Search

Figure S1: Vaccination generates T cell immunity to FR following immunization.

Ligation of programmed cell death-1 (PD-1) in the tumor microenvironment is known to inhibit effective adaptive antitumor immunity. Blockade of PD-1 in humans has resulted in impressive, durable regression responses in select tumor types. However, durable responses have been elusive in ovarian cancer patients. PD-1 was recently shown to be expressed on and thereby impair the functions of tumor-infiltrating murine and human myeloid dendritic cells (TIDC) in ovarian cancer. In the present work, we characterize the regulation of PD-1 expression and the effects of PD-1 blockade on TIDC. Treatment of TIDC and bone marrow–derived dendritic cells (DC) with IL10 led to increased PD-1 expression. Both groups of DCs also responded to PD-1 blockade by increasing production of IL10. Similarly, treatment of ovarian tumor–bearing mice with PD-1 blocking antibody resulted in an increase in IL10 levels in both serum and ascites. While PD-1 blockade or IL10 neutralization as monotherapies were inefficient, combination of these two led to improved survival and delayed tumor growth; this was accompanied by augmented antitumor T- and B-cell responses and decreased infiltration of immunosuppressive MDSC. Taken together, our findings implicate compensatory release of IL10 as one of the adaptive resistance mechanisms that undermine the efficacy of anti–PD-1 (or anti–PD-L1) monotherapies and prompt further studies aimed at identifying such resistance mechanisms. Cancer Res; 77(23); 6667–78. ©2017 AACR.

Supplementary Figure S1 describes the pre-existing antibody responses to tumor antigens in patients with surgically resected HER2+ breast cancer. Supplementary Figure S2 compares the proportion of either the metastatic or adjuvant patients that respond to therapy with increased antibody responses. Table S1 shows the demographic information for the patients in this study. Table S2 compares the levels of antibodies to tumor antigens and TT in metastatic patients and healthy controls. Table S3 compares the mean pre- and post-treatment antibody levels to all of the tumor antigens and TT in the patients with metastatic disease. Table S4 describes correlations of the antibody responses to the type of chemotherapy received in the metastatic patients. Table S5 shows the results of a cox univariate analysis correlating PFS and antibody responses. Table S6 shows the results of a cox univariate analysis correlating OS and antibody responses. Table S7 compares the mean pre- and post-treatment antibody levels to all of the tumor antigens and TT in patients with surgically-resected disease.

Expression of PD-1 on BMDCs and T cells after exposure to IL-6 and IL-10

Purpose: Folate receptor alpha (FR) is overexpressed in several cancers. Endogenous immunity to the FR has been demonstrated in patients and suggests the feasibility of targeting FR with vaccine or other immune therapies. CD4 helper T cells are central to the development of coordinated immunity, and prior work shows their importance in protecting against relapse. Our previous identification of degenerate HLA-class II epitopes from human FR led to the development of a broad coverage epitope pool potentially useful in augmenting antigen-specific immune responses in most patients.Patients and Methods: We conducted a phase I clinical trial testing safety and immunogenicity of this vaccine, enrolling patients with ovarian cancer or breast cancer who completed conventional treatment and who showed no evidence of disease. Patients were initially treated with low-dose cyclophosphamide and then vaccinated 6 times, monthly. Immunity and safety were examined during the vaccine period and up to 1 year later.Results: Vaccination was well tolerated in all patients. Vaccine elicited or augmented immunity in more than 90% of patients examined. Unlike recall immunity to tetanus toxoid (TT), FR T-cell responses developed slowly over the course of vaccination with a median time to maximal immunity in 5 months. Despite slow development of immunity, responsiveness appeared to persist for at least 12 months.Conclusions: The results demonstrate that it is safe to augment immunity to the FR tumor antigen, and the developed vaccine is testable for therapeutic activity in most patients whose tumors express FR, regardless of HLA genotype. Clin Cancer Res; 24(13); 3014–25. ©2018 AACR.

