Immunotherapy has demonstrated significant activity in a broad range of cancer types, but still the majority of patients receiving it do not maintain durable therapeutic responses. Amino acid metabolism has been proposed to be involved in the regulation of immune response. Here, we investigated in detail the role of arginase 1 (Arg1) in the modulation of antitumor immune response against poorly immunogenic Lewis lung carcinoma. We observed that tumor progression is associated with an incremental increase in the number of Arg1 + myeloid cells that accumulate in the tumor microenvironment and cause systemic depletion of ʟ-arginine. In advanced tumors, the systemic concentrations of ʟ-arginine are decreased to levels that impair the proliferation of antigen-specific T-cells. Systemic or myeloid-specific Arg1 deletion improves antigen-induced proliferation of adoptively transferred T-cells and leads to inhibition of tumor growth. Arginase inhibitor was demonstrated to modestly inhibit tumor growth when used alone, and to potentiate antitumor effects of anti-PD-1 monoclonal antibodies and STING agonist. The effectiveness of the combination immunotherapy was insufficient to induce complete antitumor responses, but was significantly better than treatment with the checkpoint inhibitor alone. Together, these results indicate that arginase inhibition alone is of modest therapeutic benefit in poorly immunogenic tumors; however, in combination with other treatment strategies it may significantly improve survival outcomes.

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Glioblastomas (GBM) are the common and aggressive primary brain tumors that are incurable by conventional therapies. Immunotherapy with immune checkpoint inhibitors is not effective in GBM patients due to the highly immunosuppressive tumor microenvironment (TME) restraining the infiltration and activation of cytotoxic T cells. Clinical and experimental studies showed the upregulation of expression of the arginase 1 and 2 (ARG1 and ARG2, respectively) in murine and human GBMs. The elevated arginase activity leads to the depletion of L-arginine, an amino-acid required for the proliferation of T lymphocytes and natural killer cells. Inhibition of ARG1/2 in the TME may unblock T cell proliferation and activate effective antitumor responses. To explore the antitumor potential of ARG1/2 inhibition, we analyzed bulk and single-cell RNA sequencing (scRNA-seq) data from human and murine gliomas. We found the upregulation of ARG1/2 expression in GBMs, both in tumor cells and in tumor infiltrating microglia and monocytes/macrophages. We employed selective arginase inhibitors to evaluate if ARG1/2 inhibition in vitro and in vivo exerts the antitumor effects. A novel, selective ARG1/2 inhibitor - OAT-1746 blocked microglia-dependent invasion of U87-MG and LN18 glioma cells in a Matrigel invasion assay better than reference compounds, without affecting the cell viability. OAT-1746 effectively crossed the blood brain barrier in mice and increased arginine levels in the brains of GL261 glioma bearing mice. We evaluated its antitumor efficacy against GL261 intracranial gliomas as a monotherapy and in combination with the PD-1 inhibition. The oral treatment with OAT-1746 did not affect the immune composition of TME, it induced profound transcriptomic changes in CD11b+ cells immunosorted from tumor-bearing brains as demonstrated by RNA sequencing analyses. Treatment with OAT-1746 modified the TME resulting in reduced glioma growth and increased antitumor effects of the anti-PD-1 antibody. Our findings provide the evidence that inhibition of ARG1/2 activity in tumor cells and myeloid cells in the TME unblocks antitumor responses in myeloid cells and NK cells, and improves the efficacy of the PD-1 inhibition.

We designed and synthesized a series of arginase inhibitors as derivatives of the well-known 2-(S)-amino-6boronohexanoic acid (ABH) with basic and neutral side chains in the α-position relative to the amino acid group. In an effort to improve the pharmacokinetic profile of literature examples and retain potent enzymatic activity, sulfamido moieties were introduced to generate hydrogen bond interaction with the aspartic acid residue in the arginase active site. The compounds with basic guanidine-containing side chains were even more potent arginase inhibitors. Both groups of compounds, as designed, demonstrated low clearance in their pharmacokinetic profile. The most active inhibitor 15aa showed high nanomolar potency with IC 50 = 32 nM toward human arginase 1 and demonstrated low clearance (4.2 mL/min/kg), long t 1/2 , and moderate volume of distribution in rat pharmacokinetic studies.

Background: The immunotherapeutic approach, adoptive cell transfer (ACT) have in malignant melanoma studies showed clinical durable responses in more than 50% of patients. However, the expansion of tumor infiltrating lymphocytes (TILs) requires extensive ex vivo culturing often at the cost of T cell differentiation and functional capacity. Most current strategies involve non-specific expansion of bulk TILs, often providing growth preference to co-infiltrated virus specific T cells and driving an exhausted phenotype of the T cell product. Methods: It is aimed to develop a new technology to expand tumor reactive T cells, through use of Major histocompatibility complex (MHC)-loaded artificial antigenpresenting scaffolds (Ag-scaffold) to provide the T cells with specific functional stimulation to obtain phenotypic and functional properties to mediate tumor regression. These scaffolds will be build using a dextran-based polysaccharide backbone associated with streptavidin molecules where biotinylated peptide-MHC class I molecules are attached to govern the specific interaction with a specific T cell, and a combination of biotinylated cytokines and co-stimulatory molecules are co-attached to provide stimulation to the T cell to achieve increased functional properties. The Ag-scaffolds interacts specifically with T cells based on recognition of the peptide-MHC molecule and effectively expand and functionally stimulate specific T cells, while leaving all other T cell specificities untouched. Results: from in vitro experiments have showed that antigen specific CD8 T cells stimulated with these Ag-scaffolds has high CD28 expression and low PD-1 expression, associated with high proliferation potential and enhanced antitumor effect in vivo. Furthermore, this expansion strategy provides a high frequency of multifunctional antigen specific CD8 T cells expressing IFN-, TNF-a, and CD107a upon target recognition. Conclusions: This expansion technology could with great advantage be used in ACT, to increase the anti-tumor effect of the transferred T cell product, as all of the achieved T cell characteristics are of significant importance for in vivo tumor cell recognition following ACT of expanded T cell products.Legal entity responsible for the study: Sine Reker Hadrup Funding: Lundbeck foundation Disclosure: All authors have declared no conflicts of interest.