MC38 Tumor-bearing Mice Research Articles

Abstract 4443: Inhibition of PGE2/EP4 pathway by ONO-4578/BMS-986310, a novel EP4 antagonist, promotes T cell activation and myeloid cell differentiation to dendritic cells

Abstract Prostaglandin E2 (PGE2) contributes to immunosuppression in the tumor microenvironment through one or more of its receptors (EP1-EP4), especially EP4 which is expressed at high levels on myeloid cells. ONO-4578/BMS-986310 is a novel EP4-selective antagonist that potently inhibits the PGE2-induced cAMP production in CHO cells expressing mouse and human EP4 receptors with single-digit nM IC50. Furthermore, ONO-4578/BMS-986310 shows excellent pharmacokinetic properties after oral dosing in mouse. ONO-4578/ BMS-986310 reverses PGE2-mediated suppression of T cell activation and promotes DC differentiation in vitro. Moreover, treatment with ONO-4578/BMS-986310 inhibits growth of CT26 and MC38 tumors in syngeneic mouse. The anti-tumor effect of ONO-4578/BMS-986310 is abrogated in immuno-deficient mice bearing CT26 tumors indicating that such effect is immune-mediated. ONO-4578/BMS-986310 treatment significantly prolongs survival of mice with highly aggressive SB28 gliomas in the brain. Flow cytometric analysis reveals that ONO-4578/BMS-986310 reduces infiltration of M2 macrophages in MC38 tumors compared with control mice. Microarray analysis of MC38 tumor tissues demonstrates up-regulation of cytotoxic T lymphocytes, natural killer cells and innate immunity-related genes after treatment with ONO-4578/BMS-986310. Finally, a combination of ONO-4578/BMS-986310 and anti-PD-1 antibody results in enhanced anti-tumor effects in GL261 and MC38 tumor-bearing mice compared with anti-PD-1 monotherapy.Our data demonstrate that ONO-4578/BMS-986310 promotes anti-tumor immunity and may be useful in the treatment of cancer patients. ONO-4578/BMS-986310 is being evaluated as monotherapy and in combination with nivolumab in an ongoing Ph1/1b clinical trials in patients with advanced solid tumors (NCT03155061, NCT03661632). Citation Format: Tohru Kotani, Hirotsugu Takano, Tomoko Yoshida, Ryota Hamasaki, Gary Kohanbash, Kazuhiko Takeda, Hideho Okada. Inhibition of PGE2/EP4 pathway by ONO-4578/BMS-986310, a novel EP4 antagonist, promotes T cell activation and myeloid cell differentiation to dendritic cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4443.

Abstract 6529: GI101, A novel CD80-IgG4-IL2 variant bispecific protein, inhibits tumor growth and induces anti-tumor immune response in multiple preclinical models

Abstract Introduction: Immune checkpoint blockades (ICBs) have revolutionized cancer treatment and broadened clinical applicability. However, the majority of patients still fail to respond to standard ICBs. To overcome such unmet needs in a clinical study, we designed GI-101, combining the extracellular domain of CD80 serve as a CTLA-4 blockade and an IL-2 variant that preferentially binds the IL-2 receptor β subunit (IL-2Rβ) together. The harmonizing mechanisms of action are projected to translate into improved clinical benefits for this first-in-class immune checkpoint inhibitor fusion protein, even in non-inflamed “cold” tumors. Methods: Binding affinity of GI101 to IL2Rs, CTLA4, and CD28 was determined by SPR. Immune cell proliferation was analyzed by CFSE assay. In vivo anti-tumor efficacy was tested by single or combination treatment on CT26, MC38 and B16F10 syngeneic tumor models. To elucidate the involvement of GI101 on tumor microenvironment (TME), immune cell population was analyzed by flow cytometry from tumor. Tumor specific T cells (surrogate marker, gp70) were measured by splenocyte proliferation assay and IFN-γ ELISPOT assay. RNA sequencing was performed to elucidate immune mechanism of GI-101. Results: GI101 highly binds to CTLA-4 (Kd, 2.9 nM) which leads to the reinforcement of endogenous CD80 and CD28 interaction resulting in the activation of T cells. Bivalent IL-2 variant of GI101 triggers both CD8+ T and NK cells proliferation in vitro and in vivo without Tregs proliferation. GI101 has no evidence for toxicity associated with IL-2 activity including vascular leakage syndrome and cytokine storm in non-GLP monkey studies whereas isolated mortality was observed in the anti-PD-1 and anti-CTLA4 combination treatment group. GI101 elicits restoration of immune functions in vitro settings using mouse splenocytes co-cultured with different PDL-1 and CTLA-4 expression level tumor cells. A dose-dependent (3 to 12 mg/kg) inhibition of tumor growth was observed in CT26 syngeneic models without toxicity. Immune profiling of tumor samples also revealed that a robust increment of M1 macrophages, CD8+ central memory T cells (Tcm) and Ki-67+ proliferating T cells but not Tregs in TME (p < 0.05). Tumor specific T cells were strongly proliferated when stimulated with CT26 neoantigens (gp70, RSPWFTTLI and MGPLIVLLL) in splenocyte. IFN-γ+ cells were significantly increased in draining lymph nodes from GI101 treated mice. Furthermore, drastic tumor regression was observed in MC38 tumor-bearing mice treated with GI101 and anti-PD-1 combination. Conclusion: GI101 facilitates the dual function of checkpoint blockade and IL2 activity that enhances the proliferation and activation of T and NK cells. This novel target drug is expected to be interpreted as superior clinical efficacy and safety as indicated even in ‘cold tumor' models. GI101 is the promising immune-oncology drug to replace the first-generation ICBs by single or combining with other immunotherapies. Our findings provide a rationale for further clinical investigations. Keywords: CD80, IL-2 variant, GI101, Bispecific fusion protein, immunotherapy Citation Format: Kyoung-Ho Pyo, Young Jun Koh, Chun-Bong Synn, Jae Chan Park, Jae-Hwan Kim, Yeongseon Byeon, Sung Eun Kim, Ji Min Lee, Ha Ni Jo, Wongeun Lee, Do Hee Kim, Sungwon Park, Yoo Jeong Song, Won Jae Lee, Ji Young Kim, Hyung Nam Ji, Sang Su Park, Kyung Wha Lee, Young Gyu Cho, Young Min Oh, Bo Gie Yang, Su Youn Nam, Myoung Ho Jang, Byoung Chul Cho. GI101, A novel CD80-IgG4-IL2 variant bispecific protein, inhibits tumor growth and induces anti-tumor immune response in multiple preclinical models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6529.

Probody Therapeutic Design of 89Zr-CX-072 Promotes Accumulation in PD-L1-Expressing Tumors Compared to Normal Murine Lymphoid Tissue.

Probody therapeutic CX-072 is a protease-activatable antibody that is cross-reactive with murine and human programmed death-ligand 1 (PD-L1). CX-072 can be activated in vivo by proteases present in the tumor microenvironment, thereby potentially reducing peripheral, anti-PD-L1-mediated toxicities. To study its targeting of PD-L1-expressing tissues, we radiolabeled CX-072 with the PET isotope zirconium-89 (89Zr). 89Zr-labeled CX-072, nonspecific Probody control molecule (PbCtrl) and CX-072 parental antibody (CX-075) were injected in BALB/c nude mice bearing human MDA-MB-231 tumors or C57BL/6J mice bearing syngeneic MC38 tumors. Mice underwent serial PET imaging 1, 3, and 6 days after intravenous injection (pi), followed by ex vivo biodistribution. Intratumoral 89Zr-CX-072 distribution was studied by autoradiography on tumor tissue sections, which were subsequently stained for PD-L1 by IHC. Activated CX-072 species in tissue lysates were detected by Western capillary electrophoresis. PET imaging revealed 89Zr-CX-072 accumulation in MDA-MB-231 tumors with 2.1-fold higher tumor-to-blood ratios at 6 days pi compared with 89Zr-PbCtrl. Tumor tissue autoradiography showed high 89Zr-CX-072 uptake in high PD-L1-expressing regions. Activated CX-072 species were detected in these tumors, with 5.3-fold lower levels found in the spleen. Furthermore, 89Zr-CX-072 uptake by lymphoid tissues of immune-competent mice bearing MC38 tumors was low compared with 89Zr-CX-075, which lacks the Probody design. 89Zr-CX-072 accumulates specifically in PD-L1-expressing tumors with limited uptake in murine peripheral lymphoid tissues. Our data may enable clinical evaluation of 89Zr-CX-072 whole-body distribution as a tool to support CX-072 drug development (NCT03013491).

NKG2D-Fc fusion protein promotes antitumor immunity through the depletion of immunosuppressive cells.

A major factor impeding the success of numerous therapeutic approaches in cancer is the immunosuppressive nature of the tumor microenvironment (TME). Hence, methods capable of reverting tumor immunosuppression through depletion or reprogramming of myeloid-derived suppressive cells (MDSCs) and regulatory T cells (Tregs) are of great clinical need. Here, we explore NKG2D-Fc as a modality to modulate antitumor immunity through the depletion of immunosuppressive MDSCs and Tregs in the TME. We have generated the NKG2D-Fc fusion protein and characterized its potential to mediate tumor control and overall survival in LL2 and MC38 murine models. Upon treatment of LL2 or MC38 tumor-bearing mice with NKG2D-Fc, we observe significant tumor control and enhanced survival compared to Fc control. When characterizing MDCSs and Tregs from tumor-bearing mice, we observe clear expression of NKG2D-ligand RAE1γ and subsequent binding of NKG2D-Fc fusion protein to both MDSCs and Tregs. Examining the immune profile of mice treated with NKG2D-Fc reveals significant depletion of MDSCs and Tregs in the TME, as well as an increase in NK cells likely due to the reversed suppressive TME. In conclusion, NKG2D-Fc induces antitumor immunity and tumor control through the depletion of MDSCs and Tregs, subsequently providing a niche for the infiltration and expansion of proinflammatory cells, such as NK cells. Strategies capable of modulating the immunosuppressive state in cancer are in high clinical demand. NKG2D-Fc is a simple, single tool capable of depleting both MDSCs and Tregs and should be further investigated as a therapeutic agent for the treatment of cancer.

Abstract A68: A casein kinase 2 inhibitor disorders myeloid cell differentiation by blocking CCAAT/enhancer-binding protein-alpha signaling pathway in tumor microenvironment

Abstract Myeloid cells are major components of the tumor microenvironment, which are known to suppress antitumor immunity. Casein kinase 2 (CK2), a serine/threonine protein kinase with diverse intracellular protein substrates, regulates several signaling pathways involved in tumor progression and cell differentiation. We examined the efficacy of a novel CK2 inhibitor on modulating the myeloid cells in the tumor microenvironment. Although CK2 inhibitors BMS-595 and BMS-211 moderately inhibited the tumor growth in LLC, 4T1, MC38 and CT26 tumor-bearing mice, these inhibitors drastically enhanced the antitumor efficacy of anti-CTLA4 antibody with 60% to 90% of complete rejection. Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) and macrophages in spleens, and tumor-associated macrophages (TAM) were found to be decreased in LLC tumor-bearing mice treated with BMS-595 for 2 weeks. Murine hematopoietic progenitor cells (HPC) from bone marrow and human progenitor cells from cord blood were cultured with BMS-595 to evaluate the differentiation. BMS-595 treatment dramatically decreased the proportion of granulocytic cells, and this decrease was not caused by the direct killing nor apoptosis. Therefore, the genes regulated by transcription factors, IRF8, C/EBPα and C/EBPβ, which are involved in PMN-MDSC differentiation, were evaluated. Many genes controlled by C/EBPα were downregulated by the BMS-595 treatment in HPCs. Active p42 subunit of C/EBPα was revealed to be reduced by the BMS-595 treatment in HPCs, which are inversely correlated with the increased level of dominant negative p30 subunit of C/EBPα, in both the cytoplasm and nucleus of HPCs. Our results suggest that CK2 inhibition leads to a reduction of immune-suppressive PMN-MDSC and TAM by inhibiting C/EBPα activity, resulting in an enormous augmentation of antitumor efficacy of immunotherapy. Citation Format: Ayumi Hashimoto, Chan Gao, Jerome Mastio, Andrew Kossenkov, Scott I. Abrams, Ashok V. Purandare, Heshani Desilva, Susan Wee, John Hunt, Maria Jure-Kunkel, Dmitry I. Gabrilovich. A casein kinase 2 inhibitor disorders myeloid cell differentiation by blocking CCAAT/enhancer-binding protein-alpha signaling pathway in tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A68.

Abstract A06: Direct and indirect effects of low-dose radiation on Tregs and antitumor efficacy among diverse tumor models

Abstract The impact of radiation on TREG cells remains controversial. Radiation has been shown by our lab, and others, to alter the phenotype of human tumor cell by enhancing their expression of the co-stimulatory molecules OX-40L and 4-1BBL. Both OX-40 and 4-1BB signaling have been reported to influence TREG frequency and function, and we hypothesized that radiation-induced changes in expression of these molecules could influence the frequency of TREG cells within tumors. We evaluated three widely used murine tumor models for these experiments. 4T1 (mammary), MC38 (colon), or CT26 (colon) cells were implanted s.c. into mice and treated locally with 8-10Gy of radiation. Several days after treatment the frequency of tumoral T cells, including CD4+Foxp3+ TREG cells, was evaluated by fluorescent IHC and flow cytometry. We found that radiation differentially impacted the frequency of TREGS among the three tumor models evaluated and appeared to correlate with expression levels of 4-1BBL and OX-40L on the tumor cells post-IR. The level of tumor control post-RT also mirrored expression levels of 4-1BBL and OX-40L on tumor cells. Interestingly, 4T1 tumors contained more total CD4+ cells within tumors, but contained less TREG cells within the CD4 population, after radiation as compared to MC38 tumors. 4T1 tumor-bearing mice also contained more CD4+41BB+ cells as compared to MC38 tumor-bearing mice. These differences may contribute to differences in the therapy responses of various murine models utilizing radiation and may serve as useful biomarker to predict antitumor responses post-IR. Citation Format: Samantha Simon, Charlie Garnett-Benson. Direct and indirect effects of low-dose radiation on Tregs and antitumor efficacy among diverse tumor models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A06.

Synthetic HDL Nanoparticles Delivering Docetaxel and CpG for Chemoimmunotherapy of Colon Adenocarcinoma.

Colon carcinomas comprise over two-thirds of all colorectal cancers with an overall 5-year survival rate of 64%, which rapidly decreases to 14% when the cancer becomes metastatic. Depending on the stage of colon carcinoma at diagnosis, patients can undergo surgery to attempt complete tumor resection or move directly to chemotherapy with one or a combination of drugs. As with most cancers, colon carcinomas do not always respond to chemotherapies, so targeted therapies and immunotherapies have been developed to aid chemotherapy. We report the development of a local combination therapy for colon carcinoma whereby chemo- and immunotherapeutic entities are delivered intratumorally to maximize efficacy and minimize off-target side effects. A hydrophobic chemotherapeutic agent, docetaxel (DTX), and cholesterol-modified Toll-like receptor 9 (TLR9) agonist CpG (cho-CpG) oligonucleotide are co-loaded in synthetic HDL (sHDL) nanodiscs. In vivo survival analysis of MC-38 tumor-bearing mice treated intratumorally with DTX-sHDL/CpG (median survival; MS = 43 days) showed significant improvement in overall survival compared to mice treated with single agents, free DTX (MS = 23 days, p < 0.0001) or DTX-sHDL (MS = 28 days, p < 0.0001). Two of seven mice treated with DTX-sHDL/CpG experienced complete tumor regression. None of the mice experienced any systemic toxicity as indicated by body weight maintenance and normal serum enzyme and protein levels. In summary, we have demonstrated that chemo- and immunotherapies can be co-loaded into sHDLs, delivered locally to the tumor, and can be used to improve survival outcomes significantly compared to chemotherapy alone.

Endogenous retroviral proteins provide an immunodominant but not requisite antigen in a murine immunotherapy tumor model

ABSTRACT Clinical observations suggest that responses to cancer immunotherapy are correlated with intra-tumoral T cell receptor (TCR) clonality, tumor mutation burden (TMB) and host HLA genotype, highlighting the importance of host T cell recognition of tumor antigens. However, the dynamic interplay between T cell activation state and changes in TCR repertoire in driving the identification of potential immunodominant antigen(s) remains largely unexplored. Here, we performed single-cell RNA-sequencing on CD8+ tumor-infiltrating T cells (TILs) using the murine colorectal tumor model MC38 to identify unique TCR sequences and validate their tumor reactivity. We found that the majority of clonally expanded TILs are tumor-reactive and their TCR repertoire is unique amongst individual MC38 tumor-bearing mice. Our query identified that multiple expanded TCR clones recognized the retroviral epitope p15E as an immunodominant antigen. In addition, we found that the endogenous retroviral genome encoding for p15E is highly expressed in MC38 tumors, but not in normal tissues, due to epigenetic derepression. Further, we demonstrated that the p15E-specific TILs exhibit an activated phenotype and an increase in frequency upon treatment with anti-41BB and anti-PD-1 combination immunotherapy. Importantly, we showed that although p15E-specific TILs are not required to mount a primary anti-tumor response, they contributed to the development of strong immune memory. Overall our results revealed that endogenous retroviral antigens expressed by tumor cells may represent an important and underappreciated category of tumor antigens that could be readily targeted in the clinic.

Development of LAG-3 nanobodies as potent cancer imaging tracers

Abstract Background Immune checkpoint blockade revolutionized anti-cancer therapy but unfortunately not all patients can benefit from it. The development of innovative and efficacious diagnostic methods that can guide treatment decisions is warranted. Nanobodies (Nbs) are small antigen-binding moieties that efficiently penetrate cell-cell interfaces in tumors and generate high contrast in noninvasive imaging, making them prime candidates for development of novel imaging tracers. In this study, we generated and characterized Nbs as tools for nuclear imaging of the immune checkpoint LAG-3. Methods Nanobody generation was initiated by immunization of llamas with recombinant LAG-3. Periplasmic extracts were generated and analyzed for their binding to mouse LAG-3 using ELISA and flow cytometry. Selected Nbs were cloned in a vector for high yield production and purified in order to analyze their affinity using SPR. A total of 9 high affinity Nbs were subsequently evaluated for their labeling efficiency with Technetium-99m (99mTc), after which the biodistribution of 99mTc labeled Nbs was assessed in mice. Additionally, we evaluated the noninvasive detection of LAG-3 expressed by immune cells in the tumor environment of murine colon adenocarcinoma (MC38) using 99mTc-Nb3132. Results Nine high affinity Nbs were selected to evaluate their potential as noninvasive imaging tracers. Subsequently, Nb3132 was reported as the most potent tracer for noninvasive imaging of the immune checkpoint LAG-3 (1). SPECT/CT of MC38 tumor-bearing mice 1h after injection of 99mTc-Nb3132 presented specific uptake in spleen, thymus, lymph nodes as well as at the tumor site. This uptake pattern coincided with the presence of LAG-3 expressed on immune cells in these organs, as determined by flow cytometry and immunohistochemistry. No specific uptake was observed when injecting mice with a radiolabeled control Nb. Conclusion Nb3132 showed excellent in vivo imaging capacities to specifically identify LAG-3 expressing immune cells within the tumor site. These findings support the predictive potential of Nb3132 to noninvasively detect LAG-3 by nuclear imaging and to guide treatment decisions in future, improving the shortcomings of using full-sized antibody formats as diagnostic tracers. Legal entity responsible for the study Geert Raes. Funding Kom op tegen kanker and FWO flanders. Disclosure All authors have declared no conflicts of interest.

PLGA Nanoparticles Codelivering siRNAs against Programmed Cell Death Protein-1 and Its Ligand Gene for Suppression of Colon Tumor Growth.

Tumor-infiltrating T lymphocytes highly express programmed cell death protein-1 (PD-1) that interacts with its ligand, programmed cell death protein ligand-1 (PD-L1) on tumors. PD-1/PD-L1 interactions cause functional exhaustion of effector T cells and impair antitumor immunity, allowing tumors to escape immune surveillance. In addition to such extrinsic interactions, tumors proliferate by transmitting intrinsic PD-L1 signals via the mTOR pathway. Here, we simultaneously silenced PD-1 and PD-L1 expressions on CTLs and colon tumors using PD-1 siRNA/PD-L1 siRNA-loaded PLGA nanoparticles and investigated functional activation of tumor-specific CTLs. When compared to a single PD-1 silencing on CTLs or a single PD-L1 silencing on tumors, cosilencing of PD-1/PD-L1 on CTLs and tumors more efficiently promoted effector functions of tumor-specific CTLs. Moreover, PD-L1-silenced tumors inhibited mTOR signaling and showed an antiproliferative response independent of the adaptive immune response. Ultimately, systemic administration of PD-1 and PD-L1 siRNA via PLGA nanoparticles restored the effector functions of tumor-specific CTLs in MC38 tumor-bearing mice. Compared with antitumor effects of single silencing of PD-1 or PD-L1 alone, cosilencing of PD-1 and PD-L1 showed more significant tumor growth suppression and long-term tumor inhibition in colon cancer. Thus, this study provides an efficient therapeutic strategy for achieving immunotherapy in colon cancer.

Abstract 2379: In vivo drug screening platform accelerated 4-1BB agonistic antibody development

Abstract 4-1BB (TNFRSF9/CD137) belongs to the TNFR superfamily. 4-1BB is expressed by activated CD4 helper T cells, B cells, natural killer cells, natural killer T cells, dendritic cells, and activated endothelium. A majority of regulatory T cells (Tregs) also express 4-1BB, but it remains unclear whether agonistic antibody treatment exerts pro- or anti-suppressive effect on these cells. 4-1BB enhances tumor rejection because it is upregulated on T cells following activation and its engagement increases T cell proliferation and pro-inflammatory cytokine production. The clinical development of 4-1BB targeting therapy was slow due to the toxicity associated with overt immune activation. Fortunately, new therapeutic modalities using 4-1BB targeting aptamers as well as therapeutic combinations with other immuno-modulatory and traditional anti-cancer treatments have revived excitement for the use of 4-1BB agonists in the clinic. We focused on discovering novel 4-1BB therapeutic antibodies for cancer patients. Candidate molecules obtained through the classical hybridoma technology were screened by high-throughput flow cytometry. Then, we took the advantages of Biocytogen’s humanized mice and screened the purified antibodies in B-h4-1BB mice bearing MC-38 tumor cells. We found some antibodies that worked as equivalent as BMS’s Urelumab. To determine which isotype should be adopted, we engrafted the antigen binding domain onto various human Fc backbones. Interestingly, we found that the human IgG1 isotype appeared to be more effective against tumors than other isotypes in B-4-1BB mice bearing MC-38 tumors. This study provides a reliable basis for our follow-up mechanism research and clinical evaluation for this target. Citation Format: Benny(Yi) Yang, Jian Ni, Yanan Li, Yuelei Shen, Qingcong Lin. In vivo drug screening platform accelerated 4-1BB agonistic antibody development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2379.

