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Despite reports of striking outcomes, immunotherapy efficacy in melanoma is limited to subsets of patients 1, 2. Combining immunotherapies with other modalities has yielded limited improvements but also adverse events requiring cessation of treatment 1. In addition to ineffective patient stratification, efficacy can be impaired by paucity of tumor-infiltrating lymphocytes (TILs). Thus, effective strategies to safely increase TILs are urgently needed to improve immunotherapies 3. Here, we report that dietary administration of the sugar L-fucose triggers CD4+T cell-mediated increases in TILs, anti-tumor immunity, and enhanced immune checkpoint blockade responses. This is induced by the fucosylation and cell surface enrichment of the MHC-II protein HLA-DRB1 in melanoma. Single-cell immunofluorescent staining analysis of patient melanoma specimens demonstrates that fucosylation and fucosylated HLA-DRB1 is associated with intratumoral T cell abundance and anti-PD1 responder status. Our findings demonstrate that fucosylation is a key mediator of anti-tumor immunity, via regulation of melanoma cell surface HLA-DRB1 and induction of anti-tumor immunity, suggesting use of melanoma fucosylation as a novel strategy to stratify patients for immunotherapies. Importantly, our study suggests that L-fucose represents a powerful, non-toxic agent for safely increasing anti-tumor immunity and immunotherapy efficacy in melanoma.

The HER3/ERBB3 receptor is an oncogenic receptor tyrosine kinase that forms heterodimers with EGFR family members and is overexpressed in numerous cancers. HER3 overexpression associates with reduced survival and acquired resistance to targeted therapies, making it a potential therapeutic target in multiple cancer types. Here, we report on immunogenic, promiscuous MHC class II–binding HER3 peptides, which can generate HER3-specific CD4+ Th1 antitumor immune responses. Using an overlapping peptide screening methodology, we identified nine MHC class II–binding HER3 epitopes that elicited specific Th1 immune response in both healthy donors and breast cancer patients. Most of these peptides were not identified by current binding algorithms. Homology assessment of amino acid sequence BLAST showed >90% sequence similarity between human and murine HER3/ERBB3 peptide sequences. HER3 peptide–pulsed dendritic cell vaccination resulted in anti-HER3 CD4+ Th1 responses that prevented tumor development, significantly delayed tumor growth in prevention models, and caused regression in multiple therapeutic models of HER3-expressing murine tumors, including mammary carcinoma and melanoma. Tumors were robustly infiltrated with CD4+ T cells, suggesting their key role in tumor rejection. Our data demonstrate that class II HER3 promiscuous peptides are effective at inducing HER3-specific CD4+ Th1 responses and suggest their applicability in immunotherapies for human HER3-overexpressing tumors.

HER2 breast cancer (BC) remains a significant problem in patients with locally advanced or metastatic BC. We investigated the relationship between T helper 1 (Th1) immune response and the proteasomal degradation pathway (PDP), in HER2-sensitive and -resistant cells. HER2 overexpression is partially maintained because E3 ubiquitin ligase Cullin5 (CUL5), which degrades HER2, is frequently mutated or underexpressed, while the client-protective co-chaperones cell division cycle 37 (Cdc37) and heat shock protein 90 (Hsp90) are increased translating to diminished survival. The Th1 cytokine interferon (IFN)-γ caused increased CUL5 expression and marked dissociation of both Cdc37 and Hsp90 from HER2, causing significant surface loss of HER2, diminished growth, and induction of tumor senescence. In HER2-resistant mammary carcinoma, either IFN-γ or Th1-polarizing anti-HER2 vaccination, when administered with anti-HER2 antibodies, demonstrated increased intratumor CUL5 expression, decreased surface HER2, and tumor senescence with significant therapeutic activity. IFN-γ synergized with multiple HER2-targeted agents to decrease surface HER2 expression, resulting in decreased tumor growth. These data suggest a novel function of IFN-γ that regulates HER2 through the PDP pathway and provides an opportunity to impact HER2 responses through anti-tumor immunity.

Preferred methods for generating mouse dendritic cells (DC) would encompass qualities of consistency, high yield, and potent function. Serum-free culture is also highly desirable, since this is the standard for cell-based therapies used in humans. We report here a serum-free modification of a culture method generating mature, activated DCs from bone marrow precursors. This is achieved through a two-stage culture comprised of 6-day expansion in Flt3 ligand and IL-6 followed by brief differentiation in a medium containing GM-CSF and IL-4, with subsequent activation using TLR ligands ODN1826 and LPS. The serum-free DCs achieve yields and surface phenotype including IL-12p70 secretion similar to standard serum-replete cultures, display a capacity to sensitize in vivo against both MHC class I- and Class II-restricted antigens, and exhibit some aspects of “killer DC” function against tumor cells. We used these DCs to help identify novel CD4pos Th epitopes on the rat ErbB2/HER-2 protein and demonstrated a subset of these as effective immunogens in a DC-based therapeutic model of HER-2pos breast cancer in Balb/c mice, where they induced powerful Th1-polarized immune responses. This method represents a useful way to efficiently produce large numbers of murine dendritic cells with excellent in vivo function well-suited for use in experimental vaccine studies.

BACKGROUND LMD occurs in ~5% of patients with breast cancer (BC) and has a median survival of 2-4 months. We found a loss of the anti-HER2 and anti-HER3 CD4 Th1 immune responses in BC patients. In pre-clinical and clinical trials the administration of class II HER2 peptide-pulsed dendritic cell vaccine (HER2-DCV) partially restores anti-HER2 Th1 immune responses with pathologic complete responses in HER2+ BC patients. Here, we examined the intrathecal (IT) delivery of HER2/HER3-DCV in BC-LMD immunocompetent animal models. MATERIALS AND METHODS Luciferase-labeled HER2+ TUBO BCs were injected into the cisterna magna of BALB/c mice to produce LMD. We used our Murine Ommaya (mimics an Ommaya reservoir clinically in patients) for the IT administration of DCVs into the cerebral spinal fluid (CSF). RESULTS AND DISCUSSION BC-LMD mice were randomized into following groups: 1) HER2-DCV IT 2) HER3-DCV IT 3) HER2/HER3-DCV IT. The median survival of untreated (control) group was 15 days. All groups given DCV IT prolonged survival (p CONCLUSION Our preclinical data supported a clinical trial (submitted) of the IT delivery of DCV in BC patients with LMD.

