Factors impacting the efficacy of the in-situ vaccine with CpG and OX40 agonist.

BACKGROUND: The in-situ vaccine using CpG oligodeoxynucleotide combined with OX40 agonist antibody (CpG + OX40) has been shown to be an effective therapy activating an anti-tumor T cell response in certain settings. The roles of tumor volume, tumor model, and the addition of checkpoint blockade in the efficacy of CpG + OX40 in-situ vaccination remains unknown. METHODS: Mice bearing flank tumors (B78 melanoma or A20 lymphoma) were treated with combinations of CpG, OX40, and anti-CTLA-4. Tumor growth and survival were monitored. In vivo T cell depletion, tumor cell phenotype, and tumor infiltrating lymphocyte (TIL) studies were performed. Tumor cell sensitivity to CpG and macrophages were evaluated in vitro. RESULTS: As tumor volumes increased in the B78 (one-tumor) and A20 (one-tumor or two-tumor) models, the anti-tumor efficacy of the in-situ vaccine decreased. In vitro, CpG had a direct effect on A20 proliferation and phenotype and an indirect effect on B78 proliferation via macrophage activation. As A20 tumors progressed in vivo, tumor cell phenotype changed, and T cells became more involved in the local CpG + OX40 mediated anti-tumor response. In mice with larger tumors that were poorly responsive to CpG + OX40, the addition of anti-CTLA-4 enhanced the anti-tumor efficacy in the A20 but not B78 models. CONCLUSIONS: Increased tumor volume negatively impacts the anti-tumor capability of CpG + OX40 in-situ vaccine. The addition of checkpoint blockade augmented the efficacy of CpG + OX40 in the A20 but not B78 model. These results highlight the importance of considering multiple preclinical model conditions when assessing the efficacy of cancer immunotherapy regimens and their translation to clinical testing.

Radiation Augments the Local Anti-Tumor Effect of In Situ Vaccine With CpG-Oligodeoxynucleotides and Anti-OX40 in Immunologically Cold Tumor Models.

Introduction: Combining CpG oligodeoxynucleotides with anti-OX40 agonist antibody (CpG+OX40) is able to generate an effective in situ vaccine in some tumor models, including the A20 lymphoma model. Immunologically "cold" tumors, which are typically less responsive to immunotherapy, are characterized by few tumor infiltrating lymphocytes (TILs), low mutation burden, and limited neoantigen expression. Radiation therapy (RT) can change the tumor microenvironment (TME) of an immunologically "cold" tumor. This study investigated the effect of combining RT with the in situ vaccine CpG+OX40 in immunologically "cold" tumor models. Methods: Mice bearing flank tumors (A20 lymphoma, B78 melanoma or 4T1 breast cancer) were treated with combinations of local RT, CpG, and/or OX40, and response to treatment was monitored. Flow cytometry and quantitative polymerase chain reaction (qPCR) experiments were conducted to study differences in the TME, secondary lymphoid organs, and immune activation after treatment. Results: An in situ vaccine regimen of CpG+OX40, which was effective in the A20 model, did not significantly improve tumor response or survival in the "cold" B78 and 4T1 models, as tested here. In both models, treatment with RT prior to CpG+OX40 enabled a local response to this in situ vaccine, significantly improving the anti-tumor response and survival compared to RT alone or CpG+OX40 alone. RT increased OX40 expression on tumor infiltrating CD4+ non-regulatory T cells. RT+CpG+OX40 increased the ratio of tumor-infiltrating effector T cells to T regulatory cells and significantly increased CD4+ and CD8+ T cell activation in the tumor draining lymph node (TDLN) and spleen. Conclusion: RT significantly improves the local anti-tumor effect of the in situ vaccine CpG+OX40 in immunologically "cold", solid, murine tumor models where RT or CpG+OX40 alone fail to stimulate tumor regression.

Identification of unique clusters of T, dendritic, and innate lymphoid cells in the peritoneal fluid of ovarian cancer patients.

