595 Inhibition of integrin αvβ8 in combination with low dose radiation induces antitumor effect in advanced immune checkpoint blockade refractory tumor model

BackgroundIntegrin αvβ8 activates TGFβ in immune cells. αvβ8 inhibitors have been shown to potentiate immune checkpoint blockade (ICB) in preclinical models [1]. Radioimmunotherapy (RIT) induces immunogenic cell death and antigen presentation, however it concurrently activates immunosuppressive pathways. Interestingly, αvβ8 immunosuppressive activity was implicated in radiotherapy resistance [2]. We have explored whether antagonizing αvβ8 overcomes the suppressive effect of TGFβ and restores anti-tumor immunity in advanced ICB and RIT resistant tumors.MethodsEfficacy was evaluated after combination treatment with low dose radiation, αvβ8 (clone C6D4) and PD-1 (clone J43) mAb in an advanced CT26 colon cancer syngeneic mouse model. Mice were treated at tumor volume of >120 mm3 and euthanized at 2,000 mm3. Flow cytometry and transcriptomic analysis were used to assess the mechanism of action. Tumor volumes are presented as mean±SEM. Statistics were performed by one-way ANOVA, or log-rank test. Bone marrow derived dendritic cell (BMdDC) cultures were isolated from C57BL/6 mice.ResultsCell death, including radiation-induced apoptosis, induced immunoregulatory and maturation program in a population of ex vivo cultured BMdDC, recently described as mregDC/DC3 [3,4]. mregDC/DC3 signature was associated with increased αvβ8 expression, suggesting a role of this integrin in inducing an immunosuppressive phenotype.A CT26 model was established to mimic the progression of late-stage tumors and was unresponsive to radiation, ICB and RIT. In CT26 implanted mice, αvβ8 is expressed on tumor stoma, and is not detectable on cancer cells. Addition of αvβ8 mAb to RIT markedly increased tumor regression (P=0.0067) and survival (P<0.0001). There were 8/10 complete responders with addition of αvβ8 mAb relative to 3/10 in RIT alone. Improved efficacy correlated with enhanced T cell activation and improved DC functionality. Consistent with a recent report in a less advanced CT26 model [5], αvβ8 mAb + radiation resulted in similar efficacy as conventional RIT although the effect was modest in more advanced tumors (Figure 1, A, B).Abstract 595 Figure 1Complete response (CR) with improved survival when αvβ8 inhibition is added to RIT in CT26 syngeneic model of colorectal cancer in an advanced, ICB and RIT unresponsive stage. (A) Effect of combination therapy with low dose radiation (small animal radiation research platform (SARRP) at 5 Gray (Gy) on the day of staging (day 10)), PD-1 mAb (10 mg/kg twice weekly for 2 weeks) and αvβ8 mAb (7 mg/kg three times weekly for 3 weeks) measured by tumor burden. 5Gy+PD-1 and 5Gy+αvβ8 has a minimal effect on tumor growth inhibition showing slight improvement relative to radiation alone (5Gy+IgG). Addition of αvβ8 antagonism (5Gy+αvβ8+PD-1) improves anti-tumor responses leading to CR in 8 of 10 mice. (B) Kaplan-Meier Curve presenting time to progression. 5Gy+IgG improved survival over monotherapy with either αvβ8 or PD1 mAb. 5Gy+αvβ8+PD-1 resulted in a profound improvement of the survival over all other treatment conditionsConclusionsInhibition of αvβ8 in combination with RIT eradicated an advanced tumor, unresponsive to the respective monotherapies or conventional RIT. The anti-tumor effect was driven by enhancement of adaptive immunity, improvement of DC function and reduced tumor tolerance. These data provide evidence that αvβ8 inhibition enhances RIT and may be effective against ICB refractory tumors.ReferencesReszka-Blanco NJ,Yadav V, Krumpoch M, Cappellucci L, Cui D, Dowling JE, et al., Inhibition of integrin αvβ8 enhances immune checkpoint induced anti-tumor immunity by acting across immunologic synapse in syngeneic models of breast cancer. AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1559.Jin S, Lee WC, Aust D, Pilarsky C, Cordes N, β8 integrin mediates pancreatic cancer cell radiochemoresistance. Mol Cancer Res. 2019; 17(10): 2126–2138.Maier B, Leader AM, Chen ST, Tung N, Chang C, LeBerichel J, et al., A conserved dendritic-cell regulatory program limits antitumour immunity. Nature. 2020; 580 (7802): 257–262.Garris CS, Arlauckas SP, Kohler RH, Trefny MP, Garren S, Piot C, Engblom C, et al., Successful anti-PD-1 cancer immunotherapy requires T cell-dendritic cell crosstalk involving the cytokines IFN-γ and IL-12. Immunity. 2018; 49(6): 1148–1161.Dodagatta-Marri E, Ma H-Y, Liang B, Li J, Meyer DS, Chen S-Y, et al., Integrin αvβ8 on T cells suppresses anti-tumor immunity in multiple models and is a promising target for tumor immunotherapy. Cell Report. 2021; 36(1): 109309Ethics ApprovalAll animal work was approved by the site Institutional Animal Care and Use Committee and was performed in conformance with the Guide for the Care and Use of Laboratory Animals within an AAALAC-accredited program. Humane euthanasia criteria were predetermined on the basis of body weight and defined clinical observations.