Background: Molecular changes underlying immune cell signaling in response to new therapy development are crucial to validate the clinical efficacy of immunotherapeutics. There are several preclinical tools including patient derived cell lines, organoid and xenograft (PDX) models to assess the efficacy of drugs. However, it is important to recapitulate the complexity of human malignancy and immune contexture within the tumor microenvironment. Here we report, an ex vivo platform (3D-EXplore) using fresh patient tumor samples with intact stromal and immune cell components to assess treatment-mediated changes in molecular and transcriptional profiles of tumor resident immune cells using a Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) platform. Methods: All tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids measuring 150 µm in size with intact tumor immune microenvironment were prepared from fresh tumor samples of colon and lung tissues using proprietary technology developed at Nilogen Oncosystems. No enzymatic digestion, propagation or reassembly was used during the preparation of the tumoroids. Hundreds of tumoroids originated from different parts of each patient’s tumor sample were pooled to represent the tumor heterogeneity and treated ex vivo with or without immunostimulatory agents for 48h. Here, we applied multi-modal CITE-seq profiling using the 10X Genomics platform to interrogate cellular responses to ex vivo treatment. Culture supernatants were collected for multiplex analysis of cytokine release in media. Additionally, flow cytometry was used to assess the activation profile of resident immune cells. Results: Following sequencing of FACS sorted viable CD45 populations, the FAST-Q output files were uploaded into Cell Ranger for analysis with Loupe Browser. Multimodal analysis of transcriptomes or proteomics at the single-cell level revealed significant changes in the top 100 gene expression levels specific to CD4 and CD8+ T cells, NK cells and B cells signaling and activation upon stimulation with immunostimulatory agents. In addition, several pro and anti-inflammatory chemokines and cytokines were upregulated in response to ex vivo treatment in both colon and lung tumoroids, which coincided with marked changes in the activation status of tumor resident immune cells, as detected by multiparameter flow cytometry analysis. Conclusions: These results demonstrate Nilogen’s 3D fresh tumoroid model reflects the significant impact on activation and proliferation of tumor resident immune cells within the intact tumor microenvironment at the transcriptional level. We believe this model system can serve as a clinically relevant tool for accelerating immuno-oncology drug development. Citation Format: Jonathan P. Waterman-Smith, Jared Ehrhart, Brittany Bunch, Krithika Kodumudi, Matt Weitzman, Olivia MacIntosh, Kelly Sussman, Soner Altiok. An ex-vivo 3D tumoroid model of fresh patient tissue (3D-EXplore) to assess transcriptional and compositional changes of the immune landscape in intact tumor microenvironment using single-cell proteogenomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 288.

BackgroundHuman epidermal growth factor receptor 2 (HER2) targeted antibodies in combination with chemotherapy has improved outcomes of HER2 positive (pos) breast cancer (BC) but toxicity of therapy remains a problem. High levels of tumor-infiltrating lymphocytes are associated with increased pathologic complete responses for patients treated with neoadjuvant therapy. Here we sought to investigate whether delivery of intratumoral (i.t.) multiepitope major histocompatibility complex (MHC) class II HER2 peptides-pulsed type I polarized dendritic cells (HER2-DC1) in combination with anti-HER2 antibodies without chemotherapy could enhance tumor regression by increasing anti-HER2 lymphocyte infiltration into the tumor.MethodsBALB/c mice bearing orthotopic TUBO tumors, BALB/c mice bearing subcutaneous (s.c.) CT26 hHER2 tumors, or BALB-HER2/neu transgenic mice were all treated with i.t. or s.c. HER2-DC1, anti-HER2 antibodies, paclitaxel, T-DM1 or in combination. Immune response, host immune cells and effector function were analyzed using flow cytometry, interferon-γ ELISA and cytokine/chemokine arrays. The contributions of CD4+ and CD8+ T cells and antibody dependent cellular cytotoxicity (ADCC) were assessed using depleting antibodies and FcγR KO mice. Molecular changes were evaluated by immunohistochemistry and western blot.ResultsHER2-DC1 combined with anti-HER2 antibodies delivered i.t. compared to s.c. induced complete tumor regression in 75–80% of treated mice, with increased tumor infiltrating CD4+ and CD8+ T, B, natural killer T cells (NKT) and natural killer cells, and strong anti-HER2 responses in all HER2pos BC models tested. The therapy caused regression of untreated distant tumors. Labeled HER2-DC1 migrated prominently into the distant tumor and induced infiltration of various DC subsets into tumors. HER2-DC1 i.t. combined with anti-HER2 antibodies displayed superior antitumor response compared to standard chemotherapy with anti-HER2 antibodies. Lasting immunity was attained which prevented secondary tumor formation. The presence of CD4+ and CD8+ T cells and ADCC were required for complete tumor regression. In the HER2pos BC models, HER2-DC1 i.t. combined with anti-HER2 antibodies effectively diminished activation of HER2-mediated oncogenic signaling pathways.ConclusionsHER2-DC1 i.t. with anti-HER2 antibodies mediates tumor regression through combined activation of T and B cell compartments and provides evidence that HER2-DC1 i.t. in combination with anti-HER2 antibodies can be tested as an effective alternative therapeutic strategy to current chemotherapy and anti-HER2 antibodies in HER2pos BC.

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the "Murine Ommaya." As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Metastatic spread in breast cancer patients is the major driver of cancer-related deaths. A unique subset of cells disseminated from pre-invasive or primary tumor lesions are recognized as the main seeds for metastatic outgrowth. Disseminated cancer cells (DCCs) can migrate to distant organs and settle in a dormant state for a prolonged period until they emerge to overt metastases. Understanding the biology of breast cancer cells dissemination, dormancy and reactivation to form overt metastases has become an important focus. In this review, we discuss the recent advancements of molecular pathways involving breast cancer cell dissemination, role of chemokine-chemokine receptor networks in DCCs migration, DCCs phenotypic heterogeneity and unique genes signatures in tumor dormancy, microenvironmental regulation and specific niches that favors DCCs homing and dormancy. In addition, we also discuss recent findings relating to the role of immune response on DCC dissemination and dormancy. With recent advances in the field of immunotherapy/targeted therapy and its beneficial effects in cancer treatment, this review will focus on their impact on DCCs, reversal of stemness, tumor dormancy and metastatic relapse.

Adoptive T cell therapy (ACT) in combination with lymphodepleting chemotherapy is an effective strategy to induce the eradication of tumors, providing long-term regression in cancer patients. Despite that lymphodepleting regimens condition the host for optimal engraftment and expansion of adoptively transferred T cells, lymphodepletion concomitantly promotes immunosuppression during the course of endogenous immune recovery. In this study, we have identified that lymphodepleting chemotherapy initiates the mobilization of hematopoietic progenitor cells that differentiate to immunosuppressive myeloid cells, leading to a dramatic increase of peripheral myeloid-derived suppressor cells (MDSCs). In melanoma and lung cancer patients, MDSCs rapidly expanded in the periphery within 1 week after completion of a lymphodepleting regimen and infusion of autologous tumor-infiltrating lymphocytes (TILs). This expansion was associated with disease progression, poor survival, and reduced TIL persistence in melanoma patients. We demonstrated that the interleukin 6 (IL-6)-driven differentiation of mobilized hematopoietic progenitor cells promoted the survival and immunosuppressive capacity of post-lymphodepletion MDSCs. Furthermore, the genetic abrogation or therapeutic inhibition of IL-6 in mouse models enhanced host survival and reduced tumor growth in mice that received ACT. Thus, the expansion of MDSCs in response to lymphodepleting chemotherapy may contribute to ACT failure, and targeting myeloid-mediated immunosuppression may support anti-tumor immune responses.

Emm55 is a bacterial gene derived from Streptococcus pyogenes (S. pyogenes) that was cloned into a plasmid DNA vaccine (pAc/emm55). In this study, we investigated the anti-tumor efficacy of pAc/emm55 in a B16 murine melanoma model. Intralesional (IL) injections of pAc/emm55 significantly delayed tumor growth compared to the pAc/Empty group. There was a significant increase in the CD8(+) T cells infiltrating into the tumors after pAc/emm55 treatment compared to the control group. In addition, we observed that IL injection of pAc/emm55 increased antigen-specific T cell infiltration into tumors. Depletion of CD4(+) or CD8(+) T cells abrogated the anti-tumor effect of pAc/emm55. Combination treatment of IL injection of pAc/emm55 with anti-PD-1 antibody significantly delayed tumor growth compared to either monotherapy. pAc/emm55 treatment combined with PD-1 blockade enhanced anti-tumor immune response and improved systemic anti-tumor immunity. Together, these strategies may lead to improvements in the treatment of patients with melanoma.

