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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.

BACKGROUND: Adoptive cell therapy (ACT) has had limited success due to low T cell infiltration in breast cancer (BC) patients. We have shown that administration of class II HER2 peptide pulsed type I polarized dendritic cell (DC1) vaccines leads to increase in CD4 T cells in the periphery of BC patients. In this study, we investigated the feasibility of expanding CD4 T cells from peripheral blood mononuclear cells (PBMC) of HER2-DC1 vaccinated BC patients. METHODS: PBMCs from BC patients receiving HER2-DC1 vaccines were used for CD4 T cell expansion. PBMCs were co-cultured with HER2-DC1 at 10:1 ratio for initial activation followed by expansion with cytokines- IL-2 and IL-7 or IL-15. Expanded T cells were assessed for immune marker phenotyping by flow cytometry and TCRvb analysis. For TCRvB analysis, pre, post DC1, IL-7 and IL-15 expanded CD4 T cells were pelleted for DNA isolation and sequenced using Immunoseq Analyzer platform. In addition, we investigated the in vivo efficacy of adoptively transferred mouse IL-7/IL-15 expanded Her2 specific CD4 T cells in HER2+ TUBO mouse model. RESULTS: Co-culturing of patient PBMCs with HER2-DC1 in the presence of cytokines IL-2, IL-7 and IL-15 expanded CD4 T cells ranging from 8-43 fold expansion in different samples with 92-98% CD4 phenotype. CD4 T cells expanded in IL-7 had stem like memory phenotype while IL-15 skewed to terminally differentiated CD4 T cells. IL-7 expanded T cells had significantly higher Tim3 and Ox40 expression, and effector memory proportion as compared to the IL-15 expanded T cells. Restimulation of expanded T cells with HER2 pulsed DCs showed HER2 specificity as measured by increased interferon-gamma production We identified top five clones in PBMCs from DC1-HER2 vaccinated BC patients compared to baseline. We observed differential abundance of TCR clones under IL-7 and IL-15 conditions and identified unique clones for IL-7 and IL-15. ACT using a combination of IL-7 and IL-15 expanded mouse CD4 T cells led to 50% tumor regression in HER2+ model. CONCLUSION: These studies demonstrate that HER2 specific CD4 T cells can be successfully expanded from HER2-DC1 vaccinated patients. Post HER2 DC1 vaccine can generate a pool of antigen specific CD4 T cells and may offer a promising ACT for HER2 BC. Further studies are warranted to demonstrate the efficacy in the clinical setting. Citation Format: Namrata Gautam, Krithika Kodumudi, Colin Snyder, Amber Beyer, Ricardo Costa, Heather Han, Brian Czerniecki. Adoptive T cell therapy using IL-7 and IL-15 expanded HER2-specific CD4 T cells for metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3197.

Breast cancer (BC) prevention remains the ultimate cost-effective method to reduce the global burden of invasive breast cancer (IBC). To date, surgery and chemoprevention remain the main risk-reducing modalities for those with hereditary cancer syndromes, as well as high-risk non-hereditary breast lesions such as ADH, ALH, or LCIS. Ductal carcinoma in situ (DCIS) is a preinvasive malignant lesion of the breast that closely mirrors IBC and, if left untreated, develops into IBC in up to 50% of lesions. Certain high-risk patients with DCIS may have a 25% risk of developing recurrent DCIS or IBC, even after surgical resection. The development of breast cancer elicits a strong immune response, which brings to prominence the numerous advantages associated with immune-based cancer prevention over drug-based chemoprevention, supported by the success of dendritic cell vaccines targeting HER2-expressing BC. Vaccination against BC to prevent or interrupt the process of BC development remains elusive but is a viable option. Vaccination to intercept preinvasive or premalignant breast conditions may be possible by interrupting the expression pattern of various oncodrivers. Growth factors may also function as potential immune targets to prevent breast cancer progression. Furthermore, neoantigens also serve as effective targets for interception by virtue of strong immunogenicity. It is noteworthy that the immune response also needs to be strong enough to result in target lesion elimination to avoid immunoediting as it may occur in IBC arising from DCIS. Overall, if the issue of vaccine targets can be solved by interrupting premalignant lesions, there is a potential to prevent the development of IBC.