PROBLEM: We hypothesize that activated peritoneal immune cells can be redirected to target ovarian tumors. Here, we obtain fundamental knowledge of the peritoneal immune environment through deep immunophenotyping of T cells, dendritic cells (DC), and innate lymphoid cells (ILC) of ovarian cancer patients. METHOD OF STUDY: T cells, DC, and ILC from ascites of ovarian cancer patients (n = 15) and peripheral blood of post-menopausal healthy donors (n = 6) were immunophenotyped on a BD Fortessa cytometer using three panels-each composed of 16 antibodies. The data were analyzed manually and by t-SNE/DensVM. CA125 levels were obtained from patient charts. RESULTS: We observed decreased CD3+ T cells and a higher proportion of activated CD4+ and effector memory CD4+ /CD8+ T cells, plasmacytoid DC, CD1c+ and CD141+ myeloid DC and CD56Hi NK cells in ascites. t-SNE/DensVM identified eight T cell, 17 DC, and 17 ILC clusters that were unique in the ascites compared to controls. Hierarchical clustering of cell frequency distinctly segregated the T-cell and ILC clusters from controls. Increased CA125 levels were associated with decreased CD8+ /CD45RA+ /CD45RO- /CCR7- T cells. CONCLUSION: The identified immune clusters serve as the basis for interrogation of the peritoneal immune environment and the development of novel immunologic modalities against ovarian cancer.

Mass Spectrometry Imaging of N-Glycans from Formalin-Fixed Paraffin-Embedded Tissue Sections Using a Novel Subatmospheric Pressure Ionization Source.

N-linked glycosylation, featuring various glycoforms, is one of the most common and complex protein post-translational modifications (PTMs) controlling protein structures and biological functions. It has been revealed that abnormal changes of protein N-glycosylation patterns are associated with many diseases. Hence, unraveling the disease-related alteration of glycosylation, especially the glycoforms, is crucial and beneficial to improving our understanding about the pathogenic mechanisms of various diseases. In past decades, given the capability of in situ mapping of biomolecules and their region-specific localizations, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been widely applied to the discovery of potential biomarkers for many diseases. In this study, we coupled a novel subatmospheric pressure (SubAP)/MALDI source with a Q Exactive HF hybrid quadrupole-orbitrap mass spectrometer for in situ imaging of N-linked glycans from formalin-fixed paraffin-embedded (FFPE) tissue sections. The utility of this new platform for N-glycan imaging analysis was demonstrated with a variety of FFPE tissue sections. A total of 55 N-glycans were successfully characterized and visualized from a FFPE mouse brain section. Furthermore, 29 N-glycans with different spatial distribution patterns could be identified from a FFPE mouse ovarian cancer tissue section. High-mannose N-glycans exhibited elevated expression levels in the tumor region, indicating the potential association of this type of N-glycans with tumor progression.

Synergy of anti-CD40, CpG and MPL in activation of mouse macrophages.

Activation of macrophages is a prerequisite for their antitumor effects. Several reagents, including agonistic anti-CD40 monoclonal antibody (anti-CD40), CpG oligodeoxynucleotides (CpG) and monophosphoryl lipid A (MPL), can stimulate activation of macrophages. Our previous studies showed synergy between anti-CD40 and CpG and between anti-CD40 and MPL in macrophage activation and antitumor efficacy in mice. In the present study, we asked whether there was synergy among these three reagents. The activation of adherent peritoneal exudate cells (PEC) obtained from mice injected with anti-CD40 and then treated with CpG and/or MPL in vitro was determined by their ability to suppress proliferation of tumor cells and to produce various cytokines and chemokines in vitro. Cell sorting and histology followed by functional testing showed that macrophages were the main cell population in PEC activated by CD40 ligation in vivo. A combination of anti-CD40, CpG or MPL activated PEC to suppress proliferation of B16 cells and produce nitric oxide far greater than the single reagents or any of the double combinations of these reagents. In addition, the combination of all three reagents activated PEC to secrete IL-12, IFN-γ and MCP-1 to a greater degree than any single reagent or any two combined reagents. These results demonstrate that macrophages can be synergistically activated by anti-CD40, CpG and MPL, suggesting that this novel combined approach might be further investigated as potential cancer therapy.