The addition of trastuzumab to chemotherapy extends survival among patients with HER2+ breast cancer. Prior work showed that trastuzumab and chemotherapy augments HER2 extracellular domain (ECD)-specific antibodies. The current study investigated whether combination therapy induced immune responses beyond HER2-ECD and, importantly, whether those immune responses were associated with survival. Pretreatment and posttreatment sera were obtained from 48 women with metastatic HER2+ breast cancer on NCCTG (now Alliance for Clinical Trials in Oncology) studies, N0337 and N983252. IgG to HER2 intracellular domain (ICD), HER2-ECD, p53, IGFBP2, CEA, and tetanus toxoid were examined. Sera from 25 age-matched controls and 26 surgically resected HER2+ patients were also examined. Prior to therapy, some patients with metastatic disease had elevated antibodies to IGFBP2, p53, HER2-ICD, HER2-ECD, and CEA, but not to tetanus toxin, relative to controls and surgically resected patients. Treatment augmented antibody responses to HER2-ICD in 69% of metastatic patients, which was highly associated with improved progression-free survival (PFS; HR = 0.5, P = 0.0042) and overall survival (OS; HR = 0.7, P = 0.038). Augmented antibody responses to HER2-ICD also correlated (P = 0.03) with increased antibody responses to CEA, IGFBP2, and p53, indicating that treatment induces epitope spreading. Paradoxically, patients who already had high preexisting immunity to HER2-ICD did not respond to therapy with increased antibodies to HER2-ICD and demonstrated poorer PFS (HR = 1.6, P < 0.0001) and OS (HR = 1.4, P = 0.0006). Overall, the findings further demonstrate the importance of the adaptive immune system in the efficacy of trastuzumab-containing regimens. Cancer Res; 76(13); 3702–10. ©2016 AACR.

PDF file - 418KB, Supplementary Methods and Results Fig. S1. In vitro blockade of PD-1 on TUBO cells does not inhibit cell growth. Fig. S2. CD4 T cells and CD4 Tregs in TUBO TILs. Fig. S3. Combination therapy enhances the antigen-specific function of tumor-infiltrating lymphocytes. Fig. S4. Antigen-specific production IL-5 and IL-4 by tumor-infiltrating CD8 T cells. Fig. S5. Combination therapy decreased the number of tumor-infiltrating myeloid derived suppressor cells (MDSCs). Fig S6. PD-1 expression on tumor DCs and splenic CD8 T cells in co-culture.

Lung adenocarcinoma (LUAD) is the most prevalent form of non–small cell lung cancer (NSCLC) and a leading cause of cancer death. Immune checkpoint inhibitors (ICIs) of programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) signaling induce tumor regressions in a subset of LUAD, but many LUAD tumors exhibit resistance to ICI therapy. Here, we identified Prkci as a major determinant of response to ICI in a syngeneic mouse model of oncogenic mutant Kras / Trp53 loss (KP)–driven LUAD. Protein kinase Cι (PKCι)–dependent KP tumors exhibited resistance to anti–PD-1 antibody therapy (α-PD-1), whereas KP tumors in which Prkci was genetically deleted (KPI tumors) were highly responsive. Prkci- dependent resistance to α-PD-1 was characterized by enhanced infiltration of myeloid-derived suppressor cells (MDSCs) and decreased infiltration of CD8 + T cells in response to α-PD-1. Mechanistically, Prkci regulated YAP1-dependent expression of Cxcl5 , which served to attract MDSCs to KP tumors. The PKCι inhibitor auranofin inhibited KP tumor growth and sensitized these tumors to α-PD-1, whereas expression of either Prkci or its downstream effector Cxcl5 in KPI tumors induced intratumoral infiltration of MDSCs and resistance to α-PD-1. PRKCI expression in tumors of patients with LUAD correlated with genomic signatures indicative of high YAP1-mediated transcription, elevated MDSC infiltration and low CD8 + T cell infiltration, and with elevated CXCL5 / 6 expression. Last, PKCι-YAP1 signaling was a biomarker associated with poor response to ICI in patients with LUAD. Our data indicate that immunosuppressive PKCι-YAP1-CXCL5 signaling is a key determinant of response to ICI, and pharmacologic inhibition of PKCι may improve therapeutic response to ICI in patients with LUAD.

