Response In Tumor-bearing Mice Research Articles

Listeria-vectored cervical cancer vaccine candidate strains reduce MDSCs via the JAK-STAT signaling pathway.

Immunosuppressive status is prevalent in cancer patients and increases the complexity of tumor immunotherapy. It has been found that Listeria-vectored tumor vaccines had the potential ability of two-side regulatory effect on the immune response during immunotherapy. The results show that the combined immunotherapy with the LM∆E6E7 and LI∆E6E7, the two cervical cancer vaccine candidate strains constructed by our lab, improves the antitumor immune response and inhibits the suppressive immune response in tumor-bearing mice in vivo, confirming the two-sided regulatory ability of the immune response caused by Listeria-vectored tumor vaccines. The immunotherapy reduces the expression level of myeloid-derived suppressor cells (MDSCs)-inducing factors and then inhibits the phosphorylation level of STAT3 protein, the regulatory factor of MDSCs differentiation, to reduce the MDSCs formation ability. Moreover, vaccines reduce the expression of functional molecules associated with MDSCs may by inhibiting the phosphorylation level of the JAK1-STAT1 and JAK2-STAT3 pathways in tumor tissues to attenuate the immunosuppressive function of MDSCs. Immunotherapy with Listeria-vectored cervical cancer vaccines significantly reduces the level and function of MDSCs in vivo, which is the key point to the destruction of immunosuppression. The study for the first to elucidate the mechanism of breaking the immunosuppression.

Maleimide as the PEG end-group promotes macrophage-targeted drug delivery of PEGylated nanoparticles in vivo by enhancing interaction with circulating erythrocytes

Radiotherapy (IR) is capable of enhancing antitumor immune responses. However, IR treatment also aggravates the infiltration of peripheral macrophages into the tumor, resulting in reversing the therapeutic effects of antitumor immunity. Thus, a strategy to effectively prevent tumor infiltration by macrophages may further improved the therapeutic efficacy of radiotherapy. Herein, we found that PEGylated solid lipid nanoparticles with maleimide as PEG end-group (SLN-PEG-Mal) show significantly enhanced adsorption onto RBCs through reacting with reactive sulfhydryl groups on RBCs’ surface both in vitro and in vivo, and caused significant changes in the surface properties and morphology of RBCs. These RBCs adsorbed by SLN-PEG-Mal were rapidly removed from circulation due to efficient engulfment by reticuloendothelial macrophages, supporting the usefulness of SLN-PEG-Mal for macrophage-targeted drug delivery. While lacking the use of radioisotope tracing (considered the gold standard for PK/BD studies), our data align with the expected pathway of host defense activation through surface-loaded RBCs. Importantly, injection of paclitaxel-loaded SLN-PEG-Mal effectively inhibited the tumor-infiltration by macrophages, and significantly improved the antitumor immune responses in tumor-bearing mice treated with low-dose irradiation. This study provides insights into the effects of maleimide as PEG end-group on enhancing the interaction between PEGylated nanoparticles and RBCs and offers an effective strategy to inhibit tumor infiltration by circulating macrophages.

Dressing Bacteria With a Hybrid Immunoactive Nanosurface to Elicit Dual Anticancer and Antiviral Immunity.

Approaches capable of simultaneously treating cancer and protecting susceptible patients from lethal infections such as coronavirus disease 2019, are highly desirable but prove to be difficult. Here, dressing bacteria with a hybrid immunoactive nanosurface is reported to elicit dual anticancer and antiviral immunity. A combination of a checkpoint blocking antibody and a virus-specific antigen is covalently conjugated to polydopamine nanoparticles, which can be anchored onto bacterial surface, by a one-step in situ polymerization of dopamine under a cell-friendly condition. By virtue of the ability to colonize and penetrate deep tumor tissue, dressed bacteria enable sustained release and expanded exposure of carried immunoactivators to stimulate immune cells. In addition to a carrier role, bacteria are able to further provoke innate immunity due to the native immunogenicity of the pathogen-associated molecular patterns. Immunization with dressed bacteria promotes the maturation, and activation of antigen-presenting cells, which induces robust humoral and cellular immune responses in tumor-bearing mice. As evidenced by efficient production of viral-antigen-specific immunoglobulin G antibody in serum and significantly suppressed tumor growth in different models, dressing bacteria with a hybrid immunoactive nanosurface paves an avenue to prepare next-generation therapeutics for synergistic treatment and prevention.