Vaccines can be a cost effective preventive measure for both primary prevention of disease and prevention of disease recurrence. Several vaccines targeting breast cancer oncodrivers are currently being tested in clinical trials. Whereas clinical response rates to breast cancer vaccines have been modest despite the induction of strong antitumor T cell responses, it is through these approaches that valuable insight and knowledge have been gained about tumor immunology. With the emergence of new immunotherapies, there is renewed excitement for effective breast cancer vaccine development.

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.

BACKGROUND Approx. 5% of BM will also develop LMD. Currently there is no effective treatment for BC-associated BM/LMD. As systemic therapies do not prevent the disease recurrence and eventual death, the better option would be direct-targeted approach. We have shown that there is a loss of the anti-HER2 and anti-HER3 CD4 Th1 immune response in BC patients (pts). In a clinical setting, administration of class II HER2 peptide-pulsed Type I polarized dendritic cell vaccine (HER2-DC1) partially restored anti-HER2 Th1 immune responses with pathologic complete response in HER2+ BC patients. In this study, we examined the IT delivery of HER2/HER3- DC1 in BC-LMD model. METHODS Luciferase-labeled HER2+ TUBO BC cell line was injected into the cisterna magna of BALB/c mice to develop BM/LMD. We developed a technique, coined the “Top Hat” (TH) for mouse model that mimics the Ommaya reservoir in BC-pts. The TH essentially allows us to administer IT treatment directly into CSF. BC-BM/LMD bearing mice were given HER2- and Her3 peptide-pulsed Type I polarized DC1 through the TH. RESULTS AND DISCUSSION BM/LMD mice were randomized into following groups: 1) systemic Her2-DC1 2) IT Her2-DC1 3) IT Her2-/Her3-DC1. The median survival (MS) of control mice was 10 days and systemically treated mice was 19 days. IT Her2-DC1 animals did significantly better than both control and systemic treated groups (MS: 63 days; p< 0.0001) and overall survival (OS): 44%. Interestingly, mice given IT Her2-/Her3-DC1 had the best OS (78%). Surviving animals were eventually disease free. Mice that had complete tumor regression were immune to subsequent rechallenge with TUBO cells. Immune cell infiltration in the of CSF, spinal cord and tissues of experimental mice are currently ongoing. CONCLUSIONS Our preclinical data supports the clinical relevance of using intrathecal delivery of DC1 vaccine as a potential treatment for BM and LMD of BC-pts.

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.

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.

In patients with HER2-expressing breast cancer many develop resistance to HER2 targeted therapies. We show that high and intermediate HER2-expressing cancer cell lines are driven toward apoptosis and tumor senescence when treated with either CD4+ Th1 cells, or Th1 cytokines TNF-α and IFN-γ, in a dose dependent manner. Depletion of HER2 activity by either siRNA or trastuzumab and pertuzumab, and subsequent treatment with either anti-HER2 Th1 cells or TNF-α and IFN-γ resulted in synergistic increased tumor senescence and apoptosis in cells both sensitive and cells resistant to trastuzumab which was inhibited by neutralizing anti-TNF-α and IFN-γ. Th1 cytokines induced minimal senescence or apoptosis in triple negative breast cancer cells (TNBC); however, inhibition of EGFR in combination with Th1 cytokines sensitized those cells causing both senescence and apoptosis. TNF-α and IFN-γ led to increased Stat1 phosphorylation through serine and tyrosine sites and a compensatory reduction in Stat3 activation. Single agent IFN-γ enhanced Stat1 phosphorylation on tyrosine 701 and similar effects were observed in combination with TNF-α and EGFR inhibition. These results demonstrate Th1 cytokines and antioncodriver blockade cooperate in causing tumor senescence and apoptosis in TNBC and HER2-expressing breast cancer, suggesting these combinations could be explored as non-cross-reactive therapy preventing recurrence in breast cancer. Fil: Rosemblit, Cinthia. H. Lee Moffitt Cancer Center; Estados Unidos. University of Pennsylvania; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Datta, Jashodeep. University of Pennsylvania; Estados Unidos Fil: Lowenfeld, Lea. University of Pennsylvania; Estados Unidos Fil: Xu, Shuwen. University of Pennsylvania; Estados Unidos Fil: Basu, Amrita. H. Lee Moffitt Cancer Center; Estados Unidos Fil: Kodumudi, Krithika. H. Lee Moffitt Cancer Center; Estados Unidos Fil: Wiener, Doris. H. Lee Moffitt Cancer Center; Estados Unidos Fil: Czerniecki, Brian J.. H. Lee Moffitt Cancer Center; Estados Unidos. University of Pennsylvania; Estados Unidos

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.