Breast cancer, one of the leading causes of death in women in the United States, challenges therapeutic success in patients due to tumor heterogeneity, treatment resistance, metastasis and disease recurrence. Knowledge of immune system involvement in normal breast development and breast cancer has led to extensive research into the immune landscape of breast cancer and multiple immunotherapy clinical trials in breast cancer patients. However, poor immunogenicity and T-cell infiltration along with heightened immunosuppression in the tumor microenvironment have been identified as potential challenges to the success of immunotherapy in breast cancer. Oncodrivers, owing to their enhanced expression and stimulation of tumor cell proliferation and survival, present an excellent choice for targeted immunotherapy development in breast cancer. Loss of anti-tumor immune response specific to oncodrivers has been reported in breast cancer patients as well. Dendritic cell vaccines have been tested for their efficacy in generating anti-tumor T-cell response against specific tumor-associated antigens and oncodrivers and have shown improved survival outcome in patients. Here, we review the current status of immunotherapy in breast cancer, focusing on dendritic cell vaccines and their therapeutic application in breast cancer. We further discuss future directions of breast cancer immunotherapy and potential combination strategies involving dendritic cell vaccines and existing chemotherapeutics for improved efficacy and better survival outcome in breast cancer.

Breast cancer, one of the leading causes of death in women in the United States, challenges therapeutic success in patients due to tumor heterogeneity, treatment resistance, metastasis and disease recurrence. Knowledge of immune system involvement in normal breast development and breast cancer has led to extensive research into the immune landscape of breast cancer and multiple immunotherapy clinical trials in breast cancer patients. However, poor immunogenicity and T-cell infiltration along with heightened immunosuppression in the tumor microenvironment have been identified as potential challenges to the success of immunotherapy in breast cancer. Oncodrivers, owing to their enhanced expression and stimulation of tumor cell proliferation and survival, present an excellent choice for targeted immunotherapy development in breast cancer. Loss of anti-tumor immune response specific to oncodrivers has been reported in breast cancer patients as well. Dendritic cell vaccines have been tested for their efficacy in generating anti-tumor T-cell response against specific tumor-associated antigens and oncodrivers and have shown improved survival outcome in patients. Here, we review the current status of immunotherapy in breast cancer, focusing on dendritic cell vaccines and their therapeutic application in breast cancer. We further discuss future directions of breast cancer immunotherapy and potential combination strategies involving dendritic cell vaccines and existing chemotherapeutics for improved efficacy and better survival outcome in breast cancer.

Immunotherapy, including adoptive cell transfer (ACT) with tumor-infiltrating lymphocytes (TIL) predominantly targets improvement in MHC Class I-mediated anti-tumor immune responses. The primary objective of this study is to better understand the role of CD4+ TIL in ACT as a complementary avenue for therapeutic efficacy. Briefly, CD4+ TIL were isolated by negative selection from metastatic melanoma patients who received TIL therapy at Moffitt Cancer Center on IRB approved protocols. Individual T cell clones were tracked by TCRbeta sequencing to quantify clonal persistence in patients. CD4+ TIL clones were found to be decidedly persistent in a candidate patient who achieved a complete response (CR) after infusion of 88% CD4+ T cells. CD4+ TIL from additional patients were stimulated with anti-CD3/CD28 in vitro and those who were clinical responders demonstrated a pleiotropic cytokine profile marked by an elevated ratio of Th1:Th2 cytokines (p=0.07, n=13). When cultured with APCs loaded with autologous tumor (AT), CD4+ TIL produced high levels of IFN-gamma in an MHC Class II-dependent manner. Induction of MHC Class II on melanoma cell lines and AT determined that CD4+ TIL secreted IFNg and TNFa directly in response to AT. ACT of tumor-reactive CD4+ TIL in immunodeficient (NSG) mice provided significant control of AT growth when compared to non-reactive CD4+ TIL. Preliminary data in syngeneic mouse models also suggests that antigen-specific CD4+ T cells aid in initial tumor rejection, memory formation and epitope spreading, resulting in an overall increased therapeutic efficacy during ACT. This data supports the conclusion that CD4+ TIL are tumor-reactive and instrumental to an effective anti-tumor immune response in cancer patients.

While immunotherapies have had a striking efficacy in melanoma patients, significant proportions of melanoma patients exhibit poor responsiveness to immunotherapies. Further understanding of immunoregulatory mechanisms is needed to improve immunotherapies. Previously, we discovered increasing tumor fucosylation in melanomas reduces tumor growth and metastasis (Lau et al., 2015). To identify tumor-infiltrating lymphocytes (TILs) affected by L-fucose, we profiled lymphocytes from syngeneic tumors of control- or fucose-fed mice tumors. Dietary supplementation of L-fucose increased TILs by ~10-50-fold. Of total TILs, CD3+ T cells (including CD4+ and CD8+ T cells) doubled. L-fucose did not trigger tumor suppression in immune-deficient mouse melanoma models, indicating the immune system’s requirement for L-fucose-triggered suppression. Immunodepletion of CD4+ or CD8+ T cells during L-fucose treatment revealed that CD4+ T cells are crucial for tumor suppression. CD4+ T-cell depletion abrogated infiltration of NK, dendritic, and CD8+ T cells in the tumors, implicating these populations as downstream effectors of fucosylation- and CD4+ T cell-triggered tumor suppression. To identify proteins on melanomas contributing to fucosylation-triggered immune responses, we performed mass spectrometric analysis of all fucosylated proteins in melanoma and identified 2 MHC proteins, HLA-A and HLA-DRB1, as fucosylated. Knockdown of HLA- DRB1 and HLA-A in vivo revealed HLA-DRB1 is required for L-fucose-mediated tumor suppression, and notably, for recruitment of CD4+ T cells. We further investigated how fucosylation affects CD4+ T-cell biology using CD4+ T cells isolated from healthy donors, which we pharmacologically modulated fucosylation. The roles of fucosylation on tumor vs. CD4+ T cells and clinical utility/implications for melanoma patients will be discussed. Our data show how increased fucosylation drives tumor suppression through CD4+ T cells, and support the potential of dietary L-fucose to boost immunity in melanomas. Citation Format: Daniel K. Lester, Pasquale Innamarato, Krithika Kodumudi, Gregory Watson, Matt Mercurio, Shari Pilon-Thomas, Jane Messina, Susan McCarthy, Eric Lau. Fucosylation in CD4+ T cell-mediated melanoma suppression [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A73.

Advanced bladder cancer patients have limited therapeutic options resulting in a median overall survival (OS) between 12 and 15 months. Adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) has been used successfully in treating patients with metastatic melanoma, resulting in a median OS of 52 months. In this study, we investigated the feasibility of expanding TIL from the tumors of bladder cancer patients. Primary bladder tumors and lymph node (LN) metastases were collected. Tumor specimens were minced into fragments, placed in individual wells of a 24-well plate, and propagated in high dose IL-2 for four weeks. Expanded TIL were phenotyped by flow cytometry and anti-tumor reactivity was assessed after co-culture with autologous tumor digest and IFN-gamma ELISA. Of the 28 transitional cell bladder or LN tumors collected, 14/20 (70%) primary tumors and all of the LN metastases demonstrated TIL expansion. Expanded TIL were predominantly CD3+ (median 63%, range 10–87%) with a median of 30% CD8 + T cells (range 5–70%). TIL secreted IFN-gamma in response to autologous tumor. Addition of agonisitic 4-1BB antibody improved TIL expansion from primary bladder tumors regardless of pre-treatment with chemotherapy. This study establishes the practical first step towards an autologous TIL therapy process for therapeutic testing in patients with bladder cancer.