Abstract 5017: MEDI1191, a novel IL-12 mRNA therapy for intratumoral injection to promote TH1 transformation of the patient tumor microenvironment

Abstract Patients who respond to PD-L1 / PD-1 immune checkpoint blockade tend to have an inflamed, TH1 polarised tumor microenvironment (TME), characterised by expression of interferon-γ (IFNγ) and PD-L1. Novel therapies that induce TH1 transformation of the patient TME therefore have the potential to enhance anti-tumor immunity. As a central mediator of TH1 immune responses, interleukin 12 (IL-12) directly induces IFNγ release from activated NK, NKT and T cells, and is known to play a key role in driving anti-tumor responses. However systemic recombinant IL-12 was poorly tolerated in early clinical trials. We therefore designed MEDI1191 as a novel IL-12-based therapy designed for injection directly into tumors, composed of a lipid nanoparticle (LNP)-formulated mRNA encoding human IL-12. We previously reported that intratumoral (IT) mouse (m) IL-12 mRNA, the surrogate for MEDI1191, promotes cytotoxic T cell-dependent anti-tumor immunity and enhances responses to PD-L1 blockade in pre-clinical models. Here, we demonstrate that IFNγ is also required for the anti-tumor activity of mIL-12 mRNA. A single dose of mIL-12 mRNA significantly increased expression of IFNγ and TH1 genes in MC38 tumor-bearing mice. Treatment with an IFNγ neutralising antibody blocked mIL-12 mRNA anti-tumor activity in this model. In addition, we report here that MC38 tumor rejection in response mIL-12 mRNA / anti-PD-L1 combination therapy correlates with increased cytotoxic T cell infiltration into tumors, and expansion of tumor-reactive T cells in the periphery. We next investigated the pharmocodynamic activity of MEDI1191 in patient tumor-derived models. A single IT dose of MEDI1191 induced human IL-12p70 expression in mice bearing four different patient-derived xenograft tumors. Furthermore, in an ex vivo patient tumor slice culture assay, MEDI1191 induced dose-dependent IL-12 release, IFNγ expression and upregulation of TH1-signature gene expression. IL-12 protein secretion was induced in slices of all patient tumors tested. However, the magnitude of the IFNγ response to MEDI1191 varied between patient tumors. Quantification of the tumoral T cell and NK cell numbers within the patient tumor samples revealed a positive correlation between MEDI1191-induced IFNγ release and baseline tumor NK infiltrate. These preclinical data demonstrate the potential for MEDI1191 to induce IFNγ-dependent TH1 transformation of the TME, and support the development of MEDI1191 as a potential treatment for patients with solid tumors, alone and in combination with inhibitors of the PD-1/PD-L1 T cell checkpoint. Citation Format: Nadia Luheshi, Susannah Hewitt, Fabien Garcon, Shannon Burke, Amanda Watkins, Kristen Arnold, John Zielinski, Philip Martin, Michael Sulikowski, Christopher Bagnall, Jean-Martin Lapointe, Gordon Moody, Han Si, Christopher Morehouse, Robert W. Wilkinson, Ronald Herbst, Joshua Frederick. MEDI1191, a novel IL-12 mRNA therapy for intratumoral injection to promote TH1 transformation of the patient tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5017.

Abstract 3241: Novel anti-CD40 antibodies demonstrate anti-tumor activity in humanized mouse models

Abstract CD40 (Cluster of Differentiation 40) is a member of the TNF-receptor superfamily. CD40 is a co-stimulatory protein found primarily on antigen presenting cells, including B cells, macrophages and dendritic cells. Its ligand, CD40L (CD154) is predominantly expressed on T helper cells, whose engagement activates antigen presenting cells and induces a variety of positive downstream effects. Whereas most immune checkpoint receptors are expressed on the surface of T cells, CD40 stands out as it is expressed by antigen presenting cells and controls the proper activation of these cells. In turn, CD40 activation exerts a significant impact on T cell-mediated activity against cancer cells. Several pharmaceutical companies are pursuing anti-CD40 therapies for treating hematological and solid cancers. Therefore, it is imperative to develop novel CD40 antibodies to boost cancer patients’ own immunity in light of other successful immune checkpoint modulators. We asked whether we could find suitable anti-CD40 reagents that successfully combat developed tumors in preclinical settings. We designed a powerful screening strategy based on Biocytogen’s in vivo drug screening platform. Candidate antibodies were generated by immunizing wild-type mice with recombinant hCD40 protein. Hybridomas were then screened by high-throughput flow cytometry. Instead of examining the immune modulating activity in cell culture, we tested these antibodies in B-hCD40 mice bearing MC38 tumors and monitored the tumor growth. Through this straightforward screening strategy, several clones stood out by their unparalleled benefits. These clones were further selected for humanization. Furthermore, we identified that one clone works in concert with Keytruda in the dual humanized model of h-CD40 and h-PD1. In conclusion, we uncovered CD40 antibody candidates with the most potent anti-tumor activity which are promising as tools for cancer treatment in the preclinical setting. Citation Format: Chaoshe Guo, Benny(Yi) Yang, Jian Ni, Yanan Guo, Tian Gan. Novel anti-CD40 antibodies demonstrate anti-tumor activity in humanized mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3241.

