it extensively. Currently, we know that the tumor microenvironment is infiltrated by different immune cells and depending on space and time they can participate in tumor initiation, promotion, progression and metastasis. Moreover, it has been shown that certain immune cell can influence tumor resistance to therapy. So far, most of the studies have been directed towards understanding tumor infiltrating T cells. However, in this thesis I focused on the interplay between different immune cells including B cells, T cells, Natural killer cells, macrophages and the tumor cells. Further from this research, we found potential predictive biomarkers associated with the checkpoint therapies, such as anti-CTLA-4, anti- PD-1 and anti-PD-L1. This thesis can be divided into two major studies investigating two immune cell types infiltrating the tumor, B cells (Paper I and II) and macrophages (Paper III and IV). More specifically, Paper I touches upon the role of B cells infiltrating two types of tumors, melanoma and breast cancer, studied in mouse models. Here, we could detect the presence of a heterogeneous B cell population that could be further divided by its surface CD5 expression. These subpopulations were phenotypically and functionally distinct. In particular, CD5+ TIBs were found to have an activated phenotype and were able to secrete proinflammatory cytokines, which were induced by the tumor milieu. Additionally, we identified a similar B cell subpopulation in human breast cancer highlighting the importance of this finding. Paper II focused on the B cell responses towards melanoma when treated with immune checkpoint therapies anti-PD-1 or anti-PD-L1. This study showed an augmented IgG response against the tumor mice treated with checkpoint antibodies. More importantly, it revealed significant differences in IgG subclasses depending on the checkpoint treatment given. In the second part of the thesis, paper III identifies MARCO, a scavenger receptor, as a novel marker for immunosuppressive TAMs subset present in three different mouse models, namely melanoma, mammary carcinoma and colon cancer. Monoclonal antibody against MARCO was found to successfully decrease the tumor growth and metastasis, while re- polarizing this subpopulation of TAMs to have an anti-tumor phenotype. Moreover, we could observe an enhanced effect of anti-CTLA-4 treatment when combined with anti-MARCO. Finally, MARCO proved to be expressed in human metastatic melanoma and in an aggressive breast cancer subtype. Thus, targeting MARCO could potentially be a combinatory treatment for these cancer types. Paper IV further provides evidence on MARCO expression in adistinct subset of immunosuppressive TAMs, but now in human non-small cell lung cancer. Moreover, this study uncovered the strategical localization of MARCO positive macrophages at the tumor-stroma border. This creates an immunosuppressive barrier that could be potentially targeted with anti-MARCO. In summary, this thesis contributes to our overall understanding of the tumor microenvironment, specifically of B cells and macrophages. It gives us new possible targets and approaches for cancer therapy as well as potential predictive biomarkers

Avidity characterization of genetically engineered T-cells with novel and established approaches

Background: Adoptive transfer of genetically engineered autologous T-cells is becoming a successful therapy for cancer. The avidity of the engineered T-cells is of crucial importance for therapy success. We have in the past cloned a T-cell receptor (TCR) that recognizes an HLA-A2 (MHC class I)-restricted peptide from the prostate and breast cancer- associated antigen TARP. Herein we perform a side-by-side comparison of the TARP-specific TCR (TARP-TCR) with a newly cloned TCR specific for an HLA-A2-restricted peptide from the cytomegalovirus (CMV) pp65 antigen. Results: Both CD8(+) T-cells and CD4(+) T-cells transduced with the HLA-A2-restricted TARP-TCR could readily be detected by multimer analysis, indicating that the binding is rather strong, since binding occured also without the CD8 co-receptor of HLA-A2. Not surprisingly, the TARP-TCR, which is directed against a self-antigen, had weaker binding to the HLA-A2/peptide complex than the CMV pp65-specific TCR (pp65-TCR), which is directed against a viral epitope. Higher peptide concentrations were needed to achieve efficient cytokine release and killing of target cells when the TARP- TCR was used. We further introduce the LigandTracer technology to study cell-cell interactions in real time by evaluating the interaction between TCR-engineered T-cells and peptide-pulsed cancer cells. We were able to successfully detect TCR-engineered T-cell binding kinetics to the target cells. We also used the xCELLigence technology to analyzed cell growth of target cells to assess the killing potency of the TCR-engineered T-cells. T-cells transduced with the pp65 - TCR exhibited more pronounced cytotoxicity, being able to kill their targets at both lower effector to target ratios and lower peptide concentrations. Conclusion: The combination of binding assay with functional assays yields data suggesting that TARP- TCR-engineered T-cells bind to their target, but need more antigen stimulation compared to the pp65-TCR to achieve full effector response. Nonetheless, we believe that the TARP- TCR is an attractive candidate for immunotherapy development for prostate and/or breast cancer

Expression of scavenger receptor MARCO defines a targetable tumor-associated macrophage subset in non-small cell lung cancer

Tumor-associated macrophages (TAMs) are attractive targets for immunotherapy. Recently, studies in animal models showed that treatment with an anti-TAM antibody directed against the scavenger receptor MARCO resulted in suppression of tumor growth and metastatic dissemination. Here we investigated the expression of MARCO in relation to other macrophage markers and immune pathways in a non-small cell lung cancer (NSCLC) cohort (n = 352). MARCO, CD68, CD163, MSR1 and programmed death ligand-1 (PD-L1) were analyzed by immunohistochemistry and immunofluorescence, and associations to other immune cells and regulatory pathways were studied in a subset of cases (n = 199) with available RNA-seq data. We observed a large variation in macrophage density between cases and a strong correlation between CD68 and CD163, suggesting that the majority of TAMs present in NSCLC exhibit a protumor phenotype. Correlation to clinical data only showed a weak trend toward worse survival for patients with high macrophage infiltration. Interestingly, MARCO was expressed on a distinct subpopulation of TAMs, which tended to aggregate in close proximity to tumor cell nests. On the transcriptomic level, we found a positive association between MARCO gene expression and general immune response pathways including strong links to immunosuppressive TAMs, T-cell infiltration and immune checkpoint molecules. Indeed, a higher macrophage infiltration was seen in tumors expressing PD-L1, and macrophages residing within tumor cell nests co-expressed MARCO and PD-L1. Thus, MARCO is a potential new immune target for anti-TAM treatment in a subset of NSCLC patients, possibly in combination with available immune checkpoint inhibitors

Reprogramming Tumor-Associated Macrophages by Antibody Targeting Inhibits Cancer Progression and Metastasis

Tumors are composed of multiple cell types besides the tumor cells themselves, including innate immune cells such as macrophages. Tumor-associated macrophages (TAMs) are a heterogeneous population of myeloid cells present in the tumor microenvironment (TME). Here, they contribute to immunosuppression, enabling the establishment and persistence of solid tumors as well as metastatic dissemination. We have found that the pattern recognition scavenger receptor MARCO defines a subtype of suppressive TAMs and is linked to clinical outcome. An anti-MARCO monoclonal antibody was developed, which induces anti-tumor activity in breast and colon carcinoma, as well as in melanoma models through reprogramming-TAM-populations to a pro-inflammatory phenotype and increasing tumor immunogenicity. This anti-tumor activity is dependent on the inhibitory Fc-receptor, Fc gamma RIIB, and also enhances the efficacy of checkpoint therapy. These results demonstrate that immunotherapies using antibodies designed to modify myeloid cells of the TME represent a promising mode of cancer treatment