Activation Of CTLs Research Articles

IL-1β Induces Human Endothelial Surface Expression of IL-15 by Relieving let-7c-3p Suppression of Protein Translation.

Expression of IL-15 on the surface of human graft endothelial cells (ECs) bound to the IL-15Rα subunit can increase the activation of CTLs, potentiating allograft rejection. Our previous work showed that surface expression of this protein complex could be induced by alloantibody-mediated complement activation through increased IL-1β synthesis, secretion, and autocrine/paracrine IL-1-mediated activation of NF-κB. In this article, we report that cultured human ECs express eight differently spliced IL-15 transcripts. Remarkably, IL-1β does not alter the expression level of any IL-15 transcript but induces surface expression independently of RNA polymerase II-mediated transcription while requiring new protein translation. Mechanistically, IL-1β causes an NF-κB-mediated reduction in the level of microRNA Let-7c-3p, thereby relieving a block of translation of IL-15 surface protein. Let7c-3p anti-miR can induce EC surface expression of IL-15/IL-15Rα in the absence of complement activation or of IL-1, enabling IL-15 transpresentation to boost CD8 T cell activation. Because of the complexity we have uncovered in IL-15 regulation, we recommend caution in interpreting increased total IL-15 mRNA or protein levels as a surrogate for transpresentation.

NK Receptor Signaling Lowers TCR Activation Threshold, Enhancing Selective Recognition of Cancer Cells by TAA-Specific CTLs.

Cytotoxic CD8+ T lymphocyte (CTL) recognition of non-mutated tumor-associated antigens (TAA), present on cancer cells and also in healthy tissues, is an important element of cancer immunity, but the mechanism of its selectivity for cancer cells and opportunities for its enhancement remain elusive. In this study, we found that CTL expression of the NK receptors (NKR) DNAM1 and NKG2D was associated with the effector status of CD8+ tumor-infiltrating lymphocytes and long-term survival of patients with melanoma. Using MART1 and NY-ESO-1 as model TAAs, we demonstrated that DNAM1 and NKG2D regulate T-cell receptor (TCR) functional avidity and set the threshold for TCR activation of human TAA-specific CTLs. Superior co-stimulatory effects of DNAM1 over CD28 involved enhanced TCR signaling, CTL killer function, and polyfunctionality. Double transduction of human CTLs with TAA-specific TCR and NKRs resulted in strongly enhanced antigen sensitivity, without a reduction in antigen specificity and selectivity of killer function. In addition, the elevation of NKR ligand expression on cancer cells due to chemotherapy also increased CTL recognition of cancer cells expressing low levels of TAAs. Our data help explain the ability of self-antigens to mediate tumor rejection in the absence of autoimmunity and support the development of dual-targeting adoptive T-cell therapies that use NKRs to enhance the potency and selectivity of recognition of TAA-expressing cancer cells.

DEC-205 receptor targeted poly(lactic-co-glycolic acid) nanoparticles containing Eucommia ulmoides polysaccharide enhances the immune response of foot-and-mouth disease vaccine in mice

Nanoparticles targeting the DEC-205 receptor were found to induce antigen-specific protective immune response. When the delivery system carries both antigens and immunomodulators, it can maximize the expected therapeutic effect of the drug and induce effective humoral and cellular immune responses to antigens.In this study, we encapsulated the Eucommia ulmoides Oliv. polysaccharides (EUPS) into PLGA nanoparticles (NPs) and conjugated it with anti-CD205 monoclonal Ab (MAb) to produce a DEC-205 receptor targeted PLGA nanoparticles (anti-DEC-205-EUPS-PLGA NPs). The physicochemical characteristics and adjuvant activity of the above NPs were evaluated in vitro and in vivo. In the in vitro setting, 200 μg·mL−1 anti-DEC-205-EUPS-PLGA could improve the proliferation of DCs and promote their antigen up-take activity. In the in vivo setting, anti-DEC-205-EUPS-PLGA NPs remarkably controlled the release of drug and antigen to induce sustained immune responses and up-regulated the levels of FMDV-specific IgG antibodies, promoted the cytotoxic activity of CTLs and NK cells, and improved the proliferation of splenocytes. Moreover, the anti-DEC-205-EUPS-PLGA NPs facilitated the maturation of DCs. The above data indicated that anti-DEC-205-EUPS-PLGA NPs employed as an targeted adjuvant induced the humoral and cellular immune activity by promoting the maturation of DCs. These findings may provide a new insight onto the development of vaccine adjuvants.

The TLR-NF-kB axis contributes to the monocytic inflammatory response against a virulent strain of Lichtheimia corymbifera, a causative agent of invasive mucormycosis.

Invasive mucormycosis (IM) is a life-threatening infection caused by the fungal order Mucorales, its diagnosis is often delayed, and mortality rates range from 40-80% due to its rapid progression. Individuals suffering from hematological malignancies, diabetes mellitus, organ transplantations, and most recently COVID-19 are particularly susceptible to infection by Mucorales. Given the increase in the occurrence of these diseases, mucormycosis has emerged as one of the most common fungal infections in the last years. However, little is known about the host immune response to Mucorales. Therefore, we characterized the interaction among L. corymbifera—one of the most common causative agents of IM—and human monocytes, which are specialized phagocytes that play an instrumental role in the modulation of the inflammatory response against several pathogenic fungi. This study covered four relevant aspects of the host-pathogen interaction: i) The recognition of L. corymbifera by human monocytes. ii) The intracellular fate of L. corymbifera. iii) The inflammatory response by human monocytes against the most common causative agents of mucormycosis. iv) The main activated Pattern-Recognition Receptors (PRRs) inflammatory signaling cascades in response to L. corymbifera. Here, we demonstrate that L. corymbifera exhibits resistance to intracellular killing over 24 hours, does not germinate, and inflicts minimal damage to the host cell. Nonetheless, viable fungal spores of L. corymbifera induced early production of the pro-inflammatory cytokine IL-1β, and late release of TNF-α and IL-6 by human monocytes. Moreover, we revealed that IL-1β production predominantly depends on Toll-like receptors (TLRs) priming, especially via TLR4, while TNF-α is secreted via C-type lectin receptors (CTLs), and IL-6 is produced by synergistic activation of TLRs and CTLs. All these signaling pathways lead to the activation of NF-kB, a transcription factor that not only regulates the inflammatory response but also the apoptotic fate of monocytes during infection with L. corymbifera. Collectively, our findings provide new insights into the host-pathogen interactions, which may serve for future therapies to enhance the host inflammatory response to L. corymbifera.

Asah2 Represses the p53-Hmox1 Axis to Protect Myeloid-Derived Suppressor Cells from Ferroptosis.

Myeloid-derived suppressor cells (MDSCs) are immune suppressive cells that massively accumulate under pathological conditions to suppress T cell immune response. Dysregulated cell death contributes to MDSC accumulation, but the molecular mechanism underlying this cell death dysregulation is not fully understood. In this study, we report that neutral ceramidase (N-acylsphingosine amidohydrolase [ASAH2]) is highly expressed in tumor-infiltrating MDSCs in colon carcinoma and acts as an MDSC survival factor. To target ASAH2, we performed molecular docking based on human ASAH2 protein structure. Enzymatic inhibition analysis of identified hits determined NC06 as an ASAH2 inhibitor. Chemical and nuclear magnetic resonance analysis determined NC06 as 7-chloro-2-(3-chloroanilino)pyrano[3,4-e][1,3]oxazine-4,5-dione. NC06 inhibits ceramidase activity with an IC50 of 10.16-25.91 μM for human ASAH2 and 18.6-30.2 μM for mouse Asah2 proteins. NC06 induces MDSC death in a dose-dependent manner, and inhibition of ferroptosis decreased NC06-induced MDSC death. NC06 increases glutathione synthesis and decreases lipid reactive oxygen species to suppress ferroptosis in MDSCs. Gene expression profiling identified the p53 pathway as the Asah2 target in MDSCs. Inhibition of Asah2 increased p53 protein stability to upregulate Hmox1 expression to suppress lipid reactive oxygen species production to suppress ferroptosis in MDSCs. NC06 therapy increases MDSC death and reduces MDSC accumulation in tumor-bearing mice, resulting in increased activation of tumor-infiltrating CTLs and suppression of tumor growth in vivo. Our data indicate that ASAH2 protects MDSCs from ferroptosis through destabilizing p53 protein to suppress the p53 pathway in MDSCs in the tumor microenvironment. Targeting ASAH2 with NC06 to induce MDSC ferroptosis is potentially an effective therapy to suppress MDSC accumulation in cancer immunotherapy.

T-cell Enriching Therapy using combination of IL-12 gene electrotransfer and anti-PD-1 attenuates the resistance of anti-PD1 in preclinical metastatic cancer

Abstract Immune checkpoint blockades, such as anti-PD-1, have demonstrated significant clinical benefits. However, both primary and acquired resistance has become major obstacles. Limited immune cell infiltration into tumors and expression of PD-1 are two reasons leading to resistance to PD-1 inhibition. Our laboratory has demonstrated that expression of plasmid IL-12 delivered intraumorally can increase immune cell infiltration as well as potentially increase levels of PD-1. In this current study, we used the intraperitoneal metastatic B16F10 tumor model and report that IL-12 gene electrotransfer (IL-12 GET) combined with anti-PD-1 induced long-lived CD8+PD-1− T cells. Strikingly, the effector cytolytic activity of CTLs was expanded and maintained for up to 60 days, indicating that these cells do not differentiate into a memory functional phenotype. In vivo imaging results, illuminate a lower flux signal in B16.F10 intraperitoneal metastases following the combination treatment. Simultaneously, the primary subcutaneous tumors are fully regressed in most of these mice (80%). Expression of CD44+ CD62L− on CD8 CTLs increased with time, suggesting that the memory response was induced at around 120 days. Depletion experiments revealed that CD8+ T cells and CD4+ T cells were required for tumor regression. Our results revealed that this treatment not only boosted CD3+ T-cell infiltration into tumor mass but also upregulated the production of proinflammatory cytokines such as IFN-γ as well as chemokines CXCL9, CXCL10 and CXCL11, promoting accumulation of T cells to the tumor microenvironment and enhancing the effector functions of infiltrated T cells and simultaneously, attenuate the resistant of immune checkpoint blockade of anti-PD1.

A Combination Therapy with Dendritic Cells, Pomalidomide and Programmed Death-Ligand 1 Blockade Exerts a Potent Antitumor Immunity in a Murine Model of Multiple Myeloma

Multiple myeloma (MM) is the second-most-common hematologic malignancy, and develops from clonal malignant plasma cells within bone marrow. Despite tremendous improvements in therapeutic strategies (e.g. stem cell transplantation, immune-modulatory drugs (IMiDs), proteasome inhibitors, and, more recently, immunotherapy), which have led to improved responses to treatment and overall survival, most patients eventually relapse. We have previously shown that the immunization with tumor antigen-loaded dendritic cells (DCs) and pomalidomide/dexamethasone synergistically potentiates the enhancing the antitumor immunity in a myeloma mouse model. In the present study, we investigated whether a DC-based vaccine combined with pomalidomide and PD-L1 blockade has a synergistic effect in a murine MM model. MOPC-315 cell lines were injected subcutaneously to establish MM-bearing mice. Four test groups were used to mimic the clinical protocol: (1) PBS control, (2) DCs + pomalidomide/dexamethasone, (3) pomalidomide/dexamethasone + PD-L1 blockade, and (4) DCs + pomalidomide/dexamethasone + PD-L1 blockade. After treatment, preclinical response and in vitro immunological responses were evaluated. The study was designed to closely mimic the clinical MM treatment protocol and clearly demonstrated that combination treatment with DCs + pomalidomide with dexamethasone + PD-L1 blockade more strongly inhibited MM tumor growth. Consequently, the mice treated with DCs + pomalidomide with dexamethasone + PD-L1 blockade displayed markedly induced tumor regression and significantly prolonged survival, as well as very strong anti-myeloma CTL responses and increased numbers of effector cells (such as CD4+ T cells, CD8+ T cells, memory T cells, NK cells and M1 macrophages) associated with antitumor effects. This treatment also effectively decreased the proportions of suppressor cells, including MDSCs, Tregs and M2 macrophages, in the spleen and tumor microenvironment of treated mice. Tregs, MDSCs and M2 macrophages play crucial roles in immunosuppression and tolerance, which are mediated by tumor-secreted cytokines. The inhibition of Tregs, MDSC and M2 macrophage accumulation may enhance systemic cell-mediated immunity through the activation of DCs or CTLs. Importantly, treatment with pomalidomide with dexamethasone + PD-L1 blockade led to decreased expression of PD-L1 and CTLA-4 in treated mice, which further induced effector cell infiltration of the tumor microenvironment. Moreover, DCs + pomalidomide with dexamethasone + PD-L1 blockade induced the activation of cell-mediated immunity by increasing Th1-specific immune responses, as evidenced by the increased production of IFN-γ and a decrease in the regulatory-specific immune response, as evidenced by the decreased production of TGF-β, IL-10 and VEGF in the spleen and tumor microenvironment. These findings show that inducing the systemic immune response represent a means of treating myeloma. Immunotherapy clearly represents a revolution in cancer care, and promising responses have been shown to various treatments, particularly immune checkpoint inhibitors, IMiDs, DCs and CAR T cells. However, not all patients are responsive to current immunotherapies, and among those patients who do respond, the effects are not always long-lasting. Thus, combination approaches are a cornerstone of cancer therapy for improving patient outcomes in MM. This study demonstrated that the combination of DC vaccination + pomalidomide with dexamethasone + PD-L1 blockade synergistically enhances myeloma immune responses to inhibit tumor growth, restores and enhances host immune effector cells, and reduces the generation of immune suppressor cells in MM. This study provides a framework for developing and understanding the role of immunotherapeutic modalities employing DCs, pomalidomide and PD-L1 blockade to inhibit tumor growth and restore immune function in myeloma-bearing mice. Figure Disclosures No relevant conflicts of interest to declare.

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.

Sensitization of immunotherapeutic efficiency in HCC by polyinosinic-polycytidylic acid-triggered CXCL10 secretion in liver sinusoidal endothelial cells.

126 Background: Nowadays, the efficiency of immune checkpoint blockades (ICBs) in the treatment of advanced hepatocellular carcinoma (HCC) was low. The previous study we published in Hepatology demonstrated that polyinosinic-polycytidylic acid (PolyIC), a TLR3 agonist, could trigger the accumulation, proliferation and activation of CTLs, which contributes to the enhanced anti-tumor efficiency of PD-L1 Ab in HCC. However, the mechanism remains unclear. Methods: The mouse HCC model was established by hydrodynamic transfection of combinational c-Myc/N-Ras oncogenes. PolyIC, CTLA-4 Ab, PD-L1 Ab, CD8 Ab and CXCR3 Ab were all intraperitoneal injected. Mice liver non-parenchymal cells (NPCs) were isolated after liver perfusion and multistep centrifugation. The proliferation, activation and cytotoxic function of CTLs were assessed by flow cytometry. Liver sinusoidal endothelial cells (LSECs) were isolated by magnetic cell sorting. Expressions of TLR3 and CXCL10 were quantitatively analyzed by qRT-PCR and ELISA. LSECs were cultured in vitro by using DMEM medium. Results: Combination of polyIC and PD-L1 Ab (or CTLA-4 Ab) had a synergistic anti-tumor effect on the treatment of mouse HCC. PolyIC induced the accumulation, proliferation, activation and cytotoxic function of CTLs in liver, which were further enhanced when combined ICBs. And CTLs depletion by CD8 Ab attenuated the synergistic anti-tumor effect of polyIC when combined with ICBs. TLR3 expression was much higher in NPCs than other cells in liver, especially in LSECs. Also, polyIC dramatically induced the expressions of CXCL10 in LSECs both in vivo and in vitro. And in vitro, after treated by polyIC, the CXCL10 expression in the medium and cells were all elevated. What’s more, CXCL10/CXCR3 signaling blockade by CXCR3 Ab attenuated the combinational effects of polyIC and ICBs. Meanwhile, after CXCL10/CXCR3 signaling blockade, the accumulation, proliferation and activation of CTLs were all inhibited. Conclusions: PolyIC specifically targets LSECs to induce CXCL10 expression and secretion, which contributes to the CTLs transformation and enhanced efficiency of ICBs in the treatment of HCC.

Rational Combination of Parvovirus H1 With CTLA-4 and PD-1 Checkpoint Inhibitors Dampens the Tumor Induced Immune Silencing.

The recent therapeutic success of immune checkpoint inhibitors in the treatment of advanced melanoma highlights the potential of cancer immunotherapy. Oncolytic virus-based therapies may further improve the outcome of these cancer patients. A human ex vivo melanoma model was used to investigate the oncolytic parvovirus H-1 (H-1PV) in combination with ipilimumab and/or nivolumab. The effect of this combination on activation of human T lymphocytes was demonstrated. Expression of CTLA-4, PD-1, and PD-L1 immune checkpoint proteins was upregulated in H-1PV-infected melanoma cells. Nevertheless, maturation of antigen presenting cells such as dendritic cells was triggered by H-1PV infected melanoma cells. Combining H-1PV with checkpoint inhibitors, ipilimumab enhanced TNFα release during maturation of dendritic cells; nivolumab increased the amount of IFNγ release. H-1PV mediated reduction of regulatory T cell activity was demonstrated by lower TGF-ß levels. The combination of ipilimumab and nivolumab resulted in a further decline of TGF-ß levels. Similar results were obtained regarding the activation of cytotoxic T cells. H-1PV infection alone and in combination with both checkpoint inhibitors caused strong activation of CTLs, which was reflected by an increased number of CD8+GranB+ cells and increased release of granzyme B, IFNγ, and TNFα. Our data support the concept of a treatment benefit from combining oncolytic H-1PV with the checkpoint inhibitors ipilimumab and nivolumab, with nivolumab inducing stronger effects on cytotoxic T cells, and ipilimumab strengthening T lymphocyte activity.

CTL-derived exosomes enhance the activation of CTLs stimulated by low-affinity peptides

Abstract Cytotoxic T cells (CTLs) of low TCR affinity play an important role in the control of intracellular pathogens and cancers; however, the mechanisms by which lower-affinity CTLs are activated and maintained are not well understood. We recently discovered that fully activated CTLs stimulated by strong-affinity peptides in the presence of IL-12 are able to secrete exosomes (Exo) that can stimulate bystander CTLs without requiring the presence of antigen. We hypothesized that the exosomes secreted by high-affinity CTLs could strengthen the activation of low-affinity CTLs. To test this hypothesis, naive OT-I CD8+ cells were stimulated with altered SIINFEKL peptides of different affinities in the presence or absence of Exo. The presence of Exo preferentially increased cell proliferation and enhanced the production of IFNγ in CTLs stimulated by low-affinity peptides. The expression of granzyme B (GZB) was augmented in all affinities, with higher GZB production in low-affinity stimulated CTLs than in high-affinity stimulated ones. Exosomes promoted the rapid activation of low-affinity CTLs, which remained responsive to exosomes for a prolonged duration. Unexpectedly, exosomes could be induced quickly (24 hours) following CTL activation and at a higher quantity per cell than later (72 hours). While exosome protein profiles vary significantly between early exosomes and their later-derived counterparts, both appear to have similar downstream functions. These results reveal a potential mechanism for fully activated CTLs in activating lower-affinity CTLs that may have important implications in boosting the function of low-affinity CTLs in immunotherapy for cancers and chronic viral infections.

BRAF Targeting Sensitizes Resistant Melanoma to Cytotoxic T Cells.

BRAF and MEK inhibitors (BRAFi and MEKi) are actively used for the treatment of metastatic melanoma in patients with BRAFV600E mutation in their tumors. However, the development of resistance to BRAFi and MEKi remains a difficult clinical challenge with limited therapeutic options available to these patients. In this study, we investigated the mechanism and potential therapeutic utility of combination BRAFi and adoptive T-cell therapy (ACT) in melanoma resistant to BRAFi. Investigations were performed in vitro and in vivo with various human melanoma cell lines sensitive and resistant to BRAFi as well as patient-derived xenografts (PDX) derived from patients. In addition, samples were evaluated from patients on a clinical trial of BRAFi in combination with ACT. Herein we report that in human melanoma cell lines, senstitive and resistant to BRAFi and in PDX from patients who progressed on BRAFi and MEKi therapy, BRAFi caused transient upregulation of mannose-6-phosphate receptor (M6PR). This sensitized tumor cells to CTLs via uptake of granzyme B, a main component of the cytotoxic activity of CTLs. Treatment of mice bearing resistant tumors with BRAFi enhanced the antitumor effect of patients' TILs. A pilot clinical trial of 16 patients with metastatic melanoma who were treated with the BRAFi vemurafenib followed by therapy with TILs demonstrated a significant increase of M6PR expression on tumors during vemurafenib treatment. BRAF-targeted therapy sensitized resistant melanoma cells to CTLs, which opens new therapeutic opportunities for the treatment of patients with BRAF-resistant disease.See related commentary by Goff and Rosenberg, p. 2682.

Abstract A79: Cancer immunotherapy with recombinant poliovirus induces IFN-dominant activation of antigen-presenting cells and tumor antigen-specific CTLs

Abstract The tumor microenvironment favors tumor immune escape by suppressing production, activation and/or function of antitumor T cells. Our group has developed a recombinant Rhino-Poliovirus chimera, PVSRIPO, currently being evaluated in Phase-II clinical trial against recurrent glioma. PVSRIPO therapy has afforded durable clinical responses in recurrent glioma patients. Clinical and pre-clinical findings suggest that PVSRIPO primarily acts through an immunological mechanism, rather than via cancer selective cytotoxicity. Here we define the immune adjuvant potential of intratumoral-delivered PVSRIPO that ultimately leads to the engagement/production of antitumor T cells. Sub-lethal infection of human dendritic cells (DCs) with PVSRIPO yields potent, sustained type I interferon dominant activation. PVSRIPO infection of macrophages induces similar activation; however, virus translation and the resulting type I Interferon/TNF-α production by macrophages is strongly enhanced in the presence of the Th2 cytokine IL-4. Compared to conventional adjuvants, e.g. poly(I:C) and LPS, PVSRIPO stimulation of APCs withstands tumor-associated immunosuppression, particularly in macrophages, and achieves more durable activation of DCs. Therapy of B16 melanoma tumors with PVSRIPO led to early production of type I interferon, IL-12, and IFN-γ; which was associated with the recruitment of neutrophils to the tumor site. Neutrophil influx was followed by DC and T cell infiltration. Mice treated with PVSRIPO developed tumor-antigen specific, cytotoxic T cells that were present in both tumor draining lymph nodes and spleens. These events correlated with delay in tumor growth and increase in survival following PVSRIPO therapy. Thus, PVSRIPO’s immune adjuvancy stimulates canonical innate inflammatory responses within the tumor microenvironment that culminates in tumor antigen-specific T cell responses. Citation Format: Michael C. Brown, Eda K. Holl, David Boczkowski, Elena Dobrikova, Mubeen Mosaheb, Vidya Chandramohan, Darell D. Bigner, Matthias Gromeier, Smita K. Nair. Cancer immunotherapy with recombinant poliovirus induces IFN-dominant activation of antigen-presenting cells and tumor antigen-specific CTLs [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A79.

Abstract 3758: TLR9 agonist SNA-induced innate and adaptive immune responses in tumor microenvironment enhance checkpoint inhibitor antitumor activity in mouse tumor models

Abstract Spherical Nucleic Acids (SNAs) are a novel class of therapeutic agents with oligonucleotides densely packed and radially oriented in 3D format around liposomal nanoparticles. As a result of the 3D-architecture, SNAs exhibit increased cellular uptake and nuclease stability compared with linear oligonucleotides. Checkpoint inhibitors (CPI) have shown antitumor efficacy in a variety of solid and liquid tumors. However, tumors with low immunogenicity and lacking pre-existing T lymphocyte infiltrates (TILs) in the tumor microenvironment (TME) are less responsive to CPI. TLR9 agonists induce potent innate and adaptive immune responses and have been shown to increase TIL and activate APC to secrete IFNs leading to activation and expansion of tumor-specific CTLs in TME increasing effectiveness of CPIs. In the present study, we have evaluated antitumor effects of SNA following intratumoral delivery in A20 lymphoma and CT26 colon cancer models. SNA was also studied in combination with anti-PD-1 or anti-PD-L1 in A20 and with IDO-1 inhibitor in CT26 models. We have also evaluated biomarkers in TME to elucidate the mechanism of SNA-mediated effects in enhancing anti-PD-1 efficacy. Monotherapy of SNA or oral dosing of IDO-1 inhibitor in CT26 tumor model resulted in a tumor growth inhibition (TGI) of up to 80% or 20%, respectively, compared with vehicle. The combination of SNA and IDO-1 inhibitor enhanced TGI up to 90% compared with either monotherapy. Mice bearing A20 tumors were treated with SNA, intraperitoneal dosing of anti-PD-1 or anti-PD-L1 or the combination of SNA/anti-PD-1 or SNA/anti-PD-L1. SNA alone showed potent dose-dependent TGI. Anti-PD-1 or anti-PD-L1 monotherapy resulted in modest TGI compared with vehicle. The combination of SNA with anti-PD-1 or anti-PD-L1 enhanced TGI compared with either monotherapy. FACS analysis of A20 tumor tissue following treatments revealed reduced T-regulatory cells and increased T-effector cells. Moreover, SNA treatment led to increased expression of IFN-inducible genes, ISG15, IRF7, MX1, and IP10 and a CD8 T cell marker CD8A in TME and maintained these gene expression levels when combined with anti-PD-1. These results establish the mode of action of TLR9 agonist SNA in TME in potentiating antitumor effects of anti-PD-1. These data demonstrate that the TLR9 agonist SNA showed potent antitumor activity as a result of increased IFN gene expression coupled with increased CD8+ T cells and decreased T-regulatory cells locally in the tumor and thereby enhancing efficacy of checkpoint inhibitors. A TLR9 agonist SNA, AST-008, is currently in clinical development. Citation Format: SubbaRao Nallagatla, Bart R. Anderson, Richard Kang, Ekambar R. Kandimalla. TLR9 agonist SNA-induced innate and adaptive immune responses in tumor microenvironment enhance checkpoint inhibitor antitumor activity in mouse tumor models [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 3758.

Using anti-CD38 immunotherapy to enhance anti-tumor T-cell immunity in chronic lymphocytic leukemia (CLL)

Abstract Increased CD38 expression on chronic lymphocytic leukemia (CLL) cells correlates with a negative prognosis, however, its utility as a therapeutic target in this malignancy remains unknown. Our investigations show that the anti-CD38 therapeutic mAb, daratumumab (Dara) induces direct apoptosis of CLL cells and promotes their immune-mediated clearance (through ADCC). Notably, CD38 expression is quite high on a subset of CLL-cells (CD19+/CD38hi/CD24+/IL-10+; B-regulatory-like phenotype), which promote Treg expansion while prohibiting CD8+ cytotoxic T-cells (cTLs). Indeed, immunosuppression is a hallmark of CLL and patients often present with increased number of Tregs and Th2 cells but decreased effector T cells and cTLs. It has recently been reported that CD38hi Tregs are significantly immunosuppressive and when examining this population in peripheral blood mononuclear cells (PBMCs) from CLL patients (n=13), we noted that >50% of CD4+/CD25+/FoxP3+ cells were CD38hi (median MFI ~600). We hypothesized that Dara would kill CD38hi CLL cells à decreased CD38hi Treg formation/direct killing à expansion of cTLs. Indeed, ex-vivo treatment of CLL-patient derived PBMCs showed that Dara was highly lethal to both CD38hi “Breg-like” CLL cells and Tregs; associated with decrease in IL-10, increase in IFN-γ, effector T cells, Th17 subsets and proliferation of cTLs. Altogether this pointed towards recovery of anti-tumor immunity which was further evidenced by enhanced tumor-specific cytolytic activity of cTLs. Our pre-clinical data demonstrates that targeting CD38 in CLL has an anti-tumor effect via 1.) lethal effect on CLL-Bregs and Tregs and 2.) tumor-specific expansion of cTLs, which we will test clinically in an upcoming trial.

Cancer immunotherapy with recombinant poliovirus induces IFN-dominant activation of dendritic cells and tumor antigen-specific CTLs.

Tumors thrive in an immunosuppressive microenvironment that impedes antitumor innate and adaptive immune responses. Thus, approaches that can overcome immunosuppression and engage antitumor immunity are needed. This study defines the adjuvant and cancer immunotherapy potential of the recombinant poliovirus/rhinovirus chimera PVSRIPO. PVSRIPO is currently in clinical trials against recurrent World Health Organization grade IV malignant glioma, a notoriously treatment-refractory cancer. Cytopathogenic infection of neoplastic cells releases the proteome and exposes pathogen- and damage-associated molecular patterns. At the same time, sublethal infection of antigen-presenting cells, such as dendritic cells and macrophages, yields potent, sustained type I interferon-dominant activation in an immunosuppressed microenvironment and promotes the development of tumor antigen-specific T cell responses in vitro and antitumor immunity in vivo. PVSRIPO's immune adjuvancy stimulates canonical innate anti-pathogen inflammatory responses within the tumor microenvironment that culminate in dendritic cell and T cell infiltration. Our findings provide mechanistic evidence that PVSRIPO functions as a potent intratumor immune adjuvant that generates tumor antigen-specific cytotoxic T lymphocyte responses.

Local Activation of p53 in the Tumor Microenvironment Overcomes Immune Suppression and Enhances Antitumor Immunity.

Mutations in tumor suppressor p53 remain a vital mechanism of tumor escape from apoptosis and senescence. Emerging evidence suggests that p53 dysfunction also fuels inflammation and supports tumor immune evasion, thereby serving as an immunological driver of tumorigenesis. Therefore, targeting p53 in the tumor microenvironment (TME) also represents an immunologically desirable strategy for reversing immunosuppression and enhancing antitumor immunity. Using a pharmacological p53 activator nutlin-3a, we show that local p53 activation in TME comprising overt tumor-infiltrating leukocytes (TILeus) induces systemic antitumor immunity and tumor regression, but not in TME with scarce TILeus, such as B16 melanoma. Maneuvers that recruit leukocytes to TME, such as TLR3 ligand in B16 tumors, greatly enhanced nutlin-induced antitumor immunity and tumor control. Mechanistically, nutlin-3a-induced antitumor immunity was contingent on two nonredundant but immunologically synergistic p53-dependent processes: reversal of immunosuppression in the TME and induction of tumor immunogenic cell death, leading to activation and expansion of polyfunctional CD8 CTLs and tumor regression. Our study demonstrates that unlike conventional tumoricidal therapies, which rely on effective p53 targeting in each tumor cell and often associate with systemic toxicity, this immune-based strategy requires only limited local p53 activation to alter the immune landscape of TME and subsequently amplify immune response to systemic antitumor immunity. Hence, targeting the p53 pathway in TME can be exploited to reverse immunosuppression and augment therapeutic benefits beyond tumoricidal effects to harness tumor-specific, durable, and systemic antitumor immunity with minimal toxicity. Cancer Res; 77(9); 2292-305. ©2017 AACR.

Hypoxia augments MHC class I antigen presentation via facilitation of ERO1-α-mediated oxidative folding in murine tumor cells.

To establish an effective cancer immunotherapy, it is crucial that cancer cells present a cancer-specific antigen in a hypoxic area, a hallmark of the tumor microenvironment. Here, we show the impact of hypoxia on MHC class I antigen presentation in vitro and in vivo in murine tumors. Activation of antigen-specific CTLs by tumor cells that had been pre-incubated under a condition of hypoxia was enhanced compared with that by tumor cells pre-incubated under a condition of normoxia. Cell surface expression of MHC class I-peptide complex on the tumor cells was increased under a condition of hypoxia, thereby leading to higher susceptibility to specific CTLs. We show that the hypoxia-inducible ER-resident oxidase ERO1-αplays an important role in the hypoxia-induced augmentation of MHC class I-peptide complex expression. ERO1-αfacilitated oxidative folding of MHC class I heavy chains, thereby resulting in the augmentation of cell surface expression of MHC class I-peptide complex under hypoxic conditions. These results suggest that since the expression of MHC class I-peptide complex is augmented in a hypoxic tumor microenvironment, strategies for inhibiting the function of regulatory T cells and myeloid-derived suppressor cells and/or immunotherapy with immune checkpoint inhibitors are promising for improving cancer immunotherapy.

Improvement of Peptide-Based Tumor Immunotherapy Using pH-Sensitive Fusogenic Polymer-Modified Liposomes.

To establish peptide vaccine-based cancer immunotherapy, we investigated the improvement of antigenic peptides by encapsulation with pH-sensitive fusogenic polymer-modified liposomes for induction of antigen-specific immunity. The liposomes were prepared by modification of egg yolk phosphatidylcholine and l-dioleoyl phosphatidylethanolamine with 3-methyl-glutarylated hyperbranched poly(glycidol) (MGlu-HPG) and were loaded with antigenic peptides derived from ovalbumin (OVA) OVA-I (SIINFEKL), and OVA-II (PSISQAVHAAHAEINEAPβA), which bind, respectively, to major histocompatibility complex (MHC) class I and class II molecules on dendritic cell (DCs). The peptide-loaded liposomes were taken up efficiently by DCs. The peptides were delivered into their cytosol. Administration of OVA-I-loaded MGlu-HPG-modified liposomes to mice bearing OVA-expressing E.G7-OVA tumors induced the activation of OVA-specific CTLs much more efficiently than the administration of free OVA-I peptide did. Mice strongly rejected E.G7-OVA cells after immunization with OVA-I peptide-loaded MGlu-HPG liposomes, although mice treated with free OVA-I peptide only slightly rejected the cells. Furthermore, efficient suppression of tumor volume was observed when tumor-bearing mice were immunized with OVA-I-peptide-loaded liposomes. Immunization with OVA-II-loaded MGlu-HPG-modified liposomes exhibited much lower tumor-suppressive effects. Results indicate that MGlu-HPG liposomes might be useful for improvement of CTL-inducing peptides for efficient cancer immunotherapy.

CCL21/IL21-armed oncolytic adenovirus enhances antitumor activity against TERT-positive tumor cells

Multigene-armed oncolytic adenoviruses are capable of efficiently generating a productive antitumor immune response. The chemokine (C-C motif) ligand 21 (CCL21) binds to CCR7 on naïve T cells and dendritic cells (DCs) to promote their chemoattraction to the tumor and resultant antitumor activity. Interleukin 21 (IL21) promotes survival of naïve T cells while maintaining their CCR7 surface expression, which increases their capacity to transmigrate in response to CCL21 chemoattraction. IL21 is also involved in NK cell differentiation and B cell activation and proliferation. The generation of effective antitumor immune responses is a complex process dependent upon coordinated interactions of various subsets of effector cells. Using the AdEasy system, we aimed to construct an oncolytic adenovirus co-expressing CCL21 and IL21 that could selectively replicate in TERTp-positive tumor cells (Ad-CCL21-IL21 virus). The E1A promoter of these oncolytic adenoviruses was replaced by telomerase reverse transcriptase promoter (TERTp). Ad-CCL21-IL21 was constructed from three plasmids, pGTE-IL21, pShuttle-CMV-CCL21 and AdEasy-1 and was homologously recombined and propagated in the Escherichia coli strain BJ5183 and the packaging cell line HEK-293, respectively. Our results showed that our targeted and armed oncolytic adenoviruses Ad-CCL21-IL21 can induce apoptosis in TERTp-positive tumor cells to give rise to viral propagation, in a dose-dependent manner. Importantly, we confirm that these modified oncolytic adenoviruses do not replicate efficiently in normal cells even under high viral loads. Additionally, we investigate the role of Ad-CCL21-IL21 in inducing antitumor activity and tumor specific cytotoxicity of CTLs in vitro. This study suggests that Ad-CCL21-IL21 is a promising targeted tumor-specific oncolytic adenovirus.