Intralesional (IL) injection of Rose Bengal (PV-10) induces regression of injected and uninjected lesions in several murine tumor models. In this study, we investigated the anti-tumor response of combining IL PV-10 with blockade of the PD-1 / PD-L1 pathway and the role of immune cell populations in eliciting this response. To investigate the role of T cell subsets in mediating an immune response, B16 or M05 melanoma-bearing mice received combination therapy as well as CD8+, CD4+, or CD25+ depleting antibodies. Tumor growth was measured. T cells were collected from spleens or tumors, and phenotype, activation markers, and reactivity were measured. Splenocytes from mice treated with combination therapy had increased OVA antigen-specific CD8+ T cells in M05-tumor-bearing mice. Depletion of CD4+ T cells or regulatory T cells (Tregs) in combination with IL PV-10 and anti-PD-1 antibody treatment resulted in an enhanced anti-tumor effect. Treatment with CD8+ depleting antibody abrogated anti-tumor immunity. These results support a clinical study for the safety and anti-tumor immune responses with combination therapy of IL PV-10 and PD-1/PD-L1 blockade.

Background: In the past few years, anti-PD-1 and anti-CTLA4 immune-based therapeutics have revolutionized melanoma therapy, but many patients do not respond. The IFX-Hu2.0 formulation consists of a vector encoding a streptococcal membrane protein Emm55 and a cationic polymer. When IFX-Hu2.0 is injected into murine or equine melanomas, tumors regress. Transfection of the emm55 vector into B16 melanoma cells resulted in expression of Emm55 as measured by RT-PCR. Systemic immune responses elicited by IFx-Hu2.0 in B16 bearing mice were CD4 and CD8 T cell dependent and antigen specific. Antigen specificity was tested utilizing a M05 tumor model (B16 cells transfected with the Ovalbumin gene). Mice received 5*106 OT-1 T cells (T cells that have transgenic TCR that recognizes OVA). Tumors were harvested (48 hours) and tetramer specific CD8+ T cells were increased in treated mice. Given this preliminary data, we proceeded to design a first in human Phase I feasibility trial (single dose level with no dose escalation) to test IFx-Hu2.0 use as an intralesional immunotherapy for melanoma to provoke the immune response by facilitating Emm55 antigen surface expression and enhancing tumor recognition. Methods: The primary objective is to assess the safety, tolerability, and feasibility of intralesional injection with IFx-Hu2.0 as a monotherapy in 6 adult patients (>18 years) with unresectable stage III or stage IV cutaneous melanoma lesions. To be eligible, a patient must have at least 1 injectable lesion and 1 un-injected lesion available for biopsies, but up to 3 lesions may be injected. Unlike other trials for melanoma, only 3 mm cutaneous lesions are needed. Patients must have failed, refused or been deemed not candidates for one form of systemic anti-PD-1-based immunotherapy as well as BRAF inhibition, if BRAF V600 mutated. In addition, patients with unresectable cutaneous, subcutaneous, and nodal melanoma lesions recurrent after initial surgery must have failed, refused or not candidates for TVEC. Patients may be enrolled with brain metastases (3 months, but may not be receiving concurrent chemotherapy/biological therapy, have uncontrolled hepatitis B/C/HIV infection, or have a history of organ allograft transplantation. Concurrent radiotherapy is allowed at distant sites from the injected lesion. 40 mL of peripheral blood will be collected prior to treatment and at the follow-up visit (28 days) for correlative studies to complement the preclinical murine models. Patients responding to therapy may continue dosing every 3 weeks thereafter. Feasibility is defined as the ability to treat five of the six patients enrolled without dose-limiting toxicity (28 days). As of January 2019, 1/6 of the planned patients have been enrolled. Clinical trial registry number: NCT03655756. Citation Format: Joseph Markowitz, Krithika N. Kodumudi, Deanryan B. De Aquino, Vernon K. Sondak, Shari Pilon-Thomas. Trial in progress: First in human Phase I study using a plasmid DNA coding for Emm55 streptococcal antigen (IFx-Hu2.0) in patients with unresectable stage III or stage IV cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT119.

HER-2/neu overexpression plays a critical role in breast cancer development, and its expression in ductal carcinoma in situ (DCIS) is associated with development of invasive breast cancer. The detection of disseminated cancer cells (DCCs) in early-stage DCIS likely to become invasive and manifests prominent role in metastasis and recurrence. The HER2-positive DCCs can easily escape targeted therapies and become a source of tumor recurrence suggesting that targeting these cells may provide eminent benefits in HER2 positive breast cancer patients. Unfortunately, the conventional therapies that target these DCCs are limited. Previous studies from our lab have shown that HER2 peptide pulsed-DC1 (HER2-DC1) vaccine induced 30% complete response (pCR) in DCIS patients. However, little is known about the effect of HER2-DC1 vaccine on DCCs. Here, we investigated the efficacy of HER2-DC1 vaccine on DCCs in a transgenic HER2 mammary cancer model (neuT). The key features of this model are each one of ten mammary glands develops an independent carcinoma that slowly progresses from a microscopic lesion to an invasive tumor and metastasis is mainly driven by DCCs. Since this model mimics some of the most critical features of human disease, we evaluated the efficacy of HER2-DC1 vaccine on DCCs. Mice received six doses of HER2-DC1 vaccine subcutaneously twice a week. The spontaneous tumor development in the mammary glands of NeuT mice was examined once a week by ultrasound until the age of week 16. HER2-DC1 vaccine significantly prevented spontaneous tumor growth in NeuT mice. Remarkably, HER2-DC1 vaccine treatment significantly decreased HER2+CYT8/18+Ki-67+ proliferative DCCs (2%, p Citation Format: Ganesan Ramamoorthi, Krithika Kodumudi, Colin Synder, William Dominguez-Viqueira, Scott Kidd, Shari Pilon-Thomas, Brian Czerniecki. HER2 peptide pulsed dendritic cell vaccine induce senescence and eliminates disseminated cancer cells in a preventive model of transgenic HER2/neu breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1451.

Triple Negative Breast Cancer (TNBC) is characterized by lack of hormone receptors and targeted therapies, resistance to existing treatment and poor survival outcome, highlighting the need for novel therapy development. Oncogene addiction in breast cancer (BC) cells to drive malignancy up presents oncodrivers as promising therapeutic candidates in BC. Our group has shown loss of anti-oncodriver CD4+-T-helper-type-1 (Th1) response in TNBC patients. Here, we studied effects of combination treatment with Th1 cytokines (IFN-γ+TNF-α) and oncodriver blockade in human and mouse TNBC cells (MDA-MB-231, MDA-MB-468, HCC1143, BT549, 4T1). We measured expression of 3 oncodriver proteins: EGFR, HER3 and cMET and observed multiple oncodrivers in majority of the cell lines, suggesting combined oncodriver inhibition may have stronger antitumor effects. Based on oncodriver status, we tested combination treatment with Th1 cytokines and a) monoclonal α-EGFR antibody cetuximab, b) cMET RTK inhibitor crizotinib and c) siRNA inhibition of three oncodrivers. No significant difference in proliferation after IFN-γ or IFN-γ+TNF-α treatment suggested IFN-γ is the primary effector cytokine. IFN-γ alone reduced proliferation >50% in majority of TNBC cells. In EGFRhigh/HER3pos/cMETlow MDA-MB-468, IFN-γ and cetuximab treatment significantly increased apoptosis compared to untreated cells. Combined EGFR and HER3 siRNA inhibition, followed by IFN-γ treatment, increased apoptosis in those cells. In EGFRpos/HER3low/cMEThigh MDA-MB-231 and EGFRlow/HER3high/cMEThigh 4T1, IFN-γ and crizotinib combination significantly decreased proliferation (p Citation Format: Amrita Basu, Yongsheng Jia, Krithika Kodumudi, Doris Wiener, Brian Czerniecki. Th1 cytokines and oncodriver inhibition: A combination treatment strategy in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4088.