Treatment of solid tumors using bispecific anti-PDL-1/ICOS antibody

PD-L1 and ICOS are immune control points in cancer and their presence in cancer tends to have a poor prognosis. WO2019122882 patent describes a bispecific antibody that targets PDL-1/ICOS with the potential application of cancer treatment. WO2019122882 patent describes a bispecific antibody with antitumor efficacy in CT26 model through of the depletion of TReg cells and improved ratio of CD8+ T cells: TReg in tumor microenvironment. The anti-PDL-1/ICOS antibody is new; however, only preclinical assays are shown using colon carcinoma model. So far, there are no reports of clinical trials to evaluate the safety, toxicity and efficacy, but it will be of great interest to analyze in the future if this antibody surpasses the action of the combinatorial therapy in cancer.

198 Combination intratumoral treatment with INTASYL™ self-delivering RNAi targeting TIGIT and PD-1/PD-L1 improves tumor control compared to monotherapy in a CT26 model of murine colorectal cancer

BackgroundDespite clinical successes of immune checkpoint blockade (ICB) antibodies blocking the inhibitory receptors CTLA-4, PD-1, or PD-L1, substantial challenges remain. Many patients do not respond, and ICB treatment is associated with serious immune-related adverse effects (irAEs) which are exacerbated by combination therapies. TIGIT blockade has been demonstrated to provide tumor control in pre-clinical studies, sparking ongoing clinical trials, including those targeting TIGIT in combination with anti-PD-1 or anti-PD-L1. The INTASYL™ platform is a self-delivering RNAi technology that (1) provides efficient delivery into target cells bypassing the need for specialized formulations, mechanical perturbation, or drug delivery systems; and (2) specifically and durably silence target gene expression when administered intratumorally (IT), providing in vivo tumor control. IT administration restricts pharmacokinetics to the tumor; an attractive strategy for mitigating ICB-mediated systemic irAEs. Additionally, using INTASYL, multiple targets can be silenced in combination. Here we demonstrate the in vivo efficacy of INTASYL specifically targeting TIGIT (PH-804), PD-1 (PH-762), PD-L1 (PH-790) alone or in combination in a CT26 model of murine colorectal carcinoma.MethodsTo assess silencing activity, activated human pan-T cells were incubated in vitro with INTASYL compounds either alone or in combination and mRNA silencing was determined by qRT-PCR and protein silencing by flow cytometry. To assess in vivo tumor efficacy CT-26 cells were implanted subcutaneously into BALB/c mice. INTASYL compounds were administered IT at 1 mg/dose on Days 1, 3, 7, and 10 either as single agents (mPH-804, mPH-762, mPH-790) or in combination (mPH-804 + mPH-762 or mPH-804 + mPH-790). Controls consisted of PBS (vehicle; (IT)), and anti-TIGIT, anti-PD-1, or anti-PD-L1 antibodies (0.2 mg/dose) administered via intraperitoneal injection (IP). Tumor volumes and body weight were recorded throughout the study. Tumors were taken at the end of the study for analysis.ResultsSingle and combination knockdown of target molecules was validated at the mRNA level (=90%) by qRT-PCR and at the protein level (=80%) in activated human pan-T cells. In vivo, combination treatment with mPH-804 + mPH-762 or mPH-790 improved tumor control compared to individual monotherapies providing evidence of potential synergy. All treatments were well tolerated.Conclusionsn/aAcknowledgementsWe demonstrate the potential of INTASYL-mediated combination therapy targeting TIGIT and PD-1/PD-L1. These findings indicate that combination of TIGIT + PD-1/PD-L1 silencing improves tumor control compared to monotherapy. As INTASYL IT is efficacious and may mitigate irAEs caused by antibody ICB, INTASYL combination therapies including PH-804, PH-762 and PH-790 warrant further investigation in patients.

Effect of Chemokine (C-C Motif) Ligand 7 (CCL7) and Its Receptor (CCR2) Expression on Colorectal Cancer Behaviors

Colorectal cancer is the source of one of the most common cancer-related deaths worldwide, where the main cause of patient mortality remains metastasis. The aim of this study was to determine the role of CCL7 (chemokine (C-C motif) ligand 7) in tumor progression and finding whether it could predict survival of colorectal cancer patients. Initially, our study focused on the crosstalk between mesenchymal stem cells (MSCs) and CT26 colon carcinoma cells and resulted in identifying CCL7 as a chemokine upregulated in CT26 colon cancer cells cocultured with MSCs, compared with CT26 in monoculture in vitro. Moreover, we showed that MSCs enhance CT26 tumor cell proliferation and migration. We analyzed the effect of CCL7 overexpression on tumor progression in a murine CT26 model, where cells overexpressing CCL7 accelerated the early phase of tumor growth and caused higher lung metastasis rates compared with control mice. Microarray analysis revealed that tumors overexpressing CCL7 had lower expression of immunoglobulins produced by B lymphocytes. Additionally, using Jh mutant mice, we confirmed that in the CT26 model, CCL7 has an immunoglobulin-, and thereby, B-cell-dependent effect on metastasis formation. Finally, higher expression of CCL7 receptor CCR2 (C-C chemokine receptor type 2) was associated with shorter overall survival of colorectal cancer patients. Altogether, we showed that CCL7 is essentially involved in the progression of colorectal cancer in a CT26 mouse model and that the expression of its receptor CCR2 could be related to a different outcome pattern of patients with colorectal carcinoma.