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

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.

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.

Oncodrivers are a promising target in novel breast cancer therapy development, owing to oncogene addiction of breast cancer cells to sustain their malignancy. We have previously shown progressive loss of CD4+ T-helper type 1 response to oncodrivers in triple negative breast cancer (TNBC) patients. TNBC is marked by lack of cell surface ER, PR and Her2 receptors and is the most aggressive subtype of breast cancer. Lack of therapeutic targets, resistance to existing hormone therapy, metastasis and poor survival in TNBC patients underline the need for novel TNBC therapy development. In this study, we investigated the effect of EGFR inhibitors (cetuximab, erlotinib and lapatinib) alone or combination with Th1 cytokines (IFN-γ and TNF-α) on growth and proliferation of MDA-MB-231 and MDA-MB-468 TNBC cells. We observed decrease in EGFR expression in TNBC cells when treated with IFN-γ alone and in combination with EGFR inhibitors. In addition, IFN-γ alone, in combination with TNF-α and all three EGFR inhibitors, markedly increased STAT1 phosphorylation, indicating suppression of growth and proliferation in TNBC cells. Decreased STAT3 phosphorylation by combination treatment in TNBC cells may induce apoptosis and inhibit proliferation further. Combination of cetuximab and IFN-γ increased cleaved caspase-3 expression in MDA-MB-231, but not in MDA-MB-468 TNBC cells. EGFR inhibition and Th1 cytokine treatment showed combination treatment resulted in severe cell loss and morphological alteration, while Th1 cytokines alone did not induce significant senescence in MDA-MB-231 cells in senescence associated β-galactosidase assay. We are currently investigating activation status of other signaling pathways in TNBC cells, following Th1 cytokines treatment with EGFR inhibition. Our data suggests a combinatorial treatment approach, including DC1 vaccination to elicit Th1 immune response and inhibition of EGFR oncodriver, may lead to an effective and novel therapeutic strategy for triple negative breast cancer. Citation Format: Amrita Basu, Krithika Kodumudi, Doris Wiener, Brian Czerniecki. Th1 cytokines and EGFR inhibition: A combinatorial therapeutic strategy in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 826.

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.

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.

Adoptive transfer of tumor infiltrating lymphocytes (TIL) has emerged as a promising approach to induce effective anti-tumor immunity and tumor regression in various types of cancers. Current studies suggest that the presence of TIL play a role in patient outcomes and can predict tumor stage, node positivity, and in some reports, disease free and overall survival in patients with colorectal cancer. TIL may be inactivated in patients due to factors in the local tumor microenvironment, such as regulatory T cells, myeloid derived suppressor cells and the expression of a number of co-inhibitory immune checkpoints including PD-L1, BTLA, PD-1, CTLA-4, Lag-3 and Tim-3. We first examined the feasibility of expanding TIL from the tumors of three colorectal patients. Tumors were minced into fragments and cultured in media supplemented with 6000 IU IL2 / mL. TIL growth was observed in tumor fragments from each of the three patients with a total of 12 out of 41 fragments demonstrating TIL expansion. TIL grown out of these fragments were predominantly CD3+ with 32% CD4+T cells and 46% CD8+ T cells and 18.5% NK cells. Seven of the 12 fragments tested were tumor specific as demonstrated by IFN-gamma production in response to autologous tumor. CD8+ T cells within the patient TIL expressed PD-1, BTLA, Lag-3 and Tim-3 and a co-stimulatory molecule, 41BB. In addition, we found high levels of PD-L1 expression on tumors of colorectal patients and human colorectal cell lines. Since we observed high levels of immune checkpoints expression in colorectal patients, we wanted to develop a preclinical colorectal model to investigate the effect of immune checkpoint blockade on tumor growth and TIL function. A previous study from our lab has shown that TIL from our preclinical MC38 colorectal murine model express high levels of co-inhibitory checkpoints and that blockade of PD-L1 leads to enhanced infiltration and anti-tumor specificity of TIL. However, given the dynamic nature of immune responses to tumors and expression of multiple immune checkpoints, it may be necessary to block additional checkpoints to improve clinical efficacy. In this study, we examined whether blockade of the immune checkpoint receptor BTLA had an effect on TIL infiltration and tumor growth. Mice bearing MC-38 tumors treated with anti-BTLA antibody demonstrated a delay in tumor growth (150mm2, p Citation Format: Krithika N. Kodumudi, James Charles, Carrie Luu, John Mullinax, Julie Li, MacLean Hall, Amy Weber, Amod A. Sarnaik, Shari Pilon-Thomas. Immune checkpoint blockade to improve T cell infiltration and function in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B12.