The proto-oncogene HER2/ErbB2 overexpression occurs in 30% of invasive breast cancer patients and its critically associated with aggressive disease, metastasis and tumor recurrence. Despite advances in the treatment of HER2+ breast cancers, resistance and metastatic disease are potential obstacles. Cullin 5 (Cul 5), a scaffold protein mediates formation of the Cullin 5-RING E3 ubiquitin ligase functional complex. This complex acts on protective molecular chaperone heat shock protein (HSP90) complex and subsequently leads to polyubiquitination and proteasomal degradation of its client protein HER2/ErbB2. Reduced expression of Cul 5 in breast cancer patients can abrogates its inhibitory function and favor HSP90 mediated stabilization of HER2/ErbB2. More recently, we showed that Th1 cytokines (IFN-γ and TNF-α) decreased HER2 expression and induced senescence and apoptosis in HER2 overexpressing breast cancer cell lines. However, the role of Th1 cytokines on Cul 5 mediated regulation of HER2/ErbB2 remains unknown. In the present study, low level expression of Cul 5 was observed in various human (SKBR3, HCC1419 and JIMT1) and mouse (Tubo) HER2+/ER- breast cancer cell lines. Interestingly, treatment of HER2+/ER- breast cancer cells with Th1 cytokines increased the expression of Cul 5 and downregulated the expression of HER2. We knocked-down Cul 5 expression by specific siRNA found that treatment of Th1 cytokines failed to decrease HER2 expression in HER2+/ER- breast cancer cells. To investigate whether the mechanism of Th1 cytokines on Cul5 is via downstream signaling molecule STAT-1, we knock-down STAT-1. Silencing of STAT-1 expression prevented Th1 cytokines mediated activation of Cul 5. We also observed no effect on the inhibition of HER2 expression after Th1 cytokines treatment in HER2+/ER- breast cancer cells in which the expression of STAT-1 was knocked-down. Collectively, our findings demonstrate a novel mechanism that Th1 cytokines promote Cul 5 expression via STAT1 signaling and enhance Cul 5 mediated proteasomal degradation of HER2 in HER2+/ER- breast cancer. Enhancing Cul 5 levels may represent a new therapeutic avenue for the inhibition of HER2 overexpression and prevention of metastasis and tumor relapse. Citation Format: Yongsheng Jia, Ganesan Ramamoorthi, Krithika Kodumudi, Amrita Basu, Doris Wiener, Brian Czerniecki. Th1 cytokines promotes E3 ubiquitin ligase Cullin 5 expression via STAT1 signaling cascade and enhance cul5 mediated proteasomal degradation of HER2 in HER2+/ER- breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4434.

Administration of nonmyeloablative chemotherapeutic agents or total body irradiation (TBI) prior to adoptive transfer of tumor-specific T cells may reduce or eliminate immunosuppressive populations such as T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSC). Little is known about these populations during immune reconstitution. This study was designed to understand the reconstitution rate and function of these populations post TBI in melanoma tumor‑bearing mice. Reconstitution rate and suppressive activity of CD4+CD25+Foxp3+ Tregs and CD11b+Gr1+ MDSC following TBI-induced lymphopenia was measured in B16 melanoma tumor‑bearing mice. To ablate the rapid reconstitution of suppressive populations, we treated mice with docetaxel, a known chemotherapeutic agent that targets MDSC, in combination with adoptive T cell transfer and dendritic cell immunotherapy. Both Treg and MDSC populations exhibited rapid reconstitution after TBI-induced lymphopenia. Although reconstituted Tregs were just as suppressive as Tregs from untreated mice, MDSC demonstrated enhanced suppressive activity of CD8+ T cell proliferation compared with endogenous MDSC from tumor-bearing mice. TBI-induced lymphopenia followed by docetaxel treatment improved the efficacy of adoptive T cell transfer and dendritic cell immunotherapy in melanoma-bearing mice, inducing a significant reduction in tumor growth and enhancing survival. Tumor regression correlated with increased CTL activity and persistence of adoptively transferred T cells. Overall, these findings suggest that TBI-induced MDSC are highly immunosuppressive and blocking their rapid reconstitution may improve the efficacy of vaccination strategies and adoptive immunotherapy.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

Intralesional (IL) therapy is under investigation to treat dermal and subcutaneous metastatic cancer. Rose bengal (RB) is a staining agent that was originally used by ophthalmologists and in liver function studies. IL injection of RB has been shown to induce regression of injected and uninjected tumors in murine models and clinical trials. In this study, we have shown a mechanism of tumor-specific immune response induced by IL RB. In melanoma-bearing mice, IL RB induced regression of injected tumor and inhibited the growth of bystander lesions mediated by CD8+ T cells. IL RB resulted in necrosis of tumor cells and the release of High Mobility Group Box 1 (HMGB1), with increased dendritic cell (DC) infiltration into draining lymph nodes and the activation of tumor-specific T cells. Treatment of DC with tumor supernatants increased the ability of DCs to stimulate T cell proliferation, and blockade of HMGB1 in the supernatants suppressed DC activity. Additionally, increased HMGB1 levels were measured in the sera of melanoma patients treated with IL RB. These results support the role of IL RB to activate dendritic cells at the site of tumor necrosis for the induction of a systemic anti-tumor immune response.

In this study filarial recombinant protein or DNA vaccine constructs encoding BmALT-2 and BmVAH as single or as cocktail antigens were evaluated. Male jirds were immunized intramuscularly with DNA vaccine constructs or were immunized intraperitoneally with protein vaccine. The single and bicistronic DNA constructs induced substantial interferon-γ responses in spleen cells; antigen-specific responses were higher following immunization with the bicistronic cocktail construct and evoked a significant protective response of 57% in jirds challenged with Brugia malayi that was similar in the antibody-dependent cellular cytotoxicity (ADCC) assay and micropore chamber experiment. The cocktail protein vaccines induced a mixture of IgG2a (Th1) and IgG1 (Th2) responses with 80% protective response when challenged with B. malayi infective larvae. However, the single protein vaccine rALT-2 induced Th2 (IgG1/IgG3) with a 70% protective response and rVAH induced Th1 (IgG2a) with a lower proliferative response with 60% protection following challenge with B. malayi infective larvae. These results suggest that filarial cocktail protein vaccines are able to elicit substantial immune and protective responses when compared with single antigen vaccination in suitably vaccinated jirds.

Designing mimetic of the interface functional groups of known receptor-ligand complexes is an attractive strategy for developing potential therapeutic agents that interfere with target protein-protein interactions. The CD80/CD86-CD28/CD152 costimulatory interactions transmit signals for CD4+ T cell activation and suppression and are critically involved in the initiation, progression, and reactivation of the immunopathology in multiple sclerosis. Differences in the pattern, levels, and kinetics of expression of CD80/CD86 molecules in conjunction with differences in the strength of the signals delivered upon binding CD28 or CD152 determine the outcome of the immune response. A temporal up-regulation of surface expression of CD80 relative to CD86 on APCs and CNS-infiltrating cells has been shown to correlate with disease progression in experimental autoimmune encephalomyelitis an animal model for multiple sclerosis. Hence blockade of the CD80 costimulatory axis has therapeutic potential in multiple sclerosis. In this study, we report the efficacy of a novel CD80-blocking agent CD80-competitive antagonist peptide (CD80-CAP) in suppressing clinical disease and relapse in experimental autoimmune encephalomyelitis. The CD80-CAP mediates protection by inhibiting proinflammatory cytokines and skewing toward anti-inflammatory response presumably by enhancing the expression of glucocorticoid-induced leucine zipper in activated CD4+ T cells.

Oral lichen planus (OLP) and burning mouth syndrome (BMS) are chronic conditions affecting the oral mucosa characterized by pain and burning sensation. Saliva plays a significant role in the maintenance of physical and functional integrity of normal oral mucosa. Identification of potential "salivary biomarkers" for early diagnosis and/or monitoring of human diseases is being explored. We investigated the soluble forms of innate immune associated proteins CD14 and toll-like receptor-2 in unstimulated whole saliva (UWS) as potential biomarkers for OLP and BMS. Our results suggest that the levels of sCD14 and sTLR-2 in UWS were upregulated in OLP and BMS respectively. In addition, oral epithelial cells in the saliva of patients with OLP and BMS exhibited elevated levels of CD14 mRNA and decreased levels of TLR-2 mRNA. Interestingly, presence of co-existent oral candidiasis nullified these changes.

A zinc containing metalloprotease, 175 kDa collagenase, purified from adult female Setaria cervi showed strong cross-reactivity with sera from putatively immune (PI) individuals (unpublished observation) and induced cytotoxicity to B. malayi L3 larvae and microfilariae by ADCC mechanism [Srivastava Y, Bhandari YP, Reddy MVR, Harinath BC, Rathaur S. An adult 175 kDa collagenase antigen of Setaria cervi in immunoprophylaxis against Brugia malayi. J Helminth 2004;78:347-52]. These preliminary observations suggested the immunoprotective nature of collagenase. To confirm the vaccine potential of this protease, a vaccine trial was conducted in jirds (Meriones unguiculatus) against human filarial parasite B. malayi. The vaccination resulted into a mean protection level of 75.86% and produced high level of protease neutralizing antibodies. Cytokine analysis in immune jirds sera suggested a mixed Th1/Th2 type cellular immune response whereas ELISA, immunoblotting and enzyme antibody inhibition assay revealed the presence of specific anti-collagenase antibodies. Taken together, all these results suggest that S. cervi 175 kDa collagenase could form the basis of an effective molecular vaccine against human lymphatic filariasis.

142 Background: Patients with advanced bladder cancer have limited therapeutic options resulting in a median overall survival (OS) between 12 and 15 months. At our institution, adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) has resulted in a durable median OS of 52 months in patients with metastatic melanoma. Immune-mediated anti-tumor responses have been previously shown in bladder cancer, therefore we investigated the phenotype and function of TIL expanded from bladder tumors to establish feasibility of ACT for the treatment of bladder cancer. Methods: Tumor specimens, including primary bladder tumors and lymph node metastases, were collected from 29 bladder cancer patients having standard of care tumor resection, who also had consented to an IRB-approved protocol for TIL generation. The tissue was minced into fragments, placed in individual wells of a 24-well plate, and propagated in high dose IL-2 for four weeks. TIL were considered expanded if they propagated to fill ≥2 wells. The remaining tumor material was digested into a single cell suspension and frozen. TIL were phenotyped by flow cytometry and assessed for autologous tumor reactivity through co-culture with tumor digest and IFN-gamma ELISA. Results: Transitional cell bladder tumors were cultured from 23 patients, of whom 19 (83%) demonstrated TIL expansion. Microbial contamination precluded TIL growth in six specimens. TIL were cultured from 9/12 (75%) patients with preceding chemotherapy and 10/11 (91%) who were chemotherapy naive. Expanded TIL were predominantly CD3+(median 63%, range 10-87%) with a median of 30% CD8+ T cells (range 5-70%). Eight of 15 tested samples (53%) contained TIL that secreted IFN-gamma in response to autologous tumor. Conclusions: The study establishes the practical first step towards an autologous TIL therapy process for therapeutic testing in patients with bladder cancer. Human bladder cancer tissue can be used to expand tumor-specific TIL in vitro. TIL were also expanded from patients that received chemotherapy prior to tumor resection. Future efforts will explore the ability to further expand bladder TIL cultures to clinically meaningful numbers to develop novel ACT strategies for patients with this diagnosis.

PV-10 is a 10% solution of Rose Bengal that is currently being examined as a novel cancer therapeutic. We have previously shown that intralesional (IL) injection of PV-10 into a single subcutaneous B16 melanoma tumor led to regression of both the injected tumor and uninjected B16 lung lesions. Tumor regression correlated with the induction of systemic anti-melanoma T cell immunity. In melanoma patients, IL injection of PV-10 has led to regression of treated lesions as well as untreated bystander lesions. In this study, we have examined whether IL PV-10 and co-inhibitory blockade could improve anti-tumor immunity and regression of melanoma. B16 cells were injected into C57BL/6 mice to establish one subcutaneous tumor. Treatment of this lesion with a single IL injection of PV-10 alone led to partial regression of the injected B16 lesion. Systemic administration of anti-CTLA-4 or anti-PD1 antibodies in combination with IL PV-10 resulted in increased tumor regression and improved survival in this model. Treatment with PV-10 also led to the induction of T cells that produced IFN-γ (495 ± 198 pg/ml) in response to B16 cells but not to irrelevant MC-38 cells. Combination therapy with IL PV-10 and anti-CTLA-4 led to increased IFN-γ responses to B16 (1235 ± 191 pg/ml, p < 0.05). In another experiment simulating heavy tumor burden using a bilateral model, systemic administration of anti-PD-L1 antibodies in combination with IL PV-10 led to regression of the injected B16 lesion as well as a bystander subcutaneous lesion on the opposite flank (p < 0.01 compared to mice treated with anti-PD-L1 antibodies or IL PV-10 alone). Together, these studies support the induction of increased tumor-specific immunity after co-inhibitory blockade in combination with IL PV-10 therapy.

Intralesional (IL) injection of PV-10 has shown to induce regression of both injected and non-injected lesions in patients with melanoma. To determine an underlying immune mechanism, the murine B16 melanoma model and the MT-901 breast cancer model were utilized. In BALB/c mice bearing MT-901 breast cancer, injection of PV-10 led to regression of injected and untreated contralateral subcutaneous lesions. In a murine model of melanoma, B16 cells were injected into C57BL/6 mice to establish one subcutaneous tumor and multiple lung lesions. Treatment of the subcutaneous lesion with a single injection of IL PV-10 led to regression of the injected lesion as well as the distant B16 melanoma lung metastases. Anti-tumor immune responses were measured in splenocytes collected from mice treated with IL PBS or PV-10. Splenocytes isolated from tumor bearing mice treated with IL PV-10 demonstrated enhanced tumor-specific IFN-gamma production compared to splenocytes from PBS-treated mice in both models. In addition, a significant increase in lysis of B16 cells by T cells isolated after PV-10 treatment was observed. Transfer of T cells isolated from tumor-bearing mice treated with IL PV-10 led to tumor regression in mice bearing B16 melanoma. These studies establish that IL PV-10 therapy induces tumor-specific T cell-mediated immunity in multiple histologic subtypes and support the concept of combining IL PV10 with immunotherapy for advanced malignancies.