Purpose: Folate receptor alpha (FR) is overexpressed in several cancers. Endogenous immunity to the FR has been demonstrated in patients and suggests the feasibility of targeting FR with vaccine or other immune therapies. CD4 helper T cells are central to the development of coordinated immunity, and prior work shows their importance in protecting against relapse. Our previous identification of degenerate HLA-class II epitopes from human FR led to the development of a broad coverage epitope pool potentially useful in augmenting antigen-specific immune responses in most patients. Patients and Methods: We conducted a phase I clinical trial testing safety and immunogenicity of this vaccine, enrolling patients with ovarian cancer or breast cancer who completed conventional treatment and who showed no evidence of disease. Patients were initially treated with low-dose cyclophosphamide and then vaccinated 6 times, monthly. Immunity and safety were examined during the vaccine period and up to 1 year later. Results: Vaccination was well tolerated in all patients. Vaccine elicited or augmented immunity in more than 90% of patients examined. Unlike recall immunity to tetanus toxoid (TT), FR T-cell responses developed slowly over the course of vaccination with a median time to maximal immunity in 5 months. Despite slow development of immunity, responsiveness appeared to persist for at least 12 months. Conclusions: The results demonstrate that it is safe to augment immunity to the FR tumor antigen, and the developed vaccine is testable for therapeutic activity in most patients whose tumors express FR, regardless of HLA genotype. Clin Cancer Res; 24(13); 3014–25. ©2018 AACR.

The increase in fatty acid metabolism is a hallmark of cancer. Stearoyl CoA desaturase (SCD) is an endoplasmic reticulum (ER) enzyme that catalyzes D9 cis désaturation of fatty acyl-CoA substrates. One of its isoforms, SCD1, is overexpressed in several cancers including breast cancer. The higher expression of SCD1 in breast cancer patients correlates with significantly shorter relapse-free and overall survival rates, indicating that SCD1 may be a therapeutic target. We have previously developed and published a novel SCD1 inhibitor, SSI4. Metabolic reprogramming facilitates tumor growth by promoting immune suppression in tumors. Fundamental metabolic processes like fatty acid (FA) metabolism are involved in T cell activation and differentiation and their modulation can differentially affect the development of T helper cell lineages. FA synthesis and oxidation are essential for the development and functioning of CD8+ T memory cells and are contributing factors that influence CD4+ T effector and Treg cell development. These associations between metabolism and immune cells lead us to our hypothesis that aberrant de novo lipogenesis is linked to immune suppression. Thus, SCD1 inhibitors should increase anti-tumor immunity. We evaluated the effect of SSI4-mediated SCD1 inhibition in two mouse triple negative breast cancer cell lines. The data showed that SSI4 stimulates calreticulin (CRT) translocation to cell membrane. CRT is an ER resident protein that translocates to the cell surface under ER stress conditions. Cell surface expression of CRT is known to induce immune responses including phagocytosis, translocation of cancer antigen to the cell surface, and apoptosis. We are currently studying the effect of CRT translocation in our model. A major focus of our project is to assess the efficacy of combination treatment of SSI4 and an immune checkpoint blockade. To this end, this combination therapy synergistically blocked E0771 tumor growth in vivo. Further, to understand the immune responses generated, we performed immunohistochemical analysis of the tumors (day 20 post-treatment) indicating that the combination therapy resulted in higher membrane localized CRT, supporting our rationale that CRT translocation is involved in mediating the anti-tumor responses. Flow cytometry analysis indicated that the combination treatment increased immune cell infiltration. Specifically, SSI4 treatment induced infiltration of double negative T cells, natural killer cells and macrophages, suggesting that it promoted a pro-inflammatory immune response. SSI4 treatment down regulated immune suppressive cells including Tregs, dendritic cells and mesenchymal derived stem cells. The response of cancers with low and intermediate tumor mutational burden such as breast cancer to immune checkpoint inhibition is limited. Our data indicates that combination treatment of SSI4 and anti PD1 is a promising therapeutic strategy. Citation Format: Sneha Vivekanandhan, Justyna Trynda, Laura A. Marlow, Barath shreeder, James L. Miller, Adam M. Kase, Winston Tan, Keith L. Knutson, John A. Copland. SCD-1 blockade sensitizes triple negative breast cancer to immune checkpoint inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1484.

