*5.2. GAGs as Immunotherapy Targets*

TME is complex and consists of immune cells (mainly lymphocytes and myeloid cells), non-immune cells (mainly endothelial cells and fibroblasts), and a complex array of structures, such as ECM, and various molecules that are either secreted or append to the cell membrane [258]. TME sustains molecules that hinder the potential effector function of NK lymphocytes. Transforming growth factor (TGF)-β and members of its superfamily downregulate NK cell cytotoxicity functions, cytokine secretion, metabolism, and proliferation. Likewise, galectins, a family of carbohydrate-binding proteins produced by different sources within the TME, downregulate NK cell functions. Various ECM components and associated enzymes (e.g., MMPs) can hinder NK cells' activation and become future therapy targets [259]. Pancreatic cancer, TME, contains various possible therapy targets, such as HA, focal adhesion kinase (FAK), connective tissue growth factor (CTGF), CD40, and chemokine (C-X-C motif) receptor 4 (CXCR-4), which could be utilized in future clinical applications [260].

Immune checkpoint inhibitor immunotherapies that had achieved broad clinical applicability in recent years [261] face the gaining of resistance. It is supposed that the HA accumulation influences tumor cells' sensitivity to chemotherapy and immunotherapy. A semiquantitative grouping of non-small lung cancer tissue demonstrated that HA deposition predicts the tumor response to pegylated hyaluronidase (PEGPH20) in animal models [262]. Thus, HA degradation facilitates tumor cells' exposure to drugs. Notably, utilization of PEGPH20, in a phase I clinical study demonstrated safety and good tolerability [263]. A phase I clinical trial, combining PEGPH20 with an immunotherapeutic agent, pembrolizumab, is currently ongoing in a cohort of metastatic gastric adenocarcinoma and non-small cell lung carcinoma patients [264], The reasoning behind this approach is the combination of facilitating drug access to tumor cells with the hypothesis that HA may modulate regulatory T cells and antitumor immune responses [265]. Clift et al. have shown that upon degrading HA, the anti-programmed death-ligand 1 (PD-L1) antibody accumulates more intensely in breast cancer tissues in vivo. An increased accumulation of T and NK cells was noticed upon HA degradation. The authors point out that decreasing HA in TME would enhance anti-tumoral immune cell infiltration and increase checkpoint inhibitor therapy efficacy [266].

Heparanase has also been linked to tumor immunology. It was shown that heparanase is implicated in chronic inflammatory bowel conditions and, consequently, in colon carcinoma initiation [222–224]. There is a clear correlation between intestinal heparanase and immune cells, mainly macrophages, which sustain the chronic inflammation and create a pro-tumoral microenvironment. Therapies that can re-equilibrate this enzyme's function and re-establish the physiological crosstalk between immune and epithelial cells would hinder colon cancer development [267]. Leukocyte-derived heparanase is versatile; therefore, subtle changes in the TME can direct the enzyme to either pro-or anti-tumoral action. Thus, in immune cancer therapy, heparanase could be a vital therapy target by either exploiting or inhibiting its activity [268].

Along these lines, heparanase inhibitors were tested in various hematological cancer models. Weissmann et al. showed in 2019 that PG545, a heparanase inhibitor, had a strong effect on human lymphoma. The inhibitor induces tumor cell apoptosis, ER stress response, and increased autophagy. PG545 did not affect naïve splenocytes but induced apoptosis even in lymphoma cells deployed of heparanase activity [239]. Another approach was utilizing heparanase-neutralizing monoclonal antibodies that strongly attenuate lymphoma cell tumor load in mouse bones due to tumor cell growth inhibition and reduced angiogenesis [269].

In chronic lymphocytic leukemia (CLL), stromal cells secrete and present CXCL12, a CXC chemokine ligand, through cellsurface-bound GAGs. By using this mechanism, CLL cells are protected from cytotoxic drugs and sustain the residual disease. The GAG mimetic, NOX-A12, binds and neutralizes CXCL12 and was tested to affect tumor cell migration. NOX-A12 inhibited CLL cell chemotaxis generated through CXCL12. Thus, NOX-A12 competes with GAGs (e.g., Hep) for CXCL12 binding and sensitizes CLL cells toward chemotherapeutic drugs [270]. An outline of the main immune-therapy targets is summarized in Table 4.