Combination TIGIT/PD-1 blockade enhances the efficacy of neoantigen vaccines in a model of pancreatic cancer.

Cancer neoantigens are important targets of cancer immunotherapy and neoantigen vaccines are currently in development in pancreatic ductal adenocarcinoma (PDAC) and other cancer types. Immune regulatory mechanisms in pancreatic cancer may limit the efficacy of neoantigen vaccines. Targeting immune checkpoint signaling pathways in PDAC may improve the efficacy of neoantigen vaccines. We used KPC4580P, an established model of PDAC, to test whether neoantigen vaccines can generate therapeutic efficacy against PDAC. We focused on two immunogenic neoantigens associated with genetic alterations in the CAR12 and CDK12 genes. We tested a neoantigen vaccine comprised of two 20-mer synthetic long peptides and poly IC, a Toll-like receptor (TLR) agonist. We investigated the ability of neoantigen vaccine alone, or in combination with PD-1 and TIGIT signaling blockade to impact tumor growth. We also assessed the impact of TIGIT signaling on T cell responses in human PDAC. Neoantigen vaccines induce neoantigen-specific T cell responses in tumor-bearing mice and slow KPC4580P tumor growth. However, KPC4580P tumors express high levels of PD-L1 and the TIGIT ligand, CD155. A subset of neoantigen-specific T cells in KPC4580P tumors are dysfunctional, and express high levels of TIGIT. PD-1 and TIGIT signaling blockade in vivo reverses T cell dysfunction and enhances neoantigen vaccine-induced T cell responses and tumor regression. In human translational studies, TIGIT signaling blockade in vitro enhances neoantigen-specific T cell function following vaccination. Taken together, preclinical and human translational studies support testing neoantigen vaccines in combination with therapies targeting the PD-1 and TIGIT signaling pathways in patients with PDAC.

Abstract 5569: CAR-T cells with αPDL1/CD28 switch-receptor synergize radiotherapy and anti-PD1 therapy in solid tumors

Abstract The therapeutic efficacy of CAR-T cells in solid tumors, unlike in hematologic tumor, is greatly limited by the insufficient infiltration and persistence of CAR-T cells as well as the immunosuppressive tumor microenvironment. The aim of this study was to overcome these obstacles by introducing an αPDL1/CD28 switch-receptor construct and by seeking combinatorial strategies for CAR-T cells in solid tumors. We found that in non-transduction T cells, the cytokine release and T cell proliferation were repressed in response to PD-L1 protein, while T cells that express αPDL1/CD28 switch-receptor showed enhanced functions, indicating that αPDL1/CD28 could revert PD-L1 inhibition into CD28 costimulation. CAR-T cells with αPDL1/CD28 switch-receptor showed better effector function than that of unitary CAR-T in vitro studies and significant responses in tumor-bearing mice, with more effector memory T cells infiltrated in the tumor. On this basis, PD-1 inhibitor can further enhance the efficacy and persistence of αPDL1/CD28 CAR-T cells, especially in PDL1+ tumor models. We found in vitro studies that radiotherapy increased the expression of T-cell recruiting chemokines and promoted CAR-T cell transmigration. In tumor-bearing mice, synergistic anti-tumor efficacy of mice treated with radiotherapy and αPDL1/CD28 CAR-T cells was further observed. Finally, triple therapy with radiotherapy and anti-PD1 plus αPDL1/CD28 CAR-T cells maximized antitumor responses and induced complete cures in the tumor-bearing mice. Our study suggests that αPDL1/CD28 augments the function of CAR-T cells, and the combinatorial regime of αPDL1/CD28 CAR-T cells, radiotherapy and anti-PD1 in solid tumors could be further investigated. Citation Format: Limei Yin, Wenbo Wang, Zhuoran Yao, Jianxin Xue, Ruizhan Tong, Hui Wang, Shuangsi Liao, Laduona Wang, Yue Zheng, Feifei Na, Min Yu, Xuanwei Zhang, Youling Gong, Meijuan Huang, Xianming Mo, Chong Chen, You Lu. CAR-T cells with αPDL1/CD28 switch-receptor synergize radiotherapy and anti-PD1 therapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5569.

Welfare Assessment on Healthy and Tumor-Bearing Mice after Repeated Ultrasound Imaging

Introduction: Ultrasound (US) imaging enables tissue visualization in high spatial resolution with short examination times. Thus, it is often applied in preclinical research. Diagnostic US, including contrast-enhanced US (CEUS), is considered to be well-tolerated by laboratory animals although no systematic study has been performed to confirm this claim. Therefore, the aim of this study was to screen for possible effects of US and CEUS examinations on welfare of healthy mice. Additionally, the potential influence of CEUS and molecular CEUS on well-being and therapy response to regorafenib was investigated in breast cancer-bearing mice. Material and Methods: Forty healthy Balb/c mice were randomly assigned for examination with US or CEUS (3×/week) for 4 weeks. Untreated healthy mice and mice receiving only isoflurane anesthesia served as controls (n = 10/group). Ninety-four 4T1 tumor-bearing Balb/c mice were allocated randomly to the following groups: no imaging, isoflurane anesthesia, CEUS, and molecular CEUS. They either received 10 mg/kg regorafenib or vehicle solution daily by oral gavage. Animals were examined three times within 2 weeks. CEUS measurements were performed using phospholipid microbubbles, and phospholipid microbubbles targeting the vascular endothelial growth factor receptor-2 were applied for molecular CEUS. Welfare evaluation was performed by daily observational score sheets, measuring the heart rate, Rotarod performance, and fecal corticosterone metabolites twice a week. On the last day, pathological changes in serum corticosterone concentrations, hemograms, and organ weights were obtained. Moreover, a potential influence of isoflurane anesthesia, CEUS, and molecular CEUS on regorafenib response in tumor-bearing mice was examined. Analysis of variance and Dunnett’s post hoc test were performed as statistical analyses. Results: Severity parameters were not altered after repeated US and CEUS examinations of healthy mice, but spleen sizes were significantly lower after isoflurane anesthesia. In tumor-bearing mice, no effect on animal welfare after repeated CEUS and molecular CEUS could be observed. However, leukocyte counts and spleen weights of tumor-bearing mice were significantly lower in animals examined with CEUS and molecular CEUS compared to the control groups. This effect was not visible in regorafenib-treated animals. Conclusions: Repeated US and (molecular) CEUS have no detectable impact on animal welfare in healthy and tumor-bearing mice. However, CEUS and molecular CEUS in combination with isoflurane anesthesia might attenuate immunological processes in tumor-bearing animals and may consequently affect responses to antitumor therapy.

Tumor microenvironment-responsive touch sensor-based pH-triggered controllable conductive hydrogel

A tumor microenvironment-responsive wireless strain-pressure hydrogel sensor based on pH-induced controllable nanoparticles was designed for cancer detection in vitro–in vivo model, with excellent ability to distinguish between cancer and normal cells. The pH-responsive nanoparticles (CD-PNB), comprising diol–diol crosslinked semiconducting carbon dots (CDs) and non-conductive polymer (PNB), are sensitive to acidic tumor microenvironments and play crucial role in demonstrating tumor-selective strain-pressure responses. Upon application of strain and pressure, CD-PNB@PVA hydrogel produced distinct electronic signals in the presence of cancer cells (HeLa, PC-3), exhibiting higher strain–pressure sensitivity compared to the normal cells (MDCK, CHO-K1). The strain and pressure gage factors for cancer-cell-treated CD-PNB@PVA hydrogel were found to be 0.7439 and 5.3052 kPa−1, which were higher than normal-cell-treated CD-PNB@PVA hydrogel (0.5009 and 4.2720 kPa−1). CD-PNB@PVA hydrogel demonstrated excellent response in tumor-bearing mice based on in situ and ex situ measurements, with no inflammation during hydrogel implantation. Moreover, wireless sensing system was used along with CD-PNB@PVA hydrogel to simplify monitoring process and obtain real-time conductivity and strain–pressure profiles on smartphone. Thus, this approach constructs a tumor microenvironment-responsive strain–pressure hydrogel sensor and presents potential for sensitive and selective tumor detection in point-of-care diagnostic applications.

Abstract 1243: Effects of ITI-214, a potent and selective phosphodiesterase type 1 inhibitor, on tumor myeloid cellular composition, tumor volume and survival in mouse models of colorectal cancer when combined with an anti-PD-1 checkpoint inhibitor