Factors such as T regulatory (Treg) cells and myeloid derived suppressor cells (MDSC) limit the effectiveness of current immunotherapy protocols. Several reports have shown that administration of chemotherapeutic agents or total body irradiation (TBI) before adoptive transfer of tumor-specific T cells reduces or eliminates the immunosuppressive populations such as Tregs and MDSC. A previous study in our lab has shown that MDSC that reconstitute after lymphodepletion are highly suppressive compared to the endogenous MDSC in a B16 melanoma model. Blockade of MDSC expansion in combination with adoptive T cell transfer delayed tumor growth and improved survival rate. However, there is very little known about the MDSC reconstitution after the adoptive transfer of tumor infiltrating lymphocytes (TIL) in melanoma patients. Melanoma patients were treated with non- myeloablative therapy that included cyclophosphamide and fludarabine. At the time of TIL transfer, no circulating lymphoid or myeloid cells were detected in the PBMC. One day after completion of chemotherapy, up to 100 billion TIL were transferred followed by high dose bolus IL-2. At weeks 1-4 after TIL transfer, PBMC were collected and we investigated the reconstitution and the suppressive function of MDSC. MDSC were phenotyped by flow cytometry as HLA-DR− Lin− CD14+CD33+ or CD11b+CD14−CD33+. While 3-15% of MDSC was observed in the PBMC of melanoma patients as their baseline, after lymphodepletion and one week post TIL infusion, the percent of MDSC increased to 10-30% and gradually dropped to baseline by week 4. To examine suppressive function, MDSC were isolated from peripheral blood at 1-2 weeks after TIL infusion and co-cultured with healthy donor T cells or patient TIL. MDSC were highly suppressive and abrogated T cell proliferation and IFN-gamma secretion. Furthermore, in patients that responded to TIL therapy, the ratio of peripheral MDSC to total CD8+ T cells was lower. These studies demonstrate that MDSC that reconstitute post- lymphodepletion are suppressive and may potentially interfere with robust T cell function. Further studies are warranted to clarify the role of MDSC in the efficacy of TIL therapy. Blocking MDSC in combination with TIL therapies represent an improved strategy for the treatment of melanoma patients. Citation Format: Krithika N. Kodumudi, Doris Wiener, Amy Weber, Erica Royster, Linda Kelley, Amod Sarnaik, Shari Pilon-Thomas. Reconstitution of myeloid derived suppressor cells after the induction of lymphopenia and TIL therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2292.

PV-10 is a 10% solution of Rose Bengal, a xanthene dye that was originally developed for ophthalmic use and later used in liver function studies. PV-10 was formulated for intralesional (IL) injection and is currently being investigated as a novel cancer therapeutic. In murine studies, we have previously shown that intralesional (IL) injection of PV-10 leads to a regression of both injected tumors and untreated bystander tumors. We have also shown that combination therapy of IL PV-10 with blockade of PD-1 and PDL-1 leads to increased anti-tumor immunity. In this study, we have examined the role played by specific immune cell populations in eliciting this response in a murine melanoma model. We first investigated the antigen specificity of CD8+ T cells in the spleens of mice treated with the combination therapy of IL PV-10 and systemic co-inhibitory blockade. We found that splenocytes from mice treated with the combination of IL PV-10 and anti PD-1 antibody have an increased mean percentage of OVA antigen-specific CD8+ T cells (5.77%) compared to single treatment with anti PD-1 antibody (3.8%) or IL PV-10 (3.60%) alone in OVA-expressing B16 tumor bearing mice. To investigate the role of T cell subsets in mediating an immune response, OVA-expressing B16 tumor bearing mice were treated with IL PV-10 followed by intraperitoneal injection of anti-PD-1 antibody. In addition, mice were given either 2.43 antibody to deplete CD8+ T cells, GK1.5 antibody to deplete CD4+ T cells, or PC61 to deplete regulatory T cells (Tregs). We found that depletion of CD4+ T cells in combination with IL PV-10 and anti-PD-1 antibody treatment resulted in an enhanced anti-tumor effect, with an average tumor size of 52.8 mm2 on day 25 compared to the control group (173.7 mm2), and depletion of Tregs resulted in an even greater anti-tumor effect, with an average tumor size of 1.2 mm2 on day 25 (p Citation Format: Amy M. Weber, Hao Liu, Krithika N. Kodumudi, Amod A. Sarnaik, Shari Pilon-Thomas. T cell mediated immunity after combination therapy with intralesional PV-10 and co-inhibitory blockade in a melanoma model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4978.

Tumor-infiltrating lymphocytes (TIL) has been associated with improved survival in cancer patients. Within the tumor microenvironment, regulatory cells and expression of co-inhibitory immune checkpoint molecules can lead to the inactivation of TIL. Hence, there is a need to develop strategies that disrupt these negative regulators to achieve robust anti-tumor immune responses. We evaluated the blockade of immune checkpoints and their effect on T cell infiltration and function. We examined the ability of TIL to induce tumor-specific immune responses in vitro and in vivo. TIL isolated from tumor bearing mice were tumor-specific and expressed co-inhibitory immune checkpoint molecules. Administration of monoclonal antibodies against immune checkpoints led to a significant delay in tumor growth. However, anti-PD-L1 antibody treated mice had a significant increase in T cell infiltration and IFN-γ production compared to other groups. Adoptive transfer of in vitro expanded TIL from tumors of anti-PD-L1 antibody treated mice led to a significant delay in tumor growth. Blockade of co-inhibitory immune checkpoints could be an effective strategy to improve TIL infiltration and function.

Objective Oral lichen planus (OLP) is a chronic inflammatory condition of the mucosa mediated by a complex signalling network between the keratinocytes and the sub-epithelial lymphocytes. Since OLP occurs in constantly renewing epithelium continuously exposed to commensals, we hypothesised that the epithelial cell microflora interactions may mediate the persistent inflammation. By virtue of their ability to respond to most oral commensal microorganisms, the toll like receptor-2 (TLR-2) and TLR-4 are the most widely investigated receptors in oral diseases. The overall objective of this study was to investigate the role of TLR-2 and TLR-4 in OLP. Design Systemically healthy OLP and control subjects were recruited after obtaining the institutional review board approval. Expression of TLR-2 and TLR-4 proteins and transcripts in the tissue epithelium and in the epithelial cells isolated from saliva were determined by immunohistochemistry and quantitative real-time polymerase chain reaction respectively. Results The tissue epithelium and the salivary epithelial cells expressed reduced TLR-2 and increased TLR-4 proteins and transcripts in OLP. The salivary epithelial cells from OLP subjects secreted elevated IL-12. However, upon stimulation with bacterial lipopolysaccharide the epithelial cells from OLP exhibited a mixed Th1 (IL-12) and Th2 (IL-4) response. Presence of dexamethasone significantly reduced inflammatory cytokines in the in vitro stimulated cultures of salivary epithelial cells from OLP subjects. Conclusion Collectively, our data support a critical role for the host–microbial interactions in the OLP pathogenesis. The potential use of exfoliated oral epithelial cells in saliva for functional analysis exponentially increases its value as biological specimen for clinical research.