Antitumor effects of anti-CD40/CpG immunotherapy combined with gemcitabine or 5-fluorouracil chemotherapy in the B16 melanoma model.

Our previous studies demonstrated that anti-CD40 mAb (anti-CD40) can synergize with CpG oligodeoxynucleotides (CpG) to mediate antitumor effects by activating myeloid cells, such as macrophages in tumor-bearing mice. Separate teams have shown that chemotherapy with gemcitabine (GEM) or 5-fluorouracil (5-FU) can reduce tumor-induced myeloid-derived suppressor cells (MDSC) in mice. In this study we asked if the same chemotherapy regimens with GEM or 5-FU will enhance the antitumor effect of anti-CD40 and CpG. Using the model of B16 melanoma growing intraperitoneally in syngeneic C57BL/6 mice, we show that these GEM or 5-FU treatment regimens reduced MDSC in the peritoneal cavity of tumor-bearing mice. Treatment of mice with GEM or 5-FU did not significantly affect the antitumor function of macrophages as assessed in vitro. In vivo, treatment with these GEM or 5-FU regimens followed by anti-CD40/CpG resulted in antitumor effects similar to those of anti-CD40/CpG in the absence of GEM or 5-FU. Likewise, reduction of MDSC by in vivo anti-Gr-1 mAb treatment did not significantly affect anti-CD40/CpG antitumor responses. Together, the results show that the GEM or 5-FU chemotherapy regimens did not substantially affect the antitumor effects induced by anti-CD40/CpG immunotherapy.

Intratumoral delivery of low doses of anti-CD40 mAb combined with monophosphoryl lipid a induces local and systemic antitumor effects in immunocompetent and T cell-deficient mice.

In this study, an agonistic anti-CD40 monoclonal antibody was combined with monophosphoryl lipid A (MPL), a nontoxic derivative of lipopolysaccharide and agonist of toll-like receptor-4, to assess the immunomodulatory and antitumor synergy between the 2 agents in mice. Anti-CD40 was capable of priming macrophages to subsequent ex vivo activation by MPL in immunocompetent and T-cell-depleted mice. Intraperitoneal injections of anti-CD40+MPL induced additive to synergistic suppression of poorly immunogenic B16-F10 melanoma growing subcutaneously in syngeneic mice. When anti-CD40+MPL were injected directly into the subcutaneous tumor, the combination treatment was more effective, even with a 25-fold reduction in dose. Low-dose intratumoral treatment also slowed the growth of a secondary tumor growing simultaneously at a distant, untreated site. Antitumor effects were also induced in severe combined immunodeficiency mice and in T-cell-depleted C57BL/6 mice. Taken together, our results show that the antitumor effects of anti-CD40 are enhanced by subsequent treatment with MPL, even in T-cell-deficient hosts. These preclinical data suggest that an anti-CD40+MPL combined regimen is appropriate for clinical testing in human patients, including cancer patients who may be immunosuppressed from prior chemotherapy.

Uterine leiomyosarcoma diffusely express disialoganglioside GD2 and bind the therapeutic immunocytokine 14.18-IL2: implications for immunotherapy.

Uterine leiomyosarcoma comprises <1 % of uterine malignancies and is known for its clinically aggressive course. Extrapelvic recurrences are common and often lethal. No adjuvant therapies have been shown to significantly improve overall survival, highlighting the need for new and novel therapies. Our objective was to determine whether GD2-specific immunocytokine therapy may be explored for the treatment for uterine leiomyosarcoma. To do so, frozen tissue sections were obtained from the Gynecologic Oncology Group tumor bank and evaluated by immunohistochemistry (IHC) for GD2 expression using both the parent mouse monoclonal antibody 14G2A and immunocytokine 14.18-IL2 generated from the 14G2A sequence. Immunoreactivity was detected by avidin-biotin complex with DAB substrate. Specimens were reviewed by a pathologist with light microscopy and classified as negative, 1+, 2+ or 3+, compared to human melanoma cells as positive control and tissue incubated in the absence of primary antibody as negative control. GD2 was diffusely present in all evaluable samples. 10 tumors (67 %) demonstrated 3+ IHC intensity for GD2, two tumors (13 %) demonstrated 2+ intensity, and 3 (20 %) tumors demonstrated 1+ intensity. Eleven cases had sufficient tissue to assess 14.18-IL2 binding. All 11 cases bound 14.18-IL2 in a pattern identical to the parent antibody. Uterine leiomyosarcoma diffusely express GD2 and bind the therapeutic immunocytokine 14.18-IL2. This warrants further exploration to determine whether immunocytokine therapy may have a clinical role in the management of these aggressive tumors.

Tumor-associated myeloid cells can be activated in vitro and in vivo to mediate antitumor effects.

Tumor growth is often accompanied by the accumulation of myeloid cells in the tumors and lymphoid organs. These cells can suppress T cell immunity, thereby posing an obstacle to T cell-targeted cancer immunotherapy. In this study, we tested the possibility of activating tumor-associated myeloid cells to mediate antitumor effects. Using the peritoneal model of B16 melanoma, we show that peritoneal cells (PEC) in tumor-bearing mice (TBM) had reduced ability to secrete nitric oxide (NO) following in vitro stimulation with interferon gamma and lipopolysaccharide, as compared to PEC from control mice. This reduced function of PEC was accompanied by the influx of CD11b(+) Gr-1(+) myeloid cells to the peritoneal cavity. Nonadherent PEC were responsible for most of the NO production in TBM, whereas in naïve mice NO was mainly secreted by adherent CD11b(+) F4/80(+) macrophages. Sorted CD11b(+) Gr-1(-) monocytic and CD11b(+) Gr-1(+) granulocytic PEC from TBM had a reduced ability to secrete NO following in vitro stimulation (compared to naïve PEC), but effectively suppressed proliferation of tumor cells in vitro. In vivo, treatment of mice bearing established peritoneal B16 tumors with anti-CD40 and CpG resulted in activation of tumor-associated PEC, reduction in local tumor burden and prolongation of mouse survival. Inhibition of NO did not abrogate the antitumor effects of stimulated myeloid cells. Taken together, the results indicate that in tumor-bearing hosts, tumor-associated myeloid cells can be activated to mediate antitumor effects.

Enhanced T-cell-independent antitumor effect of cyclophosphamide combined with anti-CD40 mAb and CpG in mice.

We have earlier demonstrated T-cell-independent antitumor effects of a combination of anti-CD40 monoclonal antibody (mAb) and CpG oligodeoxynucleotides (CpG) which involved macrophages. As some immunotherapeutic treatments can be potentiated by chemotherapy, we tested if cyclophosphamide (CY) would enhance the antitumor effect of anti-CD40 mAb+CpG. Treatment of B16 melanoma-bearing mice with CY and anti-CD40 mAb+CpG resulted in a significant reduction in tumor growth in immunocompetent mice compared with either CY alone or anti-CD40 mAb with CpG. This enhanced antitumor effect was maintained in severe combined immunodeficiency mice, as measured by both tumor growth and overall survival. Natural killer cells were not required for this antitumor effect as it was also observed in severe combined immunodeficiency/beige mice. Moreover, although CY treatment of immunocompetent mice suppressed natural killer cell activity, it did not negatively affect the antitumor activity of their macrophages when assayed in vitro. Depletion of macrophages in vivo reduced the antitumor effect of CY and anti-CD40 mAb+CpG. These results suggest that therapeutic strategies to activate macrophages may have potential for clinical application in cancer patients receiving chemotherapy.