Abstract We investigated the effect of ITI-214, a selective phosphodiesterase type 1 (PDE-1) inhibitor [Li et al., J Med Chem, 2016, 1149-64] on immune cell composition of the tumor microenvironment, tumor volume and survival in syngeneic mouse model of colorectal cancer. The studies are based on our previous preclinical work showing that PDE-1 inhibitors modulate the function of immune cells (microglia, macrophages) by reducing immune cell motility and down-regulating key cytokines (CCL2 and TNFa). We hypothesize that ITI-214 will inhibit macrophage infiltration into the tumor microenvironment and reverse macrophage suppression of immune responses in immunogenic cancers, thereby promoting antitumor immunity and tumor killing. The effects of ITI-214, alone or in combination with sub-effective doses of programmed cell death-1 (PD-1) immune checkpoint inhibitor, were assessed on the growth of CT26 tumor in BALB/c mice. Tumor responses in tumor-bearing mice treated with isotype antibodies (i.e. control group) were compared with groups of mice treated with ITI-214 monotherapy (50mg/kg, i.p. qd, from day 7), a sub-effective dose of anti-PD-1 antibody (150 µg anti-PD-1 RPMI-14, days 7, 11, 14, 17), and a combination of each dose of ITI-214 plus a sub-effective dose of the anti PD-1 antibody. Results showed that only mice receiving the combination of ITI-214 with a sub-effective dose of anti-PD-1 displayed significantly reduced tumor growth (p=0.001). Flow cytometry analysis of tumor-infiltrating immune cells revealed that tumors from ITI-214 monotherapy-treated mice showed significantly decreased numbers of tumor (infiltrating) macrophages which were further decreased in mice receiving the combination treatment. Further, the combination therapy of ITI-214 and anti-PD-1 enhanced anti-tumor immunity by eliciting increased numbers of Natural Killer (NK) cells and TNFa-producing CD4 T-cells. The results suggest that ITI-214 alone reduces numbers of tumor macrophages, an effect which we believe unmasks the anti-tumor immune response induced by anti-PD-1 antibodies. In separate experiments we assessed the effect of combined anti-PD-1 (5mg/kg anti-PD-1 RPMI-14, biwk x 2) and ITI-214 treatment (25 or 50mg/kg, oral, qd, from day 7) on survival; the effect of combined anti-PD-1 and ITI-214 treatment produced a complete response in about 50% of treated mice, translating into a statistically significant effect on survival as compared to control (p=0.001). Together, these studies demonstrate a synergistic ability of ITI-214 and anti-PD-1 to alter the tumor environment and control tumor growth. Translating these findings to humans suggests that ITI-214 may provide a means to expand the utility of immune checkpoint inhibitors in immunogenic tumors. Citation Format: Gretchen L. Snyder, Peter A. John, Marc V. Pulanco, Xingxing Zang, Allen A. Fienberg, Robert E. Davis. Effects of ITI-214, a potent and selective phosphodiesterase type 1 inhibitor, on tumor myeloid cellular composition, tumor volume and survival in mouse models of colorectal cancer when combined with an anti-PD-1 checkpoint inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1243.

Abstract 1516: Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors

Abstract Adoptive transfer of T cells is a promising anti-tumor therapy for many cancers. To enhance tumor recognition by T cells, chimeric antigen receptors (CAR) consisting of signaling domains fused to receptors that recognize tumor antigens can be expressed in T cells. One receptor that is a prospective target for a new chimeric antigen receptor is PD1 because the ligands for the PD1 receptor are expressed on many cancer types. Therefore, we developed a murine chimeric PD1 receptor (chPD1) consisting of the PD1 receptor extracellular domain and the activation domain of CD3 zeta. In addition, current chimeric antigen receptor therapies utilize various costimulatory domains to enhance anti-tumor efficacy. Therefore, we also compared the inclusion of CD28, Dap10, 4-1BB, GITR, ICOS, or OX40 costimulatory domains in the chPD1 receptor to determine which costimulatory domain induced optimal anti-tumor immunity. To determine if this novel CAR could potentially target a wide variety of tumors, the anti-tumor efficacy of chPD1 T cells against murine lymphoma, melanoma, kidney, pancreatic, liver, colon, breast, ovarian, prostate, and bladder cancer cell lines was measured. Of the eighteen cell lines tested, all expressed PD1 ligands on their cell surface, making them potential targets for chPD1 T cells. Regardless of the costimulatory domain in the CAR, all of the chPD1 T cells induced similar levels of T cell proliferation and tumor cell lysis. However, differences were observed in the cytokine secretion profiles depending on which costimulatory receptor was included in the CAR. While most of the chPD1 T cell receptor combinations secreted both pro-inflammatory (IFNγ, TNFα, IL-2, GM-CSF, IL-17, and IL-21) and anti-inflammatory cytokines (IL-10), chPD1 T cells containing a Dap10 costimulatory domain secreted high levels of proinflammatory cytokines but did not secrete a significant amount of anti-inflammatory cytokines. Furthermore, T cells expressing chPD1 receptors with a Dap10 domain also had the strongest anti-tumor efficacy in vivo. ChPD1 T cells did not survive for longer than 14 days in vivo, however treatment with chPD1 T cells induced long-lived protective host-anti-tumor immune responses in tumor-bearing mice. Therefore, adoptive transfer of chPD1 T cells could be a novel therapeutic strategy to treat multiple types of cancer and inclusion of the Dap10 costimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile for anti-tumor therapies. Citation Format: Amorette E. Barber. Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1516.