3, 5, 7-Trihydroxy-4'-methoxy-8-(3-hydroxy-3- methylbutyl)–flavone (ICT) is a novel derivative of Icariin (ICA), the major active ingredient of Herba Epimedii, a herb used in traditional Chinese and alternative medicine. We previously demonstrated its anti-inflammatory effect in murine innate immune cells and activated human PBMCs. We report herein that ICA or ICT treatment reduces the expression of MRP8/MRP14 and toll-like receptor 4 (TLR4) on human PBMCs. Administration of ICA or ICT inhibited tumor growth in 4T1-Neu tumor-bearing mice and considerably decreased MDSC numbers in the spleen of these mice. Further, we saw a restoration of IFN-γ production by CD8+ T cells in tumor bearing mice when treated with ICA or ICT. ICA and ICT significantly decreased the amounts of nitric oxide and reactive oxygen species in MDSC in vivo. When MDSC were treated in vitro with ICT, we saw a significant reduction in the percent of these cells with concomitant differentiation into dendritic cells and macrophages. Concomitant with this cell type conversion was a down-regulation of IL-10, IL-6 and TNF-α production. Decreased expression of S100A8/9 and inhibition of activation of STAT3 and AKT may in part be responsible for the observed results. In conclusion, our results showed that ICA, and more robustly, ICT, directly modulate MDSC signaling and therefore altered the phenotype and function of these cells, in vitro and in vivo.

Background: Human inflammatory bowel disease (IBD) is a chronic condition mediated by aberrant immune responses to the luminal antigens by activated CD4+ T cells. The CD80/CD86: CD28/CD152 costimulatory pathways transmit signals critical for T cell activation and suppression. Macrophages and epithelial cells are the chief antigen-presenting cells in the gut. Macrophages from the IBD colon express significantly elevated levels of CD80 and CD86 costimulatory molecules. The CD28-CD80 interaction primarily participates in breaking the tolerance and inducing the immune response in murine models of colitis. Blockade of CD80-costimulatory axis is an attractive strategy in the treatment of IBD. Methods: Incorporating the structural information of the CD80: CD152 complex together with the preferences of interface residues to form polyproline type II helix, we designed novel peptide agents that selectively blocked CD80 receptor interactions. Results: Administration of CD80 blocking agent at the time of adoptive transfer prevented the SCID mice from CD4+CD45Rbhigh T-cell mediated colitis. Significantly, CD80-CAP (competitive antagonist peptide) treatment suppressed established inflammation in TNBS-induced colitis, a model for Th1-mediated Crohn's disease. The colons of the mice receiving the CD80 blocking agent appeared unaffected macroscopically and exhibited negligible microscopic inflammation. The CD80-CAP treatment was associated with significantly reduced Th1 cytokines in the colon. Conclusions: The CD80 blocking peptide appeared to mediate protection against colitis by inducing Th2 skewing of the cytokine response. Copyright © 2008 Crohn's & Colitis Foundation of America, Inc.

Bone marrow (BM) serves as a reservoir for a unique population of memory T cells. Tumor-specific T cells have been identified in the bone marrow of cancer patients. However, few direct functional analyses of tumor-specific responses in T cells isolated from BM have been performed. In this study, we investigated the ability of bone marrow T cells to induce anti-tumor immunity in mice bearing neuro2A neuroblastoma or B16 melanoma. To demonstrate the presence of antigen-specific T cells in BM, we first examined the percent of antigen-specific CD8+ T cells in vaccinated mice. The overall percent of antigen-specific CD8+ T cells was much higher in BM compared to spleen and lymph nodes of vaccinated mice. To investigate the ability of antigen-specific BM T cells to induce cytotoxic function, BM T cells were isolated from mice vaccinated with OVASIINFEKL peptide pulsed dendritic cells (DC). Expanded BM T cells from DC vaccinated mice co-cultured with M05 or irrelevant tumor cells led to tumor-specific cytotoxicity (p Citation Format: Krithika N. Kodumudi, Amy Weber, Ellen Moore, Amod Sarnaik, Shari Pilon-Thomas. Adoptive transfer of bone marrow T cells induces antitumor immune responses in murine models of melanoma and neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3136. doi:10.1158/1538-7445.AM2015-3136

Immunotherapeutic strategies incorporating intralesional (IL) ablative therapy to elicit a tumor specific immune response are under investigation as a non-surgical option to induce tumor regression of cutaneous neoplasms. Rose Bengal (RB) is a water-soluble xanthene dye that was originally used as an intravenous liver diagnostic and is in use by ophthalmologists to stain damaged cells in the eye. In murine models of breast cancer and melanoma, we have shown that IL injection of PV-10 (10% RB in saline solution) leads to ablation of injected tumors and regression of non-injected bystander tumors. In these models, increased anti-tumor T cell responses were measured, supporting the induction of systemic anti-tumor immunity after tumor ablation with PV-10. In our ongoing phase I clinical trial exploring melanoma regression in patients, IL PV-10 has led to a significant decrease of melA positive melanoma cells in the biopsies of both PV10-injected and non-injected lesions. This regression correlated with increased circulating CD3+T cells (p=0.03) in peripheral blood mononuclear cells (PBMC). T cells purified from PBMC from a melanoma patient produced increased IFN-gamma in response to autologous tumor after treatment with PV-10. Ex vivo models implemented to investigate this phenomenon indicate that the cytotoxicity induced by PV-10 is not apoptosis-dependent as evidenced by Annexin staining of melanoma cells following PV-10 treatment. PV-10 directly induced necrosis of melanoma cells at 50 uM, but was not toxic to healthy fibroblasts at the same dose. Further preclinical translational testing has shown that treatment of murine B16 cells with PV-10 leads to release of HMGB1, a soluble Damage Associated Molecule Pattern (DAMP) that is important for activation of dendritic cells (DCs). In the murine B16 melanoma model, there is a significant increase in the number of DCs infiltrating the tumor-draining lymph nodes after IL injection of PV-10. These findings suggest that PV-10 treatment leads to the release of DC activating factors and DC recruitment. Further studies to determine the role of PV-10 on T cell activation are ongoing. In sum, these clinical and preclinical results increase our understanding of the cytotoxic and immunological mechanisms that may play a role in systemic immunity induced by PV-10 tumor ablation. Citation Format: Hao Liu, Krithika Kodumudi, Amy Weber, Amod A. Sarnaik, Shari Pilon-Thomas. Induction of anti-melanoma immunity after intralesional ablative therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 630. doi:10.1158/1538-7445.AM2014-630

9028 Background: Intralesional (IL) therapy is under investigation to treat dermal and subcutaneous metastatic cancer. In our murine model, IL injection of PV-10 (10% Rose Bengal) induced regressio...

Adoptive cell therapy with tumor infiltrating lymphocytes (TIL) is a promising immunotherapeutic approach and has resulted in durable response rates of 20-30% in patients with metastatic melanoma. However, several limitations exist to prevent the widespread implementation of adoptive cell therapy, including inadequate infiltration of tumor-specific T cells into tumors and anemic expansion of tumor-specific T cells in vitro. One such factor is PD-1 expression on T cells that plays an important role in T cell exhaustion. Agonistic co-stimulatory molecules such as 4-1BB which is transiently expressed on activated T cells can reverse T cell exhaustion and enhance anti-tumor immune response. In this study, we have examined two approaches to improve the frequency of tumor-specific T cells in tumors and TIL cultures. First, we examined whether blocking PD-L1/PD-1 signaling increased the infiltration of tumor-specific T cells in the murine B16 melanoma model. Mice bearing B16 tumors were treated with NrIgG or anti-PD-1 antibodies. T cells were isolated from B16 tumors on day 18 after tumor injection. Increased IFN-γ production following B16 restimulation was observed in TIL isolated from mice treated with anti-PD-1 antibody (1445 ± 48 pg/ml) compared to TIL isolated from mice treated with control antibody (748 ± 25 pg/ml, p Citation Format: John L. Robinson, Krithika Kodumudi, Amy Weber, Amod Sarnaik, Shari Pilon-Thomas. Effects of anti-PD-1 and anti-4-1BB antibody treatment on melanoma-specific T cells in a murine model of melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3973. doi:10.1158/1538-7445.AM2013-3973