Immunosuppression in the tumor microenvironment blunts vaccine-induced immune effectors. PD-1/B7-H1 is an important inhibitory axis in the tumor microenvironment. Our goal in this study was to determine the effect of blocking this inhibitory axis during and following vaccination against breast cancer. We observed that using anti-PD-1 antibody and a multipeptide vaccine (consisting of immunogenic peptides derived from breast cancer antigens, neu, legumain, and β-catenin) as a combination therapy regimen for the treatment of breast cancer–bearing mice prolonged the vaccine-induced progression-free survival period. This prolonged survival was associated with increase in number of Tc1 and Tc2 CD8 T cells with memory precursor phenotype, CD27+IL-7RhiT-betlo, and decrease in number of PD-1+ dendritic cells (DC) in regressing tumors and enhanced antigen reactivity of tumor-infiltrating CD8 T cells. It was also observed that blockade of PD-1 on tumor DCs enhanced IL-7R expression on CD8 T cells. Taken together, our results suggest that PD-1 blockade enhances breast cancer vaccine efficacy by altering both CD8 T cell and DC components of the tumor microenvironment. Given the recent success of anti-PD-1 monotherapy, our results are encouraging for developing combination therapies for the treatment of patients with cancer in which anti-PD-1 monotherapy alone may be ineffective (i.e., PD-L1–negative tumors). Cancer Res; 74(11); 2974–85. ©2014 AACR.

Ligation of programmed cell death-1 (PD-1) in the tumor microenvironment is known to inhibit effective adaptive antitumor immunity. Blockade of PD-1 in humans has resulted in impressive, durable regression responses in select tumor types. However, durable responses have been elusive in ovarian cancer patients. PD-1 was recently shown to be expressed on and thereby impair the functions of tumor-infiltrating murine and human myeloid dendritic cells (TIDC) in ovarian cancer. In the present work, we characterize the regulation of PD-1 expression and the effects of PD-1 blockade on TIDC. Treatment of TIDC and bone marrow–derived dendritic cells (DC) with IL10 led to increased PD-1 expression. Both groups of DCs also responded to PD-1 blockade by increasing production of IL10. Similarly, treatment of ovarian tumor–bearing mice with PD-1 blocking antibody resulted in an increase in IL10 levels in both serum and ascites. While PD-1 blockade or IL10 neutralization as monotherapies were inefficient, combination of these two led to improved survival and delayed tumor growth; this was accompanied by augmented antitumor T- and B-cell responses and decreased infiltration of immunosuppressive MDSC. Taken together, our findings implicate compensatory release of IL10 as one of the adaptive resistance mechanisms that undermine the efficacy of anti–PD-1 (or anti–PD-L1) monotherapies and prompt further studies aimed at identifying such resistance mechanisms. Cancer Res; 77(23); 6667–78. ©2017 AACR.

The addition of trastuzumab to chemotherapy extends survival among patients with HER2+ breast cancer. Prior work showed that trastuzumab and chemotherapy augments HER2 extracellular domain (ECD)-specific antibodies. The current study investigated whether combination therapy induced immune responses beyond HER2-ECD and, importantly, whether those immune responses were associated with survival. Pretreatment and posttreatment sera were obtained from 48 women with metastatic HER2+ breast cancer on NCCTG (now Alliance for Clinical Trials in Oncology) studies, N0337 and N983252. IgG to HER2 intracellular domain (ICD), HER2-ECD, p53, IGFBP2, CEA, and tetanus toxoid were examined. Sera from 25 age-matched controls and 26 surgically resected HER2+ patients were also examined. Prior to therapy, some patients with metastatic disease had elevated antibodies to IGFBP2, p53, HER2-ICD, HER2-ECD, and CEA, but not to tetanus toxin, relative to controls and surgically resected patients. Treatment augmented antibody responses to HER2-ICD in 69% of metastatic patients, which was highly associated with improved progression-free survival (PFS; HR = 0.5, P = 0.0042) and overall survival (OS; HR = 0.7, P = 0.038). Augmented antibody responses to HER2-ICD also correlated (P = 0.03) with increased antibody responses to CEA, IGFBP2, and p53, indicating that treatment induces epitope spreading. Paradoxically, patients who already had high preexisting immunity to HER2-ICD did not respond to therapy with increased antibodies to HER2-ICD and demonstrated poorer PFS (HR = 1.6, P < 0.0001) and OS (HR = 1.4, P = 0.0006). Overall, the findings further demonstrate the importance of the adaptive immune system in the efficacy of trastuzumab-containing regimens. Cancer Res; 76(13); 3702–10. ©2016 AACR.