Effects Induced In Vivo by Exposure to Magnetic Signals Derived From a Healing Technique.

Energy healing is a therapy said to manipulate and balance the flow of “energies” in the body. One such technique, the Bengston Healing Method (BHM), has shown some success in healing malignant tumors in animals and humans, but the mechanism of action and factors influencing therapeutic success of this method are poorly understood. In this study, we tested in vivo the antitumor potential of magnetic signals recorded during BHM healing. Balb/c mice engrafted with 4T1 breast cancer cells were exposed to this recording for 4 h/d on a weekly or daily basis for 28 days; control mice were not exposed at all. Tumors showed a trend to grow slower in the treatment versus control group during the fourth week of treatment. Elevated leukocyte counts, associated with an increase in blood levels of granulocyte–macrophage colony stimulating factor and interleukin-6, were observed in tumor-bearing mice exposed to the BHM recording but not in healthy animals exposed to the recording. This suggests that exposure to a recording of BHM may induce a biological response in tumor-bearing mice, but limited effects on tumor growth when observed within the predefined end point of 28 days. Studies involving longer end points are recommended to observe the progression of tumor growth.

Oncolytic Viruses Engineered to Enforce Leptin Expression Reprogram Tumor-Infiltrating T Cell Metabolism and Promote Tumor Clearance

Immunotherapy can reinvigorate dormant responses to cancer, but response rates remain low. Oncolytic viruses, which replicate in cancer cells, induce tumor lysis and immune priming, but their immune consequences are unclear. We profiled the infiltrate of aggressive melanomas induced by oncolytic Vaccinia virus using RNA sequencing and found substantial remodeling of the tumor microenvironment, dominated by effector Tcell influx. However, responses to oncolytic viruses were incomplete due to metabolic insufficiencies induced by the tumor microenvironment. We identified the adipokine leptin as a potent metabolic reprogramming agent that supported antitumor responses. Leptin metabolically reprogrammed Tcells invitro, and melanoma cells expressing leptin were immunologically controlled in mice. Engineering oncolytic viruses to express leptin in tumor cells induced complete responses in tumor-bearing mice and supported memory development in the tumor infiltrate. Thus, leptin can provide metabolic support to tumor immunity, and oncolytic viruses represent a platform to deliver metabolic therapy.

Novel tetravalent bispecific T-cell engaging antibodies for cancer immunotherapy.

e12513 Background: Current immunotherapy involves redirection of T cells against tumor either by chimeric antigen receptors (CAR-T) or bispecific T-cell engagers (BiTEs). These T cell-engaging technologies have been enabled by engineering single-chain variable fragment (scFv). Most BiTEs are designed as heterodimers, with monovalent binding to tumor cell surface antigens and to CD3+ T cells. Here, we describe a novel tetravalent bispecific (TetraBi) platform: a tumor associated antigen specific, T-cell engaging antibody that provides significant advantages over traditional BiTEs. Specifically, we showcase three unique TetraBi antibodies targeted for various cancers. Methods: ABP-100 was engineered by fusing CD3-binding scFv to the c-terminus of anti-HER2 IgG1 light chain, resulting in two binding sites each for HER2 and CD3. In preclinical studies, ABP-100 was compared with a clinical-stage heterodimeric antibody that is monovalent for HER2 and CD3. We also built TetraBi for anti-Glypican 3 (GPC3, ABP-110) and Claudin 18.2 (CLDN18.2, ABP-150) for gastric cancer therapy. Results: We observed bivalent binding to the tumor antigens (HER2, GPC3, CLDN18.2) on tumor cells like the monoclonal antibodies do. Interestingly, the CD3-binding domains in our TetraBi format showed functionally monovalent binding to CD3. ABP-100 displayed comparable cytokine release to the heterodimer molecule both in vitro and in vivo. In HER2+ cancer models, ABP-100 showed highly potent antitumor activity, resulting in complete responses in tumor bearing mice at doses as low as 0.1 mg/kg with no evidence of tumor relapse. ABP110 and ABP-150 also demonstrated potent cancer cell killing in vitro. Conclusions: This novel TetraBi antibody format enables concurrent bivalent and monovalent co-engagement of distinct target antigens in tumor microenvironment with promising therapeutic implications. Furthermore, it provides a potentially larger therapeutic index than more traditional bispecific formats that feature monovalent recognition of tumor antigens.