Pancreatic cancer is an extremely aggressive malignancy with a dismal prognosis. Cancer patients and tumor-bearing mice have multiple immunoregulatory subsets including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC) that may limit the effectiveness of anti-tumor immunotherapies for pancreatic cancer. It is possible that modulating these subsets will enhance anti-tumor immunity. The goal of this study was to explore depletion of immunoregulatory cells to enhance dendritic cell (DC)-based cancer immunotherapy in a murine model of pancreatic cancer. Flow cytometry results showed an increase in both Tregs and MDSC in untreated pancreatic cancer–bearing mice compared with control. Elimination of Tregs alone or in combination with DC-based vaccination had no effect on pancreatic tumor growth or survival. Gemcitabine (Gem) is a chemotherapeutic drug routinely used for the treatment for pancreatic cancer patients. Treatment with Gem led to a significant decrease in MDSC percentages in the spleens of tumor-bearing mice, but did not enhance overall survival. However, combination therapy with DC vaccination followed by Gem treatment led to a significant delay in tumor growth and improved survival in pancreatic cancer–bearing mice. Increased MDSC were measured in the peripheral blood of patients with pancreatic cancer. Treatment with Gem also led to a decrease of this population in pancreatic cancer patients, suggesting that combination therapy with DC-based cancer vaccination and Gem may lead to improved treatments for patients with pancreatic cancer.

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.

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. Previously, IL injection of RB induced regression of injected and uninjected tumors in murine models. However, the relevant mechanism is yet unknown. In this study, we used an OVA-expressing B16 melanoma murine model and found that IL RB treatment led to increased tumor-specific T cells with memory characteristics. CD8+ T cell are crucial for tumor-specific response elicited by IL RB. IL RB therapy also increased antigen-specific T cell proliferation and enhanced tumor regression. In addition, IL RB facilitated dendritic cells (DCs) infiltrating lymph nodes draining from tumor. Incubation of melanoma cells with RB led to necrosis and the release of High Mobility Group Box 1 (HMGB1), which activated DCs via up-regulation of CD40 expression. The blockade of HMGB1 significantly reduced the antigen-presenting ability of DCs. To determine whether this mechanism was relevant in patients treated with IL RB, we performed a pilot clinical study in melanoma patients (NCT01760499). IL RB led to tumor regression in both RB-injected and uninjected lesions, associated with an increase in circulating T cells. Increased tumor-specific response was found from those circulating T cells of 5 out of 7 tested patients after IL RB treatment. HMGB1 levels in patient sera were also elevated. Together, these results reveal a clinically relevant immunoadjuvant pathway triggered by tumor cell death secondary to ablation with RB.

Purpose: Myeloid-derived suppressor cells (MDSC) accumulate in tumor-bearing hosts and are associated with immune suppression. To date, there have only been few studies that evaluate the direct effect of chemotherapeutic agents on MDSCs. Agents that inhibit MDSCs may be useful in the treatment of patients with various cancers. Experimental Design: We investigated the in vivo effects of docetaxel on immune function in 4T1-Neu mammary tumor-bearing mice to examine if a favorable immunomodulatory effect accompanies tumor suppression. Primary focus was on the differentiation status of MDSCs and their ability to modulate T-cell responses. Results: Docetaxel administration significantly inhibited tumor growth in 4T1-Neu tumor-bearing mice and considerably decreased MDSC proportion in the spleen. The treatment also selectively increased CTL responses. Docetaxel-pretreated MDSCs cocultured with OT-II splenocytes in the presence of OVA323-339 showed OT-II–specific CD4 activation and expansion in vitro. In characterizing the phenotype of MDSCs for M1 (CCR7) and M2 [mannose receptor (CD206)] markers, MDSCs from untreated tumor bearