Background The CTLA-4 blocking antibody ipilimumab has demonstrated substantial and durable effects in patients with melanoma. While CTLA-4 therapy, both as monotherapy and in combination with PD-1 targeting therapies, has great potential in many indications, the toxicities of the current treatment regimens may limit their use. Thus, there is a medical need for new CTLA-4 targeting therapies with improved benefit-risk profile. Methods ATOR-1015 is a human CTLA-4 x OX40 targeting IgG1 bispecific antibody generated by linking an optimized version of the Ig-like V-type domain of human CD86, a natural CTLA-4 ligand, to an agonistic OX40 antibody. In vitro evaluation of T-cell activation and T regulatory cell (Treg) depletion was performed using purified cells from healthy human donors or cell lines. In vivo anti-tumor responses were studied using human OX40 transgenic (knock-in) mice with established syngeneic tumors. Tumors and spleens from treated mice were analyzed for CD8 + T cell and Treg frequencies, T-cell activation markers and tumor localization using flow cytometry. Results ATOR-1015 induces T-cell activation and Treg depletion in vitro. Treatment with ATOR-1015 reduces tumor growth and improves survival in several syngeneic tumor models, including bladder, colon and pancreas cancer models. It is further demonstrated that ATOR-1015 induces tumor-specific and long-term immunological memory and enhances the response to PD-1 inhibition. Moreover, ATOR-1015 localizes to the tumor area where it reduces the frequency of Tregs and increases the number and activation of CD8 + T cells. Conclusions By targeting CTLA-4 and OX40 simultaneously, ATOR-1015 is directed to the tumor area where it induces enhanced immune activation, and thus has the potential to be a next generation CTLA-4 targeting therapy with improved clinical efficacy and reduced toxicity. ATOR-1015 is also expected to act synergistically with anti-PD-1/PD-L1 therapy. The pre-clinical data support clinical development of ATOR-1015, and a first-in-human trial has started (NCT03782467). Electronic supplementary material The online version of this article (10.1186/s40425-019-0570-8) contains supplementary material, which is available to authorized users.

Glioblastoma multiforme is the most common malignant primary brain tumor and also one of the most therapy-resistant tumors. Because of the dismal prognosis, various therapies modulating the immune system have been developed in experimental models. Previously, we have shown a 37-70% cure in a rat glioma model where rats were peripherally immunized with tumor cells producing IFNc. On the basis of these results, we wanted to investigate whether a combination of GM-CSF and IFNc could improve the therapeutic effect in a mouse glioma model, GL261 (GL-wt). Three biweekly intraperitoneal (i.p.) immunizations with irradiated GM-CSF-transduced GL261 cells (GL-GM) induced a 44% survival in mice with intracranial glioma. While treatment of GLwt and GL-GM with IFNc in vitro induced upregulation of MHC I and MHC II on the tumor cells, it could not enhance survival after immunization. However, immunizations with GL-GM combined with recombinant IFNc at the immunization site synergistically enhanced survival with a cure rate of 88%. Tumors from mice receiving only 1 immunization on Day 10 after tumor inoculation were sectioned on Day 20 for analysis of leukocyte infiltration. Tumor volume was reduced and the infiltration of macrophages was denser in mice immunized with GL-GM combined with IFNc compared with that of both wildtype and nonimmunized mice. To our knowledge, this is the first study to demonstrate a synergy between GM-CSF and IFNc in experimental immunotherapy of tumors, by substantially increasing survival as well as inducing a potent anti-tumor response after only 1 postponed immunization. ' 2006 Wiley-Liss, Inc.

Interferon gamma (IFN‐γ) has successfully been used in immunotherapy of different experimental tumours. Mechanistically, IFN‐γ has extensive effects on the immune system including release of nitric oxide (NO) by upregulation of the inducible nitric oxide synthase (iNOS). NO has putative immunosuppressive effects but could also play a role in killing of tumour cells. Therefore, the aim of the present study was to clarify whether inhibition of iNOS in rats immunized with glioma cells (N32) producing IFN‐γ (N32‐IFN‐γ), could enhance the anti‐tumour immune response. Initially, both a selective iNOS, l‐N6‐(1‐Iminoethyl)‐l‐lysine (l‐NIL), and non‐selective, N‐nitro‐l‐arginine methyl ester (l‐NAME), inhibitor of NOS were tested in vitro. After polyclonal stimulation with LPS and SEA, both l‐NIL and l‐NAME enhanced proliferation and production of IFN‐γ from activated rat splenocytes and this effect was inversely correlated to the production of NO. However, l‐NIL had a broader window of efficacy and a lower minimal effective dose. When rats were immunized with N32‐IFN‐γ, and administered NOS inhibitors by intraperitoneal (i.p.) mini‐osmotic pumps, only splenocytes of rats treated with l‐NIL, but not l‐NAME, displayed an enhanced proliferation and production of IFN‐γ when re‐stimulated with N32 tumour cells. Based on these findings, l‐NIL was administered concurrently with N32‐IFN‐γ cells to rats with intracerebral (i.c.) tumours resulting in a prolonged survival. These results show that inhibition of iNOS can enhance an IFN‐γ‐based immunotherapy of experimental i.c. tumours implying that NO released after immunization has mainly immunosuppressive net effects.

We were the first to demonstrate that combined immunotherapy with GM-CSF producing GL261 cells and recombinant IFNc of preestablished GL261 gliomas could cure 90% of immunized mice. To extend these findings and to uncover the underlying mechanisms, the ensuing experiments were undertaken. We hypothesized that immunizations combining both GM-CSF and IFNc systemically would increase the number of immature myeloid cells, which then would mature and differentiate into dendritic cells (DCs) and macrophages, thereby augmenting tumor antigen presentation and T-cell activation. Indeed, the combined therapy induced a systemic increase of both immature and mature myeloid cells but also an increase in T regulatory cells (T-regs). Cytotoxic anti-tumor responses, mirrored by an increase in Granzyme B-positive cells as well as IFNc-producing T-cells, were augmented after immunizations with GM-CSF and IFNc. We also show that the combined therapy induced a long-term memory with rejection of intracerebral (i.c.) rechallenges. Depletion of Tcells showed that both CD4 1 and CD8 1 T-cells were essential for the combined GM-CSF and IFNc effect. Finally, when immunizations were delayed until day 5 after tumor inoculation, only mice receiving immunotherapy with both GM-CSF and IFNc survived. We conclude that the addition of recombinant IFNc to immunizations with GM-CSF producing tumor cells increased the number of activated tumoricidal T-cells, which could eradicate established intracerebral tumors. These results clearly demonstrate that the combination of cytokines in immunotherapy of brain tumors have synergistic effects that have implications for clinical immunotherapy of human malignant brain tumors.