Abstract 1474: Repurposing the yellow fever vaccine for intratumoral immunotherapy

Abstract Introduction: Yellow Fever attenuated virus vaccine (17D) induces very potent cellular and humoral immunity. Its delivery in situ in the tumor bed may induce an immune response leading to the killing of infected cells. Experimental procedures: 17D virus was propagated in Vero cells and infectious supernatants were harvested. To evaluate in vitro sensitivity of tumors to 17D infection, a panel of human and mouse tumor cell lines were infected with increasing MOIs and viability was quantified. To evaluate the antitumor efficacy of intratumor (i.t.) injection of 17D, 4x106pfu/50μl were injected (4 doses) in MC38- and in B16-OVA-tumor bearing C57BL/6 mice. Systemic antitumor effects of i.t 17D were examined in bilateral MC38 models in which one of the tumors was left uninjected. Depletion experiments were performed to assess the contribution of CD4, CD8, Gr1 and NK cells. To determine the role of conventional type-I dendritic cells (cDC1), identical experiments were performed in BATF3-/- mice. Tumor and lymph node immune infiltrates were examined by multiparametric flow cytometry. Combination with intraperitoneal anti-PD1 or anti-CD137 was performed to improve antitumor efficacy. To evaluate the effects of 17D-preexisting immunity, mice were preimmunized with 17D prior to MC38 bilateral inoculation and were subsequently treated. To assess the individual cell type contribution to the antitumor effects induced upon 17D pre-immunization, CD8, CD4, CD8+CD4 or sera from preimmunized mice were passively transferred 24h prior to first 17D i.t injection to MC38-tumor bearing mice. Results: 17D live attenuated strain is able to induce cell death of mouse and human tumor cell lines. In MC38 or B16-OVA mice models, repeated i.t. injections of 17D clearly delayed tumor progression. Regarding the systemic effect of i.t. 17D, certain contralateral therapeutic efficacy was induced upon i.t injection in bilateral MC38 models. These local and distant effects were dependent on CD8+ and cDC1 cells. 17D i.t injection increased in CD8+ T cell infiltrates and decreased in Tregs in injected tumors, while in contralateral tumors induced an increase of CD8+ and NK cells expressing key immunomodulatory receptors. I.t. 17D with anti-CD137 monoclonal antibody (mAb) induced a strong synergistic effect, while combination with anti-PD-1 mAb showed mild local additive efficacy compared to 17D monotherapy. Both combinatorial treatments delayed contralateral tumor growth. Notably, 17D preimmunization induced an important improvement of local and distant antitumor immunity. Such beneficial effects of preimmunization were transferred along with CD8+ T cells. Conclusions: 17D can be safely injected i.t. in preclinical models, giving rise to immune-mediate antitumor effects that can be combined with other immunotherapy agents. The repurposed use of this GMP-grade vaccine in preimmunized patients represents a promising and feasible immunotherapy approach. Citation Format: M. Angela Aznar, Carmen Molina, Alvaro Teijeira, Arantza Azpilikueta, Saray Garasa, Alfonso R. Sanchez-Paulete, Iñaki Etxeberria, Maite Alvarez, Pedro Berraondo, Ignacio Melero. Repurposing the yellow fever vaccine for intratumoral immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1474.

Imaging of Activated T Cells as an Early Predictor of Immune Response to Anti-PD-1 Therapy.

Compelling evidence points to immune cell infiltration as a critical component of successful immunotherapy. However, there are currently no clinically available, noninvasive methods capable of evaluating immune contexture prior to or during immunotherapy. In this study, we evaluate a T-cell-specific PET agent, [18F]F-AraG, as an imaging biomarker predictive of response to checkpoint inhibitor therapy. We determined the specificity of the tracer for activated T cells in vitro and in a virally induced model of rhabdomyosarcoma. Of all immune cells tested, activated human CD8+ effector cells showed the highest accumulation of [18F]F-AraG. Isolation of lymphocytes from the rhabdomyosarcoma tumors showed that more than 80% of the intratumoral signal came from accumulation of [18F]F-AraG in immune cells, primarily CD8+ and CD4+. Longitudinal monitoring of MC38 tumor-bearing mice undergoing anti-PD-1 treatment revealed differences in signal between PD-1 and isotype antibody-treated mice early into treatment. The differences in [18F]F-AraG signal were also apparent between responders and nonresponders to anti-PD-1 therapy. Importantly, we found that the signal in the tumor-draining lymph nodes provides key information about response to anti-PD-1 therapy. Overall, [18F]F-AraG has potential to serve as a much needed immunomonitoring clinical tool for timely evaluation of immunotherapy. SIGNIFICANCE: These findings reveal differences in T-cell activation between responders and nonresponders early into anti-PD-1 treatment, which may impact many facets of immuno-oncology, including patient selection, management, and development of novel combinatorial approaches.

Targeted Delivery of IL2 to the Tumor Stroma Potentiates the Action of Immune Checkpoint Inhibitors by Preferential Activation of NK and CD8+ T Cells.

Recombinant human IL2 is being considered as a combination partner for immune checkpoint inhibitors in cancer therapy, but the product only has a narrow therapeutic window. Therefore, we used F8-IL2, an antibody-IL2 fusion protein capable of selective localization to the tumor site, in combination with antibodies against murine CTLA-4, PD-1, and PD-L1. In immunocompetent mice bearing CT26 tumors, the combination of F8-IL2 with CTLA-4 blockade was efficacious, leading to increased progression-free survival and protective immunity against subsequent tumor rechallenges. The combination with anti-PD-1 induced substantial tumor growth retardation, but tumor clearance was rare, whereas the combination with anti-PD-L1 exhibited the lowest activity. A detailed high-parametric single-cell analysis of the tumor leukocyte composition revealed that F8-IL2 had a strong impact on NK-cell activity without collateral immune activation in the systemic immune compartment, whereas CTLA-4 blockade led to significant changes in the T-cell compartment. Leukocyte depletion studies revealed that CD8+ T and NK cells were the main drivers of the therapeutic activity. We extended the experimental observations to a second model, treating MC38 tumor-bearing mice with F8-IL2 and/or CTLA-4 blockade. Only the combination treatment displayed potent anticancer activity, characterized by an increase in cytolytic CD8+ T and NK cells in tumors and draining lymph nodes. A decrease in the regulatory T cell frequency, within the tumors, was also observed. The results provide a rationale for the combined use of engineered IL2 therapeutics with immune checkpoint inhibitors for cancer therapy.