PD-1 and PD-L1 form a major inhibitory axis that acts to suppress tumor-rejecting effector responses. In addition to its expression and inhibitory functions in T and B cells, PD-1 has also been shown to be expressed on and hence impact the functions of cells of innate arm, such as dendritic cells (DCs), primarily at the tumor site but not in the periphery. Our aim in this study was to identify tumor-associated factor(s) responsible for PD-1 regulation on DCs and investigate how DCs in the tumor microenvironment respond to anti-PD-1-based immunotherapy. Both bone marrow-derived dendritic cells (BMDCs) as well as murine ovarian tumor ascites-derived DCs were used in the studies. Using in vitro cultures, we show that the cytokine IL-10; which is expressed in abundance in many malignancies including ovarian cancer, is potent regulator of PD-1 expression on DCs. Based on inhibition and siRNA knockdown studies; we show that IL-10 mediated PD-1 expression depends on STAT3 activation. Treatment of DCs with IL-10 also led to an increase in expression of PD-L1 on surface of DCs as well as an increase in release of soluble PD-L1. Antibody-mediated blockade of PD-1 on DCs led to greatly increased production of IL-10 (> 4 fold) in cultures in vitro. Furthermore, treatment of tumor bearing mice with anti-PD-1 antibody led to a significant increase of IL-10 in ascites (> 6 fold) and periphery (> 4 fold). This compensatory release of IL-10 correlated with a further increase in PD-1 expression on DCs both in vitro and in vivo. In in vivo studies, while the blockade of PD-1 or IL-10 neutralization as monotherapy were ineffective, blockade of PD-1 and IL-10 neutralization as combination therapy augmented the anti-tumor response in ovarian tumor bearing mice; leading to a decrease in tumor growth and a significant increase in survival. These results show that the IL-10 and PD-1 pathways intersect with DCs in the tumor microenvironment. PD-1 blockade on DCs leads to compensatory release of IL-10 resulting in the maintenance of immunosuppression, and a combination regimen of PD-1 blockade and IL-10 neutralization has therapeutic benefit in ovarian tumors. These results serve as catalyst to explore the feasibility/efficacy of exploiting these targets in clinical settings for treatment of human ovarian cancer. Citation Format: Purushottam Lamichhane, Lavakumar Karyampudi, Barath Shreeder, James Krempski, Deborah Bahr, Matthew Block, Keith Knutson. Blockade of PD-1 signaling in tumor-associated dendritic cells results in compensatory IL-10 release maintaining immune suppression in ovarian cancer microenvironments. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B66.

e14028 Background: Folate receptor alpha (FRα) is overexpressed by multiple cancers, including breast and ovarian cancers. Endogenous T-cell immunity to each of five degenerate peptides from FRα (F...

587 Background: The addition of trastuzumab to chemotherapy improves response to therapy and extends survival among patients with HER2-positive (HER2+) breast cancer. Prior work showed that trastuz...

PD-1: PD-L1 is a major inhibitory axis that acts to suppress tumor-rejecting effector responses. In addition to its expression and inhibitory functions in T and B cells; PD-1 has also been shown to be expressed on and thereby impact the functions of cells of innate arm such as dendritic cells (DCs) primarily at the tumor site but not in the periphery. We show here that one of the culprits in the tumor microenvironment responsible for enhancing PD-1 expression on the tumor-associated DCs is the cytokine IL-10; which is expressed in abundance in many malignancies including ovarian cancer. Based on inhibition and knockdown studies, we show that IL-10 mediated PD-1 expression depends on STAT3 activation. Using murine model of ovarian carcinoma (ID8), we show that tumor-associated DCs express not only high levels of PD-1 on the surface but also have increased expression of STAT-3 and p-STAT3 compared to splenic DCs. Blockade of PD-1 on DCs led to increased production of IL-10 which correlated with a further increase in PD-1 expression. In in vivo studies, blockade of PD-1 and IL-10 neutralization as combination therapy augmented the anti-tumor response in ID8 ovarian tumor bearing mice; leading to a decrease in tumor mass and a significant increase in survival. These results show that IL-10 and PD-1 pathway intersect in DCs in theTME hence making them attractive targets for therapy of ovarian cancer.