Intratumoral Activity of the CXCR3 Chemokine System Is Required for the Efficacy of Anti-PD-1 Therapy

Despite compelling rates of durable clinical responses to programmed cell death-1 (PD-1) blockade, advances are needed to extend these benefits to resistant tumors. We found that tumor-bearing mice deficient in the chemokine receptor CXCR3 responded poorly to anti-PD-1 treatment. CXCR3 and its ligand CXCL9 were critical for a productive CD8+ Tcell response in tumor-bearing mice treated with anti-PD-1 but were not required for the infiltration of CD8+ Tcells into tumors. The anti-PD-1-induced anti-tumor response was facilitated by CXCL9 production from intratumoral CD103+ dendritic cells, suggesting that CXCR3 facilitates dendritic cell-T cell interactions within the tumor microenvironment. CXCR3 ligands in murine tumors and in plasma of melanoma patients were an indicator of clinical response to anti-PD-1, and their induction in non-responsive murine tumors promoted responsiveness to anti-PD-1. Our data suggest that the CXCR3 chemokine system is a biomarker for sensitivity to PD-1 blockade and that augmenting the intratumoral function of this chemokine system could improve clinical outcomes.

A powerful ICOS agonist that enhances anti-tumor immune responses restored by immune checkpoint inhibitors

Abstract The Inducible T cell Costimulator (ICOS, CD278) is a receptor in the CD28 family of B7-binding proteins expressed mostly by activated T cells. Upon binding to its ligand ICOS-L expressed on APCs,T cells are further activated by ICOS, resulting in their expansion and production of effector cytokines. Clinical data in patients treated with anti-CTLA-4 or anti-PD-1 mAbs have shown that ICOShiT cells correlated with an increased treatment response in these patients. Here, we report the design of a high avidity pentavalent form of the human ICOS-L extracellular domain fused to a short α-helical peptide from the human cartilage oligomeric matrix protein (COMP). This ICOS agonist, termed ICOS-L.COMP, spontaneously assembles into stable pentamers, binds tightly to both human and murine ICOS (SPR; Kd<10 nM) and co-stimulates (with anti CD3 mAb) the proliferation and cytokine release of both murine and human CD4+ and CD8+ T cells when added as a soluble factor (unlike ICOS-L.Fc). Soluble ICOS-L.COMP also phosphorylates Akt in activated T cells and enhances anti-tumor immune responses in tumor-bearing mice [murine MC38 or CT26 tumors] treated with either anti-murine PD-1 or anti-murine CTLA-4 mAb. Impressively, most mice treated with ICOS-L.COMP combined with an immune checkpoint inhibitor were either cured or displayed a stable tumor burden in contrast to checkpoint monotherapies (delayed tumor progression) or when ICOS-L.COMP was given as a monotherapy (comparable to no treatment). Importantly, ICOS-L.COMP lacks a Fc domain thus eliminating Fc-associated off-target adverse effects. In summary, our results suggest that ICOS-L.COMP represents a new and powerful biologic to be used in combination with existing immune checkpoint inhibitors.

120 - ROS-targeting of melanoma cells provides immuno-protection against tumor growth

Breaking barriers in science necessitates crossing of boundaries between disciplines. An intimate link is presented that connects the fields of redox research, cancer therapy, and immunology. Immunogenic cancer cell death (ICD) provides inflammatory stimuli to elicit anti-tumor adaptive immune responses. Release of adjuvant find-me (HMGB1, ATP) and eat-me (calreticulin) signals facilitates DC maturation and cross presentation of neoantigens to T cells. Key hallmarks of ICD in tumor cells are stress of the endoplasmic reticulum and the unfolded proteins response. Both are known to be subject to redox control. Potent ICD-inducing therapies are drugs such as anthracyclines, ionizing irradiation, or photodynamic therapies. Intriguingly, these antitumor approaches are reported to show concomitant generation of reactive species that can drive signaling responses through redox relays. Hence, the importance of reactive species and specifically hydrogen peroxide signaling are underappreciated in the onset of ICD in cancer cells. Recently, a novel technology capable of generating various oxidants has not only been shown to confer remarkable effects in tumor-bearing mice but is also a potent ICD inducer. This cold physical plasma, a partially ionized gas at about body temperature, can be applied to tumors topically, and first palliative cancer patients benefited from plasma therapy. Experimental evidence will be presented that i) physical plasma is an ICD inducer in various cell types including melanoma, ii) this is concomitant with a global HMOX1 signature, and iii) plasma-derived ROS evoke an antitumor T-cell response in tumor-bearing mice.

Preclinical evaluation of an innovative anti-TAM approach based on zoledronate-loaded erythrocytes.

In tumor microenvironment, tumor-associated macrophages (TAMs) are implicated in cancer sustainment, metastasis, and drug resistance, raising a growing interest as targets in cancer therapy. Since the bisphosphonate zoledronate has proven to affect TAMs' functions, the anti-tumor effect of single or repeated administrations of red blood cells (RBCs) encapsulating zoledronate was evaluated in a mouse model of mammary carcinoma. The obtained results showed that loaded RBCs, but not free zoledronate, caused a significant (p < 0.01) and time-lasting reduction of TAMs' extent in tumor mass of Balb/C mice inoculated with murine mammary carcinoma T41 cells; in addition, a significant reduction (p < 0.05) of tumor growth rate has been obtained only following repeated administrations of zoledronate-loaded RBCs. The anti-tumor effect was secondary to the early depletion of spleen macrophages. Moreover, by assessing the IgG2a/IgG1 ratio, a prevalence of Th1 cytotoxic response in tumor-bearing mice receiving zoledronate by means of RBCs has been observed. These encouraging findings provide further evidence for the central role played by macrophages in tumor setting and highlight the suitability of zoledronate-loaded RBCs as pharmacological agents in depleting, even if indirectly, TAMs and, thus, their eligibility as part of a therapeutic strategy in cancer treatment.

Abstract 3582: Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors

Abstract Adoptive transfer of tumor-reactive T cells is a promising anti-tumor therapy for many cancers. To enhance tumor recognition by T cells, chimeric antigen receptors (CAR) consisting of signaling domains fused to receptors that recognize tumor antigens can be created and expressed in T cells. One receptor that is a prospective target for a new chimeric antigen receptor is PD1 because the ligands for the PD1 receptor are expressed on many cancer types. Therefore, we developed a murine chimeric PD1 receptor (chPD1) consisting of the PD1 receptor extracellular domain and the activation domain of CD3 zeta. In addition, current chimeric antigen receptor therapies utilize various costimulatory domains to enhance anti-tumor efficacy. Therefore, we also compared the inclusion of CD28, Dap10, 41BB, GITR, ICOS, or OX40 costimulatory domains in our chPD1 receptor to determine which costimulatory domain induced optimal anti-tumor immunity. To determine if this novel CAR could potentially target a wide variety of tumors, the anti-tumor efficacy of chPD1 T cells against murine lymphoma, melanoma, kidney, pancreatic, liver, colon, breast, ovarian, prostate, and bladder cancer cell lines was measured. Of the eighteen cell lines tested, all expressed PD1 ligands on their cell surface, making them potential targets for chPD1 T cells. Regardless of the costimulatory domain in the CAR, all of the chPD1 T cells induced similar levels of T cell proliferation and tumor cell lysis. However, differences were observed in the cytokine secretion profiles depending on which costimulatory receptor was included in the CAR. While most of the chPD1 T cell receptor combinations secreted both pro-inflammatory (IFNγ, TNFα, IL-2, GM-CSF, IL-17, and IL-21) and anti-inflammatory cytokines (IL-10 and IL-5) as determined by ELISA and LegendPlex analysis, chPD1 T cells containing a Dap10 costimulatory domain secreted high levels of proinflammatory cytokines, but did not secrete a significant amount of anti-inflammatory cytokines. Furthermore, T cells expressing chPD1 receptors with a Dap10 domain also had the strongest anti-tumor efficacy in vivo. ChPD1 T cells did not survive for longer than 14 days in vivo, however treatment with chPD1 T cells induced long-lived protective host-anti-tumor immune responses in tumor-bearing mice. Therefore, adoptive transfer of chPD1 T cells could be a novel therapeutic strategy to treat multiple types of cancer and inclusion of the Dap10 costimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile for anti-tumor therapies. Citation Format: Amorette E. Barber, Geoffrey Parriott, Shane Crean, Hailey Kintz. Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3582.