Abstract IA28: Nanodisc platform technology for cancer vaccination

Abstract Recent innovations in DNA/RNA sequencing have allowed for the identification of patient-specific tumor neo-antigens, ushering in the new era of personalized cancer vaccines. Peptide vaccines in general may serve as an ideal platform for neo-antigen vaccines, but the therapeutic efficacy of peptide vaccines have been limited in clinical trials. Here we present an alternative strategy where preformed nanodiscs based on synthetic high density lipoproteins, with an established clinical manufacturing procedure and excellent safety profiles in humans, are readily formulated with adjuvants and antigen peptides. We show that these nanodiscs can efficiently deliver immunostimulatory molecules and antigens, including neo-antigens to draining lymph nodes. Notably, nanodiscs elicited up to 47-fold greater frequency of tumor neoantigen-specific CD8+ T lymphocytes (CTLs) than soluble vaccines in mice and even 31-fold greater than the standard adjuvant in clinical trials (i.e. CpG in Montanide). When nanodisc vaccination was combined with anti-PD-1 immune checkpoint inhibitor, ~88% complete response was observed in MC-38 tumor-bearing mice, compared with 25% response rate in the control group that received soluble vaccine plus anti-PD-1 therapy. In more aggressive B16F10 melanoma model, nanodiscs delivering multiple MHC class I and class II neo-epitopes were combined with α-PD-1/α-CTLA-4 therapy, leading ~90% complete response in B16F10 tumor-bearing mice, compared with ~38% response rate in the control group with soluble vaccine plus α-PD-1/α-CTLA-4 therapy. Furthermore, we have demonstrated the efficacy of nanodisc technology using shared tumor antigens in murine models of HPV+ mucosal tumors. The reproductive tract tumor model was established in C57BL/6 mice by intravaginal administration of luciferase-expressing TC-1 cells, a surrogate for HPV-induced human tumors, such as cervical carcinoma. The lung metastasis model was established by intravenous injection of luciferase-expressing TC-1 tumor cells. In mice bearing intravaginal or lung TC-1 tumors, two nanodisc vaccinations with HPV16 E7 antigen generated the peak frequency of ~35% tetramer+ CTLs in peripheral blood and eliminated established TC-1 tumors in the majority of animals (without immune checkpoint blockade). Owning to the facile production process, robust therapeutic efficacy, and good safety profiles, our nano-vaccine technology offers a powerful and convenient platform for vaccination using personalized as well as shared tumor antigens. Citation Format: James J. Moon. Nanodisc platform technology for cancer vaccination [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA28.

Formation of Microbubbles for Targeted Ultrasound Contrast Imaging: Practical Translation Considerations.

For preparation of ligand-decorated microbubbles for targeted ultrasound contrast imaging, it is important to maximize the amount of ligand associated with the bubble shell. We describe optimization of the use of a biocompatible cosurfactant in the microbubble formulation media to maximize the incorporation of targeting ligand-lipid conjugate into the microbubble shell, and thus reduce the fraction of ligand not associated with microbubbles, following amalgamation preparation. The influence of the concentration of a helper cosurfactant propylene glycol (PG) on the efficacy of microbubble preparation by amalgamation and on the degree of association of fluorescent PEG-lipid with the microbubble shell was tested. Three sets of targeted bubbles were then prepared: with VCAM-1-targeting peptide VHPKQHRGGSK(FITC)GC-PEG-DSPE, cyclic RGDfK-PEG-DSPE, selective for αVβ3, and control cRADfK-PEG-DSPE, without such affinity. Microbubbles were prepared by 45 s amalgamation, with DSPC and PEG stearate as the main components of the shell, with 15% PG in aqueous saline. In vitro microbubble targeting was assessed with a parallel plate flow chamber with a recombinant receptor coated surface. In vivo targeting was assessed in MC-38 tumor-bearing mice (subcutaneous tumor in hind leg), 10 min after intravenous bolus of microbubble contrast agent (20 million particles per injection). Ultrasound imaging of the tumor and control nontarget muscle tissue in a contralateral leg was performed with a clinical scanner. Amalgamation technique with PG cosurfactant produced microbubbles at concentrations exceeding 2 × 109 particles/mL, and ∼50-60% or more of the added fluorescein-PEG-DSPE or VCAM-1-targeted fluorescent peptide was associated with microbubbles, about 2 times higher than that in the absence of PG. After intravenous injection, peptide-targeted bubbles selectively accumulated in the tumor vasculature, with negligible accumulation in nontumor contralateral leg muscle, or with control nontargeted microbubbles (assessed by contrast ultrasound imaging). For comparison, administration of RGD-decorated microbubbles prepared by traditional sonication, and purified from free peptide-PEG-lipid by repeated centrifugation, resulted in the same accumulation pattern as for translatable amalgamated microbubbles. Following amalgamation in the presence of PG, efficient transfer of ligand-PEG-lipid to microbubble shell was achieved and quantified. Purification of microbubbles from free peptide-PEG-lipid was not necessary, as proven by in vitro and in vivo targeting studies, so PG cosurfactant amalgamation technique generated peptide-targeted microbubbles are amenable for bedside preparation and clinical translation. The pathway to clinical translation is simplified by the fact that most of the materials used in this study either are on the United States Food and Drug Administration GRAS list or can be procured as pharmaceutical grade substances.

