Exploring immunotherapeutic targets in the tumor microenvironment

The immune system has developed along with the evolution of increasingly complex cellular organisms to sustain homeostasis and protect from threats. Cancer, a detrimental side effect of increasing organismic complexity, typically sequesters the immune system and hijacks its functions for its own prosperity. Cancer immunotherapy aims to harness the intrinsic potential of the immune system for the therapeutic benefit of cancer patients. The focus of this thesis is to identify and evaluate new immunotherapeutic targets in the tumor microenvironment, which can be modulated to restrict tumor growth and metastasis. Paper I describes a novel mechanism of interaction between marginal zone macrophages (MZMs) and marginal zone B cells (MZBs) in the spleen, which can be modulated by antibodies (Abs) to scavenger receptor MARCO on MZMs. This study demonstrates that MARCO targeting diminishes antigen (Ag) uptake by MZBs, which results in reduced Ag deposition in the splenic follicles. As anti-MARCO Abs can also be found in systemic lupus erythematosus (SLE), this interaction may affect subsequent adaptive immune responses to both self- and foreign antigen. Paper II identifies MARCO as a specific marker for a tumor-promoting macrophage subtype in the tumor microenvironment of mammary carcinoma, melanoma and colon carcinoma tumor models. Targeting MARCO on tumor-associated macrophages (TAMs) by Abs, inhibits tumor growth and metastasis and reprograms TAMs to an anti-tumor phenotype. Treatment with anti-MARCO mAbs in combination with checkpoint inhibitor, anti-CTLA-4 ab, may provide a promising approach for cancer immunotherary with clinical relevance for human breast cancer and melanoma. Paper III reveals the molecular mechanism through which tumor cells imitate immune cells, during epithelial-mesenchymal transition (EMT), prompting their targeted metastasis through the lymphatics. This study demonstrates that TGF-β, a known inducer of EMT, regulates the chemotactic axis CCR7/CCL21, directing preferential lymphatic dissemination of breast cancer cells. Paper IV evaluates dendritic cell-derived exosomes in a vaccination approach to reactivate adaptive anti-tumor responses. This study shows that dendritic cell-derived CD1d expressing exosomes loaded with α-Galactosylceramide (α-GalCer) can sensitize NKT cells and lead to subsequent activation of B cell and effector T cell responses, restricting tumor growth. In summary, the work presented in this thesis describes novel targets in the tumor microenvironment that can be used in immunotherapeutic approaches to re-activate endogenous mechanisms of innate and adaptive immunity against cancer. Additionally, it gives new insight into gene regulatory pathways controlling metastatic tumor spread, as well as utilizes custom designed biological molecules in anti-tumor vaccination strategies. Increasing our understanding of the intricate mechanisms regulating the immunosuppressive tumor microenvironment will reveal new knowledge and novel targets that can contribute to the design of prospective cancer immunotherapies

Adoptive nk cell therapy: A promising treatment prospect for metastatic melanoma

Adoptive cell therapy (ACT) represents a promising alternative approach for patients with treatment-resistant metastatic melanoma. Lately, tumor infiltrating lymphocyte (TIL) therapy and chimeric antigen receptor (CAR)-T cell therapy have shown improved clinical outcome, compared to conventional chemotherapy or immunotherapy. Nevertheless, they are limited by immune escape of the tumor, cytokine release syndrome, and manufacturing challenges of autologous therapies. Conversely, the clinical use of Natural Killer (NK) cells has demonstrated a favorable clinical safety profile with minimal toxicities, providing an encouraging treatment alternative. Unlike T cells, NK cells are activated, amongst other mechanisms, by the downregulation of HLA class I molecules, thereby overcoming the hurdle of tumor immune escape. However, impairment of NK cell function has been observed in melanoma patients, resulting in deteriorated natural defense. To overcome this limitation, “activated” autologous or allogeneic NK cells have been infused into melanoma patients in early clinical trials, showing encouraging clinical benefit. Furthermore, as several NK cell-based therapeutics are being developed for different cancers, an emerging variety of approaches to increase migration and infiltration of adoptively transferred NK cells towards solid tumors is under preclinical investigation. These developments point to adoptive NK cell therapy as a highly promising treatment for metastatic melanoma in the future

Early TRAIL-engagement elicits potent multimodal targeting of melanoma by CD34+ progenitor cell-derived NK cells

Summary: Umbilical cord blood (UCB) CD34+ progenitor cell-derived natural killer (NK) cells exert efficient cytotoxicity against various melanoma cell lines. Of interest, the relative cytotoxic performance of individual UCB donors was consistent throughout the melanoma panel and correlated with IFNγ, TNF, perforin and granzyme B levels. Importantly, intrinsic perforin and Granzyme B load predicts NK cell cytotoxic capacity. Exploring the mode of action revealed involvement of the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46 and most importantly of TRAIL. Strikingly, combinatorial receptor blocking led to more pronounced inhibition of cytotoxicity (up to 95%) than individual receptor blocking, especially in combination with TRAIL-blocking, suggesting synergistic cytotoxic NK cell activity via engagement of multiple receptors which was also confirmed in a spheroid model. Importantly, lack of NK cell-related gene signature in metastatic melanomas correlates with poor survival highlighting the clinical significance of NK cell therapies as a promising treatment for high-risk melanoma patients

Boosting Natural Killer Cell Therapies in Glioblastoma Multiforme Using Supramolecular Cationic Inhibitors of Heat Shock Protein 90