Anti-tumor Activity of Agaricus bisporus Extracts and its Relation with IL-2 in Tumor-Bearing Mice

Mushroom is widely used as a traditional medicinal fungus, and it has been known to exhibit immune-stimulatory and anti-cancer activity. However, the exact immune response in tumor-bearing mice after exposure to mushroom extract was unclear. The aim of this study was to investigate whether or not crude preparations extracted from Agaricus bisporus can promote type 1 immune response in tumor-bearing mice. Watery and ethanolic extracts of mushroom were intraperitoneally administered into tumor-bearing mice for 28 alternative days, tumor volume was measured twice weekly, also IL-2 level was determined in the sera of mice one day before the beginning of treatment and after 2 and 4 weeks of treatment. The results showed that both extracts significantly inhibit the growth of tumor up to 72.2% at the end of experiment, while only the ethanolic extract significantly increased the level of IL-2 production from 11.2 and 16.9 pg/ml up to 26.4 and 42.5 pg/ml after 2 and 4 weeks of treatment respectively. To confirm these results , further studies are required to investigate the ability of these extracts to promote the production of IL-12 &amp; IFN-γ which are the major cytokines involved in immune response type 1 activation.

Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting TERT.

Immune checkpoint blockade antibodies are setting a new standard of care for cancer patients. It is therefore important to assess any new immune-based therapies in the context of immune checkpoint blockade. Here, we evaluate the impact of combining a synthetic consensus TERT DNA vaccine that has improved capacity to break tolerance with immune checkpoint inhibitors. We observed that blockade of CTLA-4 or, to a lesser extent, PD-1 synergized with TERT vaccine, generating more robust anti-tumor activity compared to checkpoint alone or vaccine alone. Despite this anti-tumor synergy, none of these immune checkpoint therapies showed improvement in TERT antigen-specific immune responses in tumor-bearing mice. αCTLA-4 therapy enhanced the frequency of T-bet+/CD44+ effector CD8+ Tcells within the tumor and decreased the frequency of regulatory Tcells within the tumor, but not in peripheral blood. CTLA-4 blockade synergized more than Treg depletion with TERT DNA vaccine, suggesting that the effect of CTLA-4 blockade is more likely due to the expansion of effector T cells in the tumor rather than a reduction in the frequency of Tregs. These results suggest that immune checkpoint inhibitors function to alter the immune regulatory environment to synergize with DNA vaccines, rather than boosting antigen-specific responses at the site of vaccination.

Toll-like receptor 2 ligand and interferon-γ suppress anti-tumor T cell responses by enhancing the immunosuppressive activity of monocytic myeloid-derived suppressor cells

ABSTRACTCD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) suppress activation/proliferation of cytotoxic T cells, thereby hindering cancer immunotherapy. MDSCs are increased after adjuvant therapy with toll-like receptor (TLR) 2 ligands, such as Pam2CSK4, in tumor-bearing mice. However, it remains unknown if the activation of TLR2 in MDSCs affects their function and the therapeutic efficacy of TLR2 ligand. Here, we show that TLR2 signaling in CD11b+Ly6G−Ly6Chigh monocytic MDSCs (M-MDSCs), but not CD11b+Ly6G+Ly6Clow granulocytic MDSCs (G-MDSCs), enhances their immunosuppressive activity, thereby limiting anti-tumor T cell responses induced by TLR2-activated dendritic cells (DCs). iNOS induction was critical for Pam2CSK4-enhanced T cell suppression by M-MDSCs. iNOS was expressed in M-MDSC-derived macrophages, but not undifferentiated M-MDSCs, in cocultures with CD8+ T cells, CD11c+ DCs, antigen peptide and Pam2CSK4. Pam2CSK4 increased the differentiation frequency of M-MDSCs to macrophages, and iNOS expression required interferon-γ (IFN-γ) production by CD8+ T cells that had been transiently stimulated by M-MDSC-derived macrophages in an antigen/TLR2-dependent manner. Although Pam2CSK4 triggered DC maturation and tumor regression via induction of tumor antigen-specific cytotoxic T lymphocyte (CTL) responses in tumor-bearing mice, Pam2CSK4 plus antigen increased the frequency of iNOS+ macrophages in the tumor. Treatment with iNOS inhibitor enhanced the therapeutic efficacy of Pam2CSK4. Hence, the results suggest that TLR2 ligand and T cell-derived IFN-γ enhance M-MDSC-mediated immunosuppression, which may negatively regulate anti-tumor CTL response.