Abstract 998: Radiation-induced immunosuppressive macrophages limit CD8 T-cell mediated tumor killing

Abstract Emerging pre-clinical data suggests that radiation both stimulates and suppresses tumor immunity. The role of tumor associated macrophages (TAMs) has been extensively investigated in the non-irradiated tumor microenvironment. We aimed to investigate the role of macrophages in the immune response to radiation. Single dose 10Gy irradiation to tumors generated from colorectal (MC38) and pancreatic (KPC) cell lines induced Colony Stimulating Factor (CSF-1). Coincident with the elevation in CSF1, significant increases of CD11b+ F480+ macrophages within the tumor microenvironment peaked five days following irradiation returning to baseline during tumor regrowth. These TAMs were skewed toward an immunosuppressive phenotpye (CD206hi iNOSlo) compared to those from controls. Furthermore, the TAMs from irradiated tumors were more effective in suppression of CD8 T cell expansion in ex vivo assays. As a result of these data, we investigated the effect of anti-Colony Stimulating Factor (αCSF-1), which reduced macrophages in both control and irradiated tumors. αCSF had no effect on the growth of control tumors, yet led to a significant enhancement of tumor growth delay after radiation. TAMs sorted from irradiated tumors after aCSF treatment had reduced inflammatory gene expression and increased expression of some genes associated with immune suppression (Arg-1, IL-10, CCL2). Following aCSF, there was a significant increase in CD8 T lymphocytes with greater expression of cytotoxic markers. Radiation is also immune-stimulatory to tumors consistent with enhanced recognition of tumor cell antigens by T cells isolated from irradiated tumors. CD8 depletion however abrogated the growth delay from aCSF further documenting the immunosuppressive effect of the macrophages within irradiated tumors. aCSF1 was given to mice bearing two tumors, one of which was irradiated. An increased growth delay was only observed in the irradiated tumours. These data document the immunosuppressive nature of macrophages generated in irradiated tumors. TAMs expressed high levels of PD-L1 and aCSF treatment resulted in a reduction in tumor PD-L1. As a result we determined the effect of checkpoint inhibition using anti-PD-L1 in combination with aCSF following 10Gy irradiation. Whilst combination treatment did not augment outcomes in mice bearing MC38 tumors, there was complete tumor regression in a minority of mice bearing KPC tumors. These results suggest that infiltrating macrophages limit the adaptive immune response initiated following of radiation. Whilst it has been demonstrated that tumor cell PD-L1 expression plays an important role in checkpoint blockade, our data suggests macrophages are an important additional source. This is of particular importance in the context of radiation which can elicit a potent immune response but in which the TAMs from irradiated tumors also suppress this in part via altered PD-L1 expression. Citation Format: Keaton I. Jones, Jiske Tiersma, Jon Buzzelli, Alex Gordon-Weeks, Arseniy Yuzhalin, Jaehong Im, Ruth J. Muschel. Radiation-induced immunosuppressive macrophages limit CD8 T-cell mediated tumor killing [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 998.

Abstract 4547: Characterization of the LAG-3 targeting antibody BI 754111 in monotherapy and in combination with the anti-PD-1 antibody BI 754091

Abstract Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor involved in maintaining immunological tolerance via regulation of T-cell activation, proliferation and response. Ligand-mediated activation of LAG-3 negatively regulates T-cell activity that is thought to actively contribute to tumor immune evasion beyond the established programmed cell death-1 (PD-1) pathway. BI 754111, a humanized IgG4 monoclonal antibody (mAb) with high affinity against LAG-3, blocks the interaction between LAG-3 and MHC II. BI 754111 was characterized in a panel of binding, blocking and functional cell-based assays; safety assessment was done in cynomolgus monkeys. BI 754111 is not mouse cross-reactive; therefore a surrogate mLAG-3 antibody was used for in vivo mouse efficacy studies. The ability of BI 754111 to stimulate cytokine production by exhausted human T cells in vitro was tested in an autologous assay system with antigen specific memory T cells being re-stimulated by antigen-pulsed dendritic cells in the presence of increasing amounts of BI 754111 or BI 754091 (anti-hPD-1 mAb) or a combination of increasing amounts of BI 754111 and a saturating dose of BI 754091. Under these assay conditions the majority of T cells co-expressed the exhaustion markers PD-1 and LAG-3 on the surface. At the end of the experiment supernatants were harvested and analyzed for IFNγ secretion as a measure of T-cell activation. Monotherapy of BI 754111 moderately increased IFNγ secretion (average of 1.8 fold) compared to BI 754091 monotherapy (6.9-fold average increase). Combining BI 754111 with BI 754091 was synergistic and led to a 13.2-fold increase in IFNγ secretion. MC-38 tumor-bearing mice (C57BL/6NTac-PDCD1tm(PDCD1)Arte mice expressing parts of the human instead of the murine extracellular domain of PD-1) were used to determine the in vivo activity of the anti-LAG3 and anti-PD-1 combination. A mouse tool antibody against mLAG-3 (IgG1 D265A) and BI 905725 (a mouse IgG1 D265A version of BI 754091, anti-hPD-1 mAb) were tested at a dose of 10 mg/kg in a q3or4d schedule as monotherapy or in combination. No anti-tumor activity was observed under mLAG-3 mAb treatment, whereas treatment with anti-PD-1 resulted in a median TGI of 100% and 4 out of 10 tumors showed a complete response. The combination of anti-PD-1 and anti-LAG-3 resulted in a median TGI of 103% and doubled the number of complete responses (8/10). BI 754111 binds to cynomolgus monkey LAG-3 with comparable affinity as to hLAG-3 thus allowing pharmacokinetic and toxicological assessment in this species. Repeated high doses of BI 754111 were well tolerated without adverse immune-related side effects. The above results describe synergistic effects upon combination of PD-1 and LAG-3 treatment thus supporting the ongoing clinical trial in which BI 754111 is being tested in combination with BI 754091 (NCT03156114). Citation Format: Markus Zettl, Melanie Wurm, Otmar Schaaf, Iñigo Tirapu, Sven Mostböck, Markus Reschke, Stephan-Michael Schmidbauer, Lee Frego, Ivo C. Lorenz, Michael Thibodeau, Diann Blanset, Elisa Oquendo Cifuentes, Jürgen Moll, Norbert Kraut, Eric Borges, Anne Vogt, Jonathon Sedgwick, Irene C. Waizenegger. Characterization of the LAG-3 targeting antibody BI 754111 in monotherapy and in combination with the anti-PD-1 antibody BI 754091 [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 4547.