Allogeneic natural killer (aNK) cell adoptive therapy has the potential to dramatically impact clinical outcomes of glioblastoma multiforme (GBM). However, in order to exert therapeutic activity, NK cells require tumor expression of ligands for activating receptors, such as MHC Class I peptide A/B (MICA/B) and ULBPs. Here, we describe the use of a blood–brain barrier (BBB) permissive supramolecular cationic drug vehicle comprising an inhibitor of the chaperone heat shock protein 90 (Hsp90), which sustains a cytotoxic effect on GBM cells, boosts the expression of MICA/B and ULBPs on the residual population, and augments the activity of clinical-grade aNK cells (GTA002). First, we identify Hsp90 mRNA transcription and gain of function as significantly upregulated in GBM compared to other central nervous system tumors. Through a rational chemical design, we optimize a radicicol supramolecular prodrug containing cationic excipients, SCI-101, which displays &amp;gt;2-fold increase in relative BBB penetration compared to less cationic formulations in organoids, in vitro. Using 2D and 3D biological models, we confirm SCI-101 sustains GBM cytotoxicity 72 h after drug removal and induces cell surface MICA/B protein and ULBP mRNA up to 200% in residual tumor cells compared to the naked drug alone without augmenting the shedding of MICA/B, in vitro. Finally, we generate and test the sequential administration of SCI-101 with a clinical aNK cell therapy, GTA002, differentiated and expanded from healthy umbilical cord blood CD34+ hematopoietic stem cells. Using a longitudinal in vitro model, we demonstrate &amp;gt;350% relative cell killing is achieved in SCI-101–treated cell lines compared to vehicle controls. In summary, these data provide a first-of-its-kind BBB-penetrating, long-acting inhibitor of Hsp90 with monotherapy efficacy, which improves response to aNK cells and thus may rapidly alter the treatment paradigm for patients with GBM.</jats:p

Additional file 2 of Natural killer cells in clinical development as non-engineered, engineered, and combination therapies

Additional file 2: Table S2. List of clinical trials with engineered NK cell therapies. N = 53 clinical trials (Phase I, II, or I/II) evaluating the infusion of engineered NK cell therapies in hematological or solid tumor patients were registered on ClinicalTrials.gov until 31–12–2021. Studies are sorted by NK cell source (PB-NK, UCB-NK, iPSCs, NK-92, Unknown). The type of engineered NK products (scFv-CAR-NK, Receptor-CAR-NK, CD16-engineered, or CD16- and scFv-engineered CAR-NK), the engineering method, transgene structure, and the clinical trial design and outcome (when available) are presented. Trial status is updated to August 2022. PB: peripheral blood; UCB: umbilical cord blood; iPSCs: induced pluripotent stem cells; scFv: single-chain variable fragment

Additional file 3 of Natural killer cells in clinical development as non-engineered, engineered, and combination therapies

Additional file 3: Table S3. List of clinical trials with non-engineered combination NK cell therapies. N = 62 clinical trials (Phase I, II, or I/II) evaluating the infusion of non-engineered allogeneic NK cells in combination with other agents were registered on ClinicalTrials.gov until 31–12–2021. Studies are sorted by types of combination therapy (NK cell priming agents, Adoptive cell therapy, Antibodies, Co-stimulation, Multiple combinations, Molecular inhibitors and NK cell engagers). Details of the combination approach and the clinical trial design and outcome (when available) are presented. Trial status is updated to August 2022

Additional file 4 of Natural killer cells in clinical development as non-engineered, engineered, and combination therapies

Additional file 4: Table S4. List of clinical trials with engineered combination NK cell therapies. N = 34 clinical trials (Phase I, II, or I/II) evaluating the infusion of engineered allogeneic NK cells in combination with other agents were registered on ClinicalTrials.gov until 31–12–2021. Studies are sorted by types of combination therapy (Antibodies, Multiple combinations). Details of the combination approach and the clinical trial design and outcome (when available) are presented. Trial status is updated to August 2022

Additional file 1 of Natural killer cells in clinical development as non-engineered, engineered, and combination therapies

Additional file 1: Table S1. List of clinical trials with non-engineered NK cell therapies. N = 36 clinical trials (Phase I, II, or I/II) evaluating the infusion of non-engineered allogeneic NK cell therapies in hematological or solid tumor patients were registered on ClinicalTrials.gov between March 2017 and December 2021. Studies are sorted by NK cell source (PB-NK, UCB-CD34, UCB-NK, iPSCs). The most relevant product characteristics and the clinical trial design and outcome (when available) are presented. Trial status is updated to August 2022. PB: peripheral blood; UCB: umbilical cord blood; iPSCs: induced pluripotent stem cells

Engineering antigen‐specific NK cell lines against the melanoma‐associated antigen tyrosinase via TCR gene transfer

Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T‐lymphocytes (CTL) targeted against intracellular antigens. However, a major barrier in the development of safe CTL‐TCR therapies exists, wherein the mispairing of endogenous and genetically transferred TCR subunits leads to formation of TCRs with off‐target specificity. To overcome this and enable specific intracellular antigen targeting, we have tested the use of NK cells for TCR gene transfer to human cells. Our results show that ectopic expression of TCR α/β chains, along with CD3 subunits, enables the functional expression of an antigen‐specific TCR complex on NK cell lines NK‐92 and YTS, demonstrated by using a TCR against the HLA‐A2‐restricted tyrosinase‐derived melanoma epitope, Tyr368‐377. Most importantly, the introduction of a TCR complex to NK cell lines enables MHC‐restricted, antigen‐specific killing of tumor cells both in vitro and in vivo. Targeting of NK cells via TCR gene delivery stands out as a novel tool in the field of adoptive immunotherapy which can also overcome the major hurdle of “mispairing” in TCR gene therapy