Tumor Antigen-specific CD8 Research Articles

Abstract 2615: Preclinical evaluation and mechanistic characterization of M7824 (MSB0011359C), a novel bifunctional fusion protein targeting the PD-L1 and TGFβ pathways

Abstract Dual targeting of the programmed death ligand 1 (PD-L1) and transforming growth factor β (TGFβ) pathways, 2 key mediators of tumor immune evasion, may provide synergistic antitumor activity. The novel bifunctional fusion protein M7824 (MSB0011359C) is a fully human IgG1 monoclonal antibody against PD-L1, fused via a glycine-serine linker at the CH3-C terminus to the extracellular domain of human TGFβ receptor II, which functions as a TGFβ “trap.” Thus, M7824 is designed to exert independent and complementary anti-immunosuppressive functions by simultaneously blocking the PD-L1 and TGFβ pathways. The purpose of this study was to evaluate M7824 preclinically. M7824 was efficacious in multiple syngeneic tumor models (EMT-6, MC38, PANC02, 4T1, CT26, and GL261). In orthotopic EMT-6 and intramuscular MC38 models, M7824 showed superior additive antitumor effects compared to anti-PD-L1 antibody or TGFβ trap monotherapy. M7824 also inhibited spontaneous metastases in EMT-6 and 4T1 models. Furthermore, M7824 extended survival and conferred long-term protective antitumor immunity, as demonstrated by protection of cured mice against tumor cell rechallenge. Mechanistically, the dual anti-immunosuppressive activity of M7824 in the MC38 model resulted in unique activation of both the adaptive and innate immune systems, including increased tumor infiltration, proliferation, and cytotoxicity of CD8+ T cells; increased prevalence of splenic tumor antigen-specific CD8+ T cells; increased CD8+ TEM infiltration and CD8+ TEM/naïve ratio; increased tumor-infiltrated natural killer (NK) cells; increased ratio of M1/M2 macrophages; and decreased infiltration of tumor-associated neutrophils and myeloid-derived suppressor cells. Similar immunophenotypic changes were observed in the EMT-6 model. Activation of both the adaptive and innate immune systems contributed to the antitumor activity of M7824 in the MC38 model, as depletion of CD8+ T cells and NK cells led to a substantial decrease in M7824-mediated tumor regression; CD4+ T cells and antibody-dependent cellular cytotoxicity, however, appeared dispensable. Toxicologic evaluation in cynomolgus monkeys indicated M7824 was well tolerated, with only minor hematologic effects observed. Although M7824 is a bifunctional molecule, its antitumor activity can be further improved by combination with standard-of-care therapies: in the MC38 model, combining M7824 with radiotherapy or chemotherapy resulted in more effective inhibition of tumor growth and an increased frequency of tumor antigen-specific IFN-γ-producing CD8+ T cells vs either monotherapy. Collectively, our preclinical data suggest that M7824 elicits potent and synergistic antitumor activity via the simultaneous blockade of the PD-L1 and TGFβ pathways. Phase 1 clinical trials with M7824 are under way in patients with solid tumors. Citation Format: Yan Lan, Dong Zhang, Chunxiao Xu, Bo Marelli, Jin Qi, Huakui Qi, Guozhong Qin, Xiaomei Xu, Hong Wang, Aroop Sircar, Beatrice Brunkhorst, Eric Austin, Laszlo Radvanyi, Kin-Ming Lo. Preclinical evaluation and mechanistic characterization of M7824 (MSB0011359C), a novel bifunctional fusion protein targeting the PD-L1 and TGFβ pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2615. doi:10.1158/1538-7445.AM2017-2615

Abstract LB-198: Combination of PEGylated recombinant hyaluronidase PH20 (PEGPH20) with live-attenuated, double-deleted (LADD) Listeria enhances tumor infiltrating CD8+ T cell response and antitumor efficacy in mice

Abstract Successful immunotherapy is highly dependent on inducing an efficient CD8+ T cell-mediated antitumor immune response. The trafficking and activation of these effector CD8+ T cells are often inhibited by various immunosuppressive mechanisms in the tumor microenvironment (TME). Hyaluronan (HA), a glycosaminoglycan that accumulates in the TME of many solid tumors, is associated with rapid tumor progression, poor prognosis and increased immunosuppression. We have previously demonstrated in nonclinical models that enzymatic degradation of TME HA by intravenous PEGylated recombinant human hyaluronidase PH20 (PEGPH20) facilitates delivery of chemotherapeutics to tumors and enhances checkpoint inhibitor efficacy. Based on these observations we hypothesized that PEGPH20 may improve current cancer immunotherapies by increasing immune cell infiltration into tumors. To test this hypothesis we used CT26/HAS3 tumors, CT26 murine colon carcinoma cells engineered to over-express hyaluronan synthase-3, and treated them with PEGPH20 in combination with recombinant live-attenuated double-deleted strains of Listeria monocytogenes expressing AH1 (gp70423-431, SPSYVYHQF), the immunodominant CD8+ T cell epitope of CT26 (LADD-AH1). PEGPH20 (1mpk), which results in nearly complete removal of HA at 24hrs, was administered 24hrs prior to LADD-AH1. In this model, LADD-AH1 alone inhibited tumor growth which correlated with an increase in IFNγ+ (p=0.0019) and granzyme B+ CD8+ tumor infiltrating lymphocytes (TILs) (p=0.0006). Tumor-associated macrophages (TAMs) from LADD-AH1 treated tumors were more immunostimulatory with a decrease in MHCIIlow (p=0.0205) and CD206+ TAMs (p=0.0003). Moreover, the combination of PEGPH20 and LADD-AH1 resulted in enhanced anti-tumor activity compared to either PEGPH20 (p=0.0047) or LADD-AH1 alone (p=0.0002). Significant TME changes were observed in the combination treatment with an increase in CD8+ TILs (p=0.0200, of which 88% were granzyme B+), NK cells (p=0.0205) and an increase in CD8:Treg ratio (p=0.0007).These findings were corroborated by immunohistochemical analysis demonstrating an increase in CD3+ T cells with PEGPH20 treatment. Taken together, these data suggest that HA accumulation in tumors may act as a barrier to immune cell access and that reduction of HA by PEGPH20 facilitates increased accumulation of tumor antigen-specific CD8+ effector T cells induced by LADD immunotherapy. Our findings reveal a benefit of PEGPH20 in enhancing tumor infiltration of cytotoxic CD8+ T cells induced by LADD that translate into a significant improvement in efficacy in a murine model of HA-high tumors. Citation Format: Jisook Lee, Meredith Leong, Yujun Huang, Benjamin J. Thompson, Sanna Rosengren, Barbara Blouw, Dirk G. Brockstedt, Curtis B. Thompson. Combination of PEGylated recombinant hyaluronidase PH20 (PEGPH20) with live-attenuated, double-deleted (LADD) Listeria enhances tumor infiltrating CD8+ T cell response and antitumor efficacy in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-198. doi:10.1158/1538-7445.AM2017-LB-198

A novel combination strategy for effectively targeting cancer stem-like cells.

Immunotherapy is now established as the fourth pillar of cancer therapy alongside surgery, radiation and chemotherapy. The major success of cancer immunotherapy to date has been the use of therapies targeting immune checkpoints such as PD-1 and CTLA-4, which act to enhance the ability of tumor-antigen specific CD8+ T cells to target cancer cells.1 Similarly, adoptive cellular therapy of expanded tumor-infiltrating lymphocytes or vaccines based on recognition of tumor (neo)antigens are also promising approaches, which make use of the cytotoxic activity of CD8+ T cells. Although these strategies have shown considerable promise in some patients, the effectiveness of anti-tumor CD8+ T cells is limited by several factors, including local tumor-induced immunosuppression and the requirement of tumors to express MHC-I, which is often downregulated as a tumor escape mechanism.2 Furthermore, the cancer stem cell (CSC) population, reported to be important for tumor progression and metastasis, is known to be resistant to anti-tumor T cells (Figure 1). Therefore, there is a pressing need to develop strategies to further enhance the activity of CD8+ T cells in patients who would otherwise be non-responsive to immunotherapy.

A STING Agonist Given with OX40 Receptor and PD-L1 Modulators Primes Immunity and Reduces Tumor Growth in Tolerized Mice.

Stimulator of interferon genes (STING) signaling induces IFNβ production by intratumoral dendritic cells (DC), driving T-cell priming and recruitment into the tumor microenvironment (TME). We examined to what extent preexisting antigen-specific tolerance influenced the efficacy of in situ delivery of a potent STING-activating cyclic dinucleotide (CDN), ADU S-100, against established HER-2+ breast tumors. ADU S-100 induced HER-2-specific CD8+ T-cell priming and durable tumor clearance in 100% of nontolerant parental FVB/N mice. In contrast, ADU S-100 did not sufficiently prime HER-2-specific CD8+ T cells in tolerant neu/N mice, resulting in only delayed tumor growth and tumor clearance in 10% of the mice. No differences in IFNβ production, DC priming, or HER-2-specific CD8+ T-cell trafficking were detected between FVB/N and neu/N mice. However, activation and expansion of HER-2-specific CD8+ T cells were defective in neu/N mice. Immune cell infiltrates of untreated tumor-bearing neu/N mice expressed high numbers of PD1 and OX40 receptors on their CD8+ T cells, and PD-L1 was highly expressed on both myeloid and tumor cells. Modulating PD-L1 and OX40 receptor signaling combined with intratumoral ADU S-100 administration enhanced HER-2-specific CD8+ T-cell activity, clearing tumors in 40% of neu/N mice. Thus, intratumoral STING agonists could potently prime tumor antigen-specific CD8+ T-cell responses, and adding PD-L1 blockade and OX40 receptor activation can overcome antigen-enforced immune tolerance to induce tumor regression. Cancer Immunol Res; 5(6); 468-79. ©2017 AACR.

Abstract TMEM-016: DENDRITIC CELL VACCINATION AND INHIBITION OF TUMOR–ASSOCIATED IMMUNE SUPPRESSION

Abstract PURPOSE: Th17 cell infiltration correlates with markedly prolonged overall survival among ovarian cancer patients, prompting the question of whether Th17 cells could be induced or expanded to therapeutic advantage. To address this question, a phase I clinical trial to test the safety and immunogenicity of Th17-inducing dendritic cell (DC) vaccination for stage IIIC–IV ovarian cancer patients (Clinicaltrials.gov NCT02111941) is nearing its accrual goal (n = 22). Interim analyses show that 8 of 9 patients completing the induction phase of five DC vaccinations mounted Th17 and antibody responses to FRα target antigen. These results are encouraging, but we found that ovarian tumor-associated CD14+ macrophages strongly suppress Th17-inducing DC-stimulated tumor antigen-specific Th1/Th17 effector CD4+ T cells responses. The purpose of this project was to elucidate the mechanisms by which ovarian tumor ascites CD14+ cells suppress DC-stimulated T cell responses, and to explore innovative strategies for alleviation of CD14+ cell-mediated immune suppression, or redirection of CD14+ cells from a suppressive phenotype to a pro-inflammatory phenotype. EXPERIMENTAL PLAN: Primary ovarian ascites CD14+ myeloid cells were tested for their ability suppress DC-stimulated tumor antigen-specific CD4+ T cell responses in contact or transwell coculture assays. Ascites fluid and ascites CD14+ cells were screened for expression of key chemokines and cytokines associated with T cell migration and immune suppression. Ascites CD14+ cells were also screened for PD-L1 expression, indoleamine 2,3-dioxygenase (IDO) activity, and key signaling pathways that determine the balance between M1 and M2 function. We also tested the ability of small molecule inhibitors to block key functions and alleviate immune suppression. SUMMARY OF RESULTS: Ovarian ascites CD14+ cells suppress DC-stimulated CD4+ T cell responses at least in part through expression of IL-10 and IDO. PD-L1 and PD-1 are consistently expressed by ascites CD14+ cells. Flow cytometric phosphoprotein assays with ascites CD14+ cells (n = 9) revealed consistent activation of AktS473, ERK1/2T202/Y204, CREBS133 and STAT3S727. Ovarian tumor-associated CD14+ macrophages consistently show high activity of a non-canonical STAT3 signaling pathway revealed by phosphorylation of S727 rather than the canonical Y705 phosphorylation seen in the classical JAK-STAT3 pathway. This innovative finding is consistent for every patient sample tested to date - canonical STAT3Y705 is absent. Inhibition of p38T180/Y182 MAPK signaling reduced expression of IL-10, RANTES, MIP-1β and IP-10. CCL22 (MDC) expression was also limited by p38 inhibition, but markedly elevated by blockade of JAK1/2 signaling. Inhibition of c-Kit and PI3K/Akt signaling showed a trend towards reduced K/T ratios, whereas inhibition of STAT3 reduced kynurenine production, suggesting that IDO activity is regulated at least in part by non-canonical STAT3S727 signaling. CONCLUSIONS: Multiple pathways regulate mechanisms of immune suppression by ovarian tumor ascites CD14+ myeloid cells. The p38 MAPK pathway regulates IL-10 expression, consistent with our published work on DC function. The non-canonical STAT3S727 pathway may also play a central role in regulating immune function by myeloid cells in the tumor microenvironment. These studies are of fundamental importance for development of adjuvant treatments to enhance the clinical efficacy of Th-17-inducing DC vaccination for ovarian cancer. Citation Format: Martin J Cannon, PhD. DENDRITIC CELL VACCINATION AND INHIBITION OF TUMOR–ASSOCIATED IMMUNE SUPPRESSION [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-016.

Development and characterization of naive single-type tumor antigen-specific CD8+ T lymphocytes from murine pluripotent stem cells

ABSTRACTOptimal approaches to differentiate tumor antigen-specific cytotoxic T lymphocytes (CTLs) from pluripotent stem cells (PSCs) remain elusive. In the current study, we showed that combination of in vitro priming through Notch ligands and in vivo development facilitated the generation of tumor Ag-specific CTLs that effectively inhibited tumor growth. We co-cultured the murine induced PSCs (iPSCs) genetically modified with tyrosinase-related protein 2 (TRP2)-specific T cell receptors with OP9 cell line expressing both Notch ligands Delta-like 1 and 4 (OP9-DL1/DL4) for a week before adoptively transferred into recipient C67BL/6 mice. Three weeks later, B16 melanoma cells were inoculated subcutaneously, and the antitumor activity of the iPSC-derived T cells was assessed. We observed the development of the TRP2-specific iPSC-CD8+ T cells that responded to Ag stimulation and infiltrated into melanoma tissues, significantly inhibited the tumor growth, and improved the survival of the tumor-bearing mice. Thus, this approach may provide a novel effective strategy to treatment of malignant tumors.

Blockade of only TGF-β 1 and 2 is sufficient to enhance the efficacy of vaccine and PD-1 checkpoint blockade immunotherapy

ABSTRACTCheckpoint inhibition has established immunotherapy as a major modality of cancer treatment. However, the success of cancer immunotherapy is still limited as immune regulation of tumor immunity is very complicated and mechanisms involved may also differ among cancer types. Beside checkpoints, other good candidates for immunotherapy are immunosuppressive cytokines. TGF-β is a very potent immunosuppressive cytokine involved in suppression of tumor immunity and also necessary for the function of some regulatory cells. TGF-β has three isoforms, TGF-β 1, 2 and 3. It has been demonstrated in multiple mouse tumor models that inhibition of all three isoforms of TGF-β facilitates natural tumor immunosurveillance and tumor vaccine efficacy. However, individual isoforms of TGF-β are not well studied yet. Here, by using monoclonal antibodies (mAbs) specific for TGF-β isoforms, we asked whether it is necessary to inhibit TGF-β3 to enhance tumor immunity. We found that blockade of TGF-β1 and 2 and of all isoforms provided similar effects on tumor natural immunosurveillance and therapeutic vaccine-induced tumor immunity. The protection was CD8+ T cell-dependent. Blockade of TGF-β increased vaccine-induced Th1-type response measured by IFNγ production or T-bet expression in both tumor draining lymph nodes and tumors, although it did not increase tumor antigen-specific CD8+ T cell numbers. Therefore, protection correlated with qualitative rather than quantitative changes in T cells. Furthermore, when combined with PD-1 blockade, blockade of TGF-β1 and 2 further increased vaccine efficacy. In conclusion, blocking TGF-β1 and 2 is sufficient to enhance tumor immunity, and it can be further enhanced with PD-1 blockade.

Enhanced antitumor effects by combining an IL-12/anti-DNA fusion protein with avelumab, an anti-PD-L1 antibody.

The combined therapeutic potential of an immunocytokine designed to deliver IL-12 to the necrotic regions of solid tumors with an anti-PD-L1 antibody that disrupts the immunosuppressive PD-1/PD-L1 axis yielded a combinatorial benefit in multiple murine tumor models. The murine version of the immunocytokine, NHS-muIL12, consists of an antibody (NHS76) recognizing DNA/DNA-histone complexes, fused with two molecules of murine IL-12 (NHS-muIL12). By its recognition of exposed DNA, NHS-muIL12 targets IL-12 to the necrotic portions of tumors; it has a longer plasma half-life and better antitumor efficacy against murine tumors than recombinant murine IL-12. It is shown here that NHS-muIL12, in an IFN-γ‒dependent mechanism, upregulates mPD-L1 expression on mouse tumors, which could be construed as an immunosuppressive action. Yet concurrent therapy with NHS-muIL12 and an anti-PD-L1 antibody resulted in additive/synergistic antitumor effects in PD-L1‒expressing subcutaneously transplanted tumors (MC38, MB49) and in an intravesical bladder tumor model (MB49). Antitumor efficacy correlated with (a) with a higher frequency of tumor antigen-specific splenic CD8+ T cells and (b) enhanced T cell activation over a wide range of NHS-muIL12 concentrations. These findings suggest that combining NHS-muIL12 and an anti-PD-L1 antibody enhances T cell activation and T cell effector functions within the tumor microenvironment, significantly improving overall tumor regression. These results should provide the rationale to examine the combination of these agents in clinical studies.

LYG1 exerts antitumor function through promoting the activation, proliferation, and function of CD4+ T cells

ABSTRACTIdentification of novel stimulatory cytokines with antitumor function would have great value in tumor immunotherapy investigations. Here, we report LYG1 (Lysozyme G-like 1) identified through the strategy of Immunogenomics as a novel classical secretory protein with tumor-inhibiting function. LYG1 recombinant protein (rhLYG1) could significantly suppress the growth of B16 tumors in WT B6 mice, but not in SCID-beige mice, Rag1−/− mice, CD4+- or CD8+ T cell-deleted mice. It could increase the number of CD4+ and CD8+ T cells in tumor-infiltrating lymphocytes, tumor-draining lymph nodes, and spleens, and promote IFNγ production by T cells in tumor-bearing mice. In vitro experiments demonstrated that rhLYG1 could directly enhance IFNγ secretion by CD4+ T cells, but not CD8+ T cells. Moreover, it could promote the activation, proliferation, and IFNγ production of tumor antigen-specific CD4+ T cells. The tumor-inhibiting effect of LYG1 was eliminated in Ifng−/− mice. Furthermore, LYG1 deficiency accelerated B16 and LLC1 tumor growth and inhibited the function of T cells. In summary, our findings reveal a tumor-inhibiting role for LYG1 through promoting the activation, proliferation, and function of CD4+ T cells in antitumor immune responses, offering implications for novel tumor immunotherapy.

Targeting the glucose metabolism of monocytic myeloid-derived suppressor cells to stimulate cancer immunity.

126 Background: Myeloid derived suppressor cells (MDSCs) inhibit the expansion of tumor antigen-specific effector CD8+ T cells via different mechanisms including increased expression of arginase, transforming growth factor – β (TGF – β) and indoleamine 2,3-dioxygenase (IDO). Recently, MDSCs were found to over-express hypoxia inducible factor 1 alpha (HIF-1α) which is required for their differentiation. An essential transcriptional target of HIF-1α is 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) which synthesizes fructose 2,6-bisphosphate, an allosteric stimulator of glycolysis and of proliferation via stimulation of cyclin dependent kinase-1 (CDK1). We hypothesized that MDSCs might over-express PFKFB3 which in turn might be required for their function as T cell suppressors. Methods: We used monocytic MDSCs (M-MDSCs) induced by co-culture with A375 melanoma cells, M-MDSCs from metastatic melanoma patients, murine bone marrow MDSCs and splenic M-MDSCs from B16 F10 tumor bearing mice for our studies. T cell suppression assays were performed to analyze M-MDSC suppression and reversal following PFKFB3 blockade. Results: We found that M-MDSCs have increased PFKFB3 expression. We also found that PFK-158 administration in B16 (wild-type) melanoma-bearing mice results in a marked reduction in MDSCs and a simultaneous increase in CD8+ T cell infiltration in the tumors. We analyzed three advanced cancer patients for circulating MDSCs before and after PFK-158 administration as part of a multi-center phase 1 clinical trial. And, we found that the MDSCs were markedly reduced in each patient. In addition, we have generated data for MDSC suppressive activity following in vitro treatment with PFK-158 showing reversal of suppressive activity. Conclusions: Taken together, these data indicate that selective inhibition of PFKFB3 may be a novel approach to target MDSCs and combinations of PFKFB3 inhibitors with immunotherapies may be a rational strategy to promote durable immune-mediated remissions in cancer patients.

Abstract PR03: Prophylactic nodal irradiation abrogates the synergy of tumor radiotherapy and immune checkpoint blockade

Abstract Background: Prophylactic nodal irradiation (PNI) is a strategy used to treat early stage cancers with the potential to enhance local control and prevent metastatic spread. It remains unclear whether irradiation of tumor-associated draining lymph nodes (LN) influences anti-tumor T cell responses. Our aim was to investigate the in vivo effects of nodal irradiation on the phenotype and function of tumor infiltrating lymphocytes (TIL) and the impact of PNI on the anti-tumor effects of immunotherapy. Methods: The Small Animal Radiation Research Platform (SARRP) delivered imaged-guided stereotactic radiation (RT) to tumor (T-only) or tumor and LN (T+LN). Syngeneic tumors (MC38 colon, B16 melanoma) were implanted in C57BL/6 mice and irradiated (12Gy x1) and/or treated with immune checkpoint blockade with αCTLA-4 IgG2a (depleting) or αPD-1 IgG1. The composition of the tumor microenvironment (TME) was assayed by flow cytometry. To query tumor-antigen specific T cell responses, adoptive transfer experiments were performed using OVA-specific CD8+ T-cells from Rag-/- OT-1 mice and OVA-MHC class I tetramer (SIINFEKL). Tumor lysate was also collected for chemokine analysis, and tumor outgrowth was quantified over time. Results: T-only RT resulted in a significant increase (P<0.01) in the proportion and absolute number of tumor infiltrating CD8+ effector T cells in comparison with T+LN RT. Immunosuppressive subsets (CD11b+ Gr-1hi MDSCs and FoxP3+ CD4+ Tregs) were also significantly increased in the T-only group. Ultimately, T-only RT significantly enhanced the CD8 effector:Treg ratio relative to untreated and T+LN treated tumors. Using the OVA-MHC class I tetramer to identify tumor-antigen specific CD8 T-cells, we observed that T-only RT significantly increased (P<0.05) the number of tumor-specific CD8-T cells in the TME compared with T+LN RT. Interestingly, T-only RT resulted in a a significant expansion of non-OVA specific CD8 T cells which was not observed with T+LN RT, suggesting a polyclonal anti-tumor immune response. Functionally, a significant increase (P<0.01) in the absolute number of IFNγ+ and TNFα+ antigen-specific TIL were noted with T-only RT. Mechanistically, a distinct chemokine signature correlated with robust TME immune infiltration and significantly elevated levels of CCL3/4/5 and CXCL10 (P<0.05) were observed in tumor lysate collected from T-only RT tumors relative to T+LN RT samples. In an effort to understand the implications of the RT target on potential synergy with immune checkpoint blockade we performed survival experiments with T-only and T+LN RT in combination with αPD-1 or αCTLA-4. T-only RT in combination with αCTLA-4 yielded the best outcome with a 86% long-term survival (day 90 post-RT) compared with 30-36% long-term survival in mice treated with other combinations of RT + immune checkpoint blockade. Intriguingly, favorable CD8 effector:Treg ratio was able to predict treatment response and was dramatically higher (P<0.001) among mice treated with T-only RT in combination with αCTLA-4. Conclusions: We have successfully developed a SARRP-based early stage cancer model with the ability to target or spare the tumor-associated LN. Results to date demonstrate significant immunological differences that are contingent upon inclusion/exclusion of the LN. Long-term survival experiments suggest response and survival advantages with T-only RT, particularly in combination with αCTLA-4 blockade. Taken together, these data suggest that PNI may dampen anti-tumor immune responses and that Tumor-only RT might be a better strategy in combination immunotherapy regimens, although these results should be explored in carefully designed clinical trials. This abstract is also being presented as Poster B41. Citation Format: Ariel E. Marciscano, Ali Ghasemzadeh, Thomas R. Nirschl, Brian J. Francica, Debebe Theodros, Esteban Velarde, J Wong, Daniel LJ Thorek, Theodore L. DeWeese, Charles G. Drake. Prophylactic nodal irradiation abrogates the synergy of tumor radiotherapy and immune checkpoint blockade. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR03.

Generation of dendritic cell-based vaccine using high hydrostatic pressure for non-small cell lung cancer immunotherapy.

High hydrostatic pressure (HHP) induces immunogenic death of tumor cells which confer protective anti-tumor immunity in vivo. Moreover, DC pulsed with HHP-treated tumor cells induced therapeutic effect in mouse cancer model. In this study, we tested the immunogenicity, stability and T cell stimulatory activity of human monocyte-derived dendritic cell (DC)-based HHP lung cancer vaccine generated in GMP compliant serum free medium using HHP 250 MPa. DC pulsed with HHP-killed lung cancer cells and poly(I:C) enhanced DC maturation, chemotactic migration and production of pro-inflammatory cytokines after 24h. Moreover, DC-based HHP lung cancer vaccine showed functional plasticity after transfer into serum-containing media and stimulation with LPS or CD40L after additional 24h. LPS and CD40L stimulation further differentially enhanced the expression of costimulatory molecules and production of IL-12p70. DC-based HHP lung cancer vaccine decreased the number of CD4+CD25+Foxp3+ T regulatory cells and stimulated IFN-γ-producing tumor antigen-specific CD4+ and CD8+ T cells from non-small cell lung cancer (NSCLC) patients. Tumor antigen specific CD8+ and CD4+ T cell responses were detected in NSCLC patient’s against a selected tumor antigens expressed by lung cancer cell lines used for the vaccine generation. We also showed for the first time that protein antigen from HHP-killed lung cancer cells is processed and presented by DC to CD8+ T cells. Our results represent important preclinical data for ongoing NSCLC Phase I/II clinical trial using DC-based active cellular immunotherapy (DCVAC/LuCa) in combination with chemotherapy and immune enhancers.

The EGR2 targets LAG-3 and 4-1BB describe and regulate dysfunctional antigen-specific CD8+ T cells in the tumor microenvironment.

Although the presence of tumor-infiltrating lymphocytes (TILs) indicates an endogenous antitumor response, immune regulatory pathways can subvert the effector phase and enable tumor escape. Negative regulatory pathways include extrinsic suppression mechanisms, but also a T cell-intrinsic dysfunctional state. A more detailed study has been hampered by a lack of cell surface markers defining tumor-specific dysfunctional TILs, and PD-1 alone is not sufficient. Recently, we identified the transcription factor Egr2 as a critical component in controlling the anergic state in vitro. In this study, we show that the Egr2-driven cell surface proteins LAG-3 and 4-1BB can identify dysfunctional tumor antigen-specific CD8+ TIL. Co-expression of 4-1BB and LAG-3 was seen on a majority of CD8+ TILs, but not in lymphoid organs. Functional analysis revealed defective IL-2 and TNF production yet retained expression of IFN-γ and regulatory T cell-recruiting chemokines. Transcriptional and phenotypic characterization revealed coexpression of multiple additional co-stimulatory and co-inhibitory receptors. Administration of anti-LAG-3 plus anti-4-1BB mAbs was therapeutic against tumors in vivo, which correlated with phenotypic normalization. Our results indicate that coexpression of LAG-3 and 4-1BB characterize dysfunctional T cells within tumors, and that targeting these receptors has therapeutic utility.

Tumor antigen-specific CD8+ T cells are negatively regulated by PD-1 and Tim-3 in human gastric cancer

Cytotoxic CD8 T lymphocytes that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally important in eliciting tumor rejection. NY-ESO-1 is a major target of CD8+ T cell recognition in gastric cancer (GC) and is among the most immunogenic tumor antigens defined to date. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8+ T cell dysfunction may reveal effective strategies for immunotherapy. In an effort to understand in vivo tolerance mechanisms, we assessed the phenotype and function of NY-ESO-1-specific CD8+ T cells derived from peripheral blood lymphocytes (PBLs) and tumor-associated lymphocytes (TALs) of GC patients. Here, we report that Tim-3 expression defines a subpopulation of PD-1+ exhausted NY-ESO-1-specific CD8+ T cell and PD-1+Tim-3+ CD8+ T cells represented the largest subset of NY-ESO-1-specific CD8+ T cells in GC patients. Functionally, CD8+PD-1+Tim-3+ T cells were more impaired in IFN-γ, TNF-α and IL-2 production compared with PD-1+Tim-3− or PD-1−Tim-3− subsets. Dual blockade of Tim-3 and PD-1 during T-cell priming efficiently augmented proliferation and cytokine production by NY-ESO-1-specific CD8+ T cells could potentially be improved by therapeutic targeting of these inhibitory receptors, indicating that antitumor function of NY-ESO-1-specific CD8+ T cells could potentially be improved by therapeutic targeting of these inhibitory receptors.

Super Cross-Presentation of Tumor Antigens to Elicit Anti-Lymphoma Immunity By Synthetic Design of an Anti-Phosphatidylserine Bridge Protein

Cell loss by apoptosis is a common feature in tumors. Dying tumor cells induce immune tolerance within the tumor microenvironment largely through highly conserved homeostatic clearance programs that restore tissue immune homeostasis and contribute to the formation of an immunosuppressive niche. The translocation of phosphatidylserine (PS) on cellular membranes, during the initial phases of apoptosis, functions as a recognition and removal signal that limits the immunogenicity of cell death. We examined whether altering clearance of dying cancer cells to elicit inflammatory turnover potentiates immune responses against lymphoma cells. To remove inhibitory signals in the homeostatic clearance pathway we utilized a molecular bridge scaffold to engineer a modified phosphatidylserine bridge protein (FA58C2-hIgG1 or C2-hIgG1) that works as a bridge between apoptotic cells expressing aminophospholipids and phagocytes bearing Fc receptors. In vitro administration of C2-hIgG1 to murine bone marrow derived macrophages promotes engulfment of apoptotic murine lymphoma cells (38C13 cell line) and ablates the secretion of the anti-inflammatory cytokine interleukin-10 (IL-10) and suppression of pro-inflammatory cytokines tumor necrosis factor (TNF-α) and IL-12p40 to the presence of apoptotic cells. Similarly, uptake of C2-hIgG1 treated lymphoma cells triggers upregulation of the costimulatory markers CD80, CD86, and MHC class II on macrophages and promotes secretion of Th1-recruiting lymphocyte chemokines CXCL9, CXCL10, and CCL5. Accordingly, in vivo administration of C2-hIgG1 partially restores immune responses to dead lymphoma cells in antigen cross presentation assays and promotes recruitment and retention of tumor antigen specific CD8+ T cells, dendritic cells, and natural killer cells into tumors. These effects combine to elicit anti-lymphoma immunity, improve responses to immune checkpoint inhibitors, and enhance the effectiveness of adoptive T cell transfers using engineered T Cell Receptors (TCRs) but not CD19-directed chimeric antigen receptor engineered (CAR-T) T cells. DisclosuresNo relevant conflicts of interest to declare.

A Novel Bioluminescent Mouse Model for Optimization of Adoptive T Cell Therapy

Adoptive T cell therapy (ATT) is a promising option for the treatment of solid cancers. However, various defense mechanisms acquired by the tumor during evolution prevent transferred T cells (TC) to unfold their full potential. A combination of ATT with accessory therapeutic approaches including checkpoint inhibition and targeted therapy could lift TC inhibition and efficiently shift the immune balance towards tumor rejection. An in-vivo analysis of the impact of combination strategies on the outcome of ATT would greatly enhance the search for an optimal accessory to ATT therapy.We generated the transgenic mouse line BLITC (bioluminescence imaging of T cells) expressing an NFAT (nuclear factor of activated T cell)-dependent Click-beetle luciferase (Na et. al, 2010) and a constitutive Renilla Luciferase, allowing us to monitor migration and activation of transferred TCs in vivo. In order to analyze crucial ATT parameters in a clinically relevant tumor model, BLITC mice were crossed to the two HY-TCR transgenic mice Marilyn (CD4: H-2Ab-Dby) and MataHari (CD8: H-2Db-Uty) to generate TCs that could be monitored for in-vivo infiltration, local activation and rejection of established (> 0,5 cm x 0,5 cm / ≥10 days growth) H-Y expressing MB49 tumors. In order to better reflect the clinical situation, we lymphodepleted tumor-bearing immunocompetent albino B6 mice with fludarabine (FLu) and/or cyclophosphamide (CTX) prior to ATT. Transferred TCs were FACSorted and injected after an optional culture expansion phase.As shown before for freshly injected tumor cells (Perez-Diez, 2007), we observed a superior response of tumor-antigen specific CD4+ TCs compared to CD8+ TCs against established tumors. Whereas 5*106 CD8+ T cells hardly attenuated tumor growth, even as few as 5000 H-Y TCR-transgenic CD4+ T cells rejected tumors in most mice, depending on the lymphodepleting treatment (Figure A - remission rates in parentheses). Tumor infiltration and activation of adoptively transferred TCs was monitored in-vivo by the respective bioluminescent reporters. Around day 4 and 6, CD4+ TCs migrated from tumor-draining lymph nodes into the tumor environment and persisted until rejection. Interestingly, activation of CD4+ TCs was only transient (between days 4 and 7) in all mice, independent of therapy outcome (in Figure B shown for refractory tumor). Whereas loss of activation signal during remission was correlated with tumor clearance and decline of effector function, in refractory tumors it suggests a rapid inactivation of infiltrating TCs by the tumor microenvironment. Our data indicate that the failure of tumor rejection is not caused by impaired peripheral expansion or tumor homing but rather by inhibition of TC effector function. Responsible mechanisms and counter-acting therapeutic interventions are the focus of ongoing studies.In summary, the BLITC reporter system facilitates analysis of therapeutic parameters for ATT in a well-established solid tumor model. Using BLITC mice for transduction with TCR or CAR expression cassettes could allow rapid monitoring of on-target as well as undesired off-target effects in virtually any tumor setting. Future experiments will focus on the beneficial effects of combination treatments on the activation of adoptively transferred TCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells

Despite the impressive rates of clinical response to programmed death 1 (PD-1) blockade in multiple cancers, the majority of patients still fail to respond to this therapy. The CT26 tumor in mice showed similar heterogeneity, with most tumors unaffected by anti-PD-1. As in humans, response of CT26 to anti-PD-1 correlated with increased T- and B-cell infiltration and IFN expression. We show that intratumoral injection of a highly interferogenic TLR9 agonist, SD-101, in anti-PD-1 nonresponders led to a complete, durable rejection of essentially all injected tumors and a majority of uninjected, distant-site tumors. Therapeutic efficacy of the combination was also observed with the TSA mammary adenocarcinoma and MCA38 colon carcinoma tumor models that show little response to PD-1 blockade alone. Intratumoral SD-101 substantially increased leukocyte infiltration and IFN-regulated gene expression, and its activity was dependent on CD8+ T cells and type I IFN signaling. Anti-PD-1 plus intratumoral SD-101 promoted infiltration of activated, proliferating CD8+ T cells and led to a synergistic increase in total and tumor antigen-specific CD8+ T cells expressing both IFN-γ and TNF-α. Additionally, PD-1 blockade could alter the CpG-mediated differentiation of tumor-specific CD8+ T cells into CD127lowKLRG1high short-lived effector cells, preferentially expanding the CD127highKLRG1low long-lived memory precursors. Tumor control and intratumoral T-cell proliferation in response to the combined treatment is independent of T-cell trafficking from secondary lymphoid organs. These findings suggest that a CpG oligonucleotide given intratumorally may increase the response of cancer patients to PD-1 blockade, increasing the quantity and the quality of tumor-specific CD8+ T cells.

Abstract A017: Method for molecular characterization of antigens, epitopes and T cell receptors for adoptive CD4 immunotherapy

Abstract Recent advances in cellular immunotherapy show that cancer cells can be efficiently eliminated by adoptive transfer of modified patient T cells. The main effector functions can be provided by genetically engineered lymphocytes expressing T cell receptors (TCR) isolated from tumor antigen-specific cytotoxic CD8+ lymphocytes, which recognize tumor-associated antigens presented on HLA class I molecules. Nevertheless, for efficient and sustained cytotoxic and memory CD8+ T cell responses, T cell help delivered by CD4+ T lymphocytes plays a critical role. Multifunctional CD4+ T cells are essential to activate, control and maintain immune responses. CD4+ T cells recognize antigen-derived peptides presented by HLA class II molecules, thereby HLA class II tumor antigen-positive cells may also be efficiently eliminated by direct CD4+ cytotoxic mechanisms. Recently it has been shown that adoptive transfer of tumor antigen-specific CD4+ T cells alone can lead to substantial regression of epithelial tumors (Science. 2014 May 9;344(6184):641-5). To appropriately employ CD4+ T cells in tumor defense, detailed molecular knowledge regarding the antigens and corresponding epitopes they recognize is essential. We recently expanded our TCR platform by introducing a fast and efficient method for direct identification of antigens and respective epitopes recognized by CD4+ T cells. During method development, elucidated viral antigens allowed rapid clinical application, and the methodology was also applied for rapid identification of epitopes of CD4+ T cell clones recognizing multiple cancer-testis antigens (CT). For this, PBL from a healthy donor were stimulated with a mixture of autologous APCs transfected with in vitro transcribed RNA encoding various CT antigens fused to targeting signals for directed HLA class II cross-presentation. Antigen-reactive CD4+ T cells were isolated and expanded. In order to identify the antigen and epitope specificity of each T cell clone, DNA sequences of each CT antigen were randomly digested and DNA fragments introduced in a vector mixture that allows expression of the fragments in all possible reading frames fused to a bacterial selection marker. These vectors were then expressed in XL1 blue bacteria and fed as pools to autologous APCs. Thereby the APCs phagocytosed and directly presented random CT epitopes on their HLA class II molecules. Subsequently, these pool-loaded APCs were tested with CT-antigen-specific CD4+ T cell clones. In a second step, the single bacterial colonies of a positive pool were re-tested individually and the antigen fragments recognized were identified by DNA sequencing, and confirmed by testing of synthetic peptides. HLA restriction was determined by testing responses against antigen-positive HLA allogenic cells. Using this approach, TCR sequences, epitope sequences and respective MHC-restriction elements were identified for multiple antigens in a single round procedure. This method has broad application for identification of: 1) antigens and epitopes recognized by CD4 tumor infiltrating lymphocytes, 2) immune relevant patient-specific mutations recognized by CD4+ T cells, 3) defined epitopes recognized by T helper cells for generation of tumor vaccines, 4) epitope-specificity of CD4+ T cells, against any selected antigen. This technology enables the generation and molecular characterization of TCRs originating from tumor-specific CD4+ helper T cells, complementing our already established approach for isolation of high affinity TCRs derived from cytotoxic CD8+ T cells. Citation Format: Milosevic Slavoljub, Ellinger Christian, Wehner Carina, Raffegerst Silke, Wilde Susanne, Weis Manon, Sailer Nadja, Schendel Dolores. Method for molecular characterization of antigens, epitopes and T cell receptors for adoptive CD4 immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A017.

Abstract B051: Super cross-presentation of tumor antigens by synthetic design of an anti-phosphatidylserine bridge protein

Abstract Cell loss by apoptosis is a common feature in tumors. Dying tumor cells induce immune tolerance within the tumor microenvironment largely through highly conserved homeostatic clearance programs that restore tissue immune homeostasis and contribute to the formation of an immunosuppressive niche. The translocation of phosphatidylserine (PS) on cellular membranes, during the initial phases of apoptosis, functions as a recognition and removal signal that limits the immunogenicity of cell death. We examined whether altering clearance of dying cancer cells to elicit inflammatory turnover can allow for and potentiate immune responses against tumor cells. To remove inhibitory signals in the homeostatic clearance pathway we utilized a molecular bridge scaffold to engineer a modified phosphatidylserine bridge protein (FA58C2-hIgG1 or C2-hIgG1) that works as a bridge between apoptotic cells expressing aminophospholipids and phagocytes bearing Fc receptors. In vivo administration of C2-hIgG1 partially restores immune responses to dead tumor cells in antigen cross presentation assays and promotes recruitment and retention of tumor antigen specific CD8+ T cells, dendritic cells, and natural killer cells into tumors. These effects combine to elicit anti-lymphoma immunity, improve responses to immune checkpoint inhibitors, and enhance the effectiveness of adoptive T cell transfers using engineered T Cell Receptors (TCRs) but not chimeric antigen receptor engineered (CAR-T) T cells. Citation Format: Daniel Corey, Aaron Ring, Melissa McCracken, Masanori Miyanishi, Sydney Gordon, Irving Weissman. Super cross-presentation of tumor antigens by synthetic design of an anti-phosphatidylserine bridge protein [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B051.

Abstract A054: Inhibition of TGF-beta isoforms 1 and 2 enhances therapeutic tumor vaccine efficacy

Abstract Transforming growth factor-beta (TGF-beta) is a pleiotropic cytokine comprised of three isoforms, TGF-beta1, 2, and 3, that exhibits immunosuppressive functions. In the context of immunosuppression, TGF-beta dampens activation and maturation of both innate and adaptive immune cells, including natural killer cells, macrophages, and dendritic cells, as well as CD4+ and CD8+ T cells. Thus, TGF-beta inhibition, like blockade of other negative regulators of immunity, provides a potential modality of cancer treatment that can be used in combination therapy. Although we have previously demonstrated that blockade of all three isoforms of TGF-beta using a monoclonal antibody increases tumor vaccine efficacy, as measured by the reduction in tumor growth and increase in the number of antigen-specific CTLs, the function of individual isoforms of TGF-beta in the framework of tumor immunity is not well understood. In this study, we examined whether it is necessary to block TGF-beta3, in addition to the two other isoforms of TGF-beta, to improve vaccine-induced tumor immunity against the syngeneic TC1 tumor model, a C57BL/6 lung epithelial cell line transfected with HPV-16 E6 and E7 genes. When the subcutaneous tumors became approximately 5 mm in diameter, the mice were immunized with a peptide-based vaccine that targets the E7 oncogene with or without the simultaneous administration of anti-TGF-beta antibodies with unique specificities for the three isoforms. Mice immunized with the vaccine alone exhibited slightly delayed tumor growth relative to the untreated control. This effect could be enhanced by the combination of anti-TGF-beta antibodies that neutralize all three isoforms of TGF-beta. Interestingly, blockade of only TGF-beta1 & 2 also improved vaccine efficacy to a similar degree as the blockade of all three isoforms, though neutralization of TGF-beta alone could not suppress tumor growth. Therefore, it is not necessary to inhibit TGF-beta3 to generate significant vaccine-induced anti-tumor immunity. Systemic depletion of CD8+ T cells using anti-CD8-depleting antibodies completely abrogated the tumor protection observed in mice immunized with vaccine alone or vaccine in combination with TGF-beta blockade, suggesting that the mechanism of anti-tumor immunity in these conditions relies entirely on CD8+ T cells. In vivo CTL assays revealed that anti-TGF-beta antibodies tended to increase the quality of CTL activity induced by the vaccine. To better understand the effect of treatment on the tumor microenvironment, we used flow cytometric analysis to examine T cells in the tumor and tumor draining lymph nodes. While the vaccine significantly increased the number of tumor antigen-specific CD8+ T cells and IFN-gamma producing T cells in both lymph nodes and tumors, TGF-beta inhibition at two weeks, but not one week, after vaccination further augmented the increase in the number of T cells infiltrating tumors. Vaccine in combination with blockade of TGF-beta1 & 2, regardless of TGF-beta3 inhibition, also led to the greatest increase in the number of T-bet-expressing CD4+ and CD8+ T cells in tumors. This observation implies that blockade of TGF-beta1 & 2 alone may be sufficient to improve therapeutic tumor vaccines by facilitating the infiltration of more Th1/Tc1-type T cells into tumors. Because several studies suggest that TGF-beta3 actually plays a protective role against tumorigenesis in a range of tissues, developing TGF-beta blockade strategies that do not inhibit this isoform could be beneficial in certain settings. Citation Format: Katharine Clark, Faith C. Robertson, Emma De Ravin, Shingo Kato, Anja Bloom, Amer Mirza, Jay A. Berzofsky, Masaki Terabe. Inhibition of TGF-beta isoforms 1 and 2 enhances therapeutic tumor vaccine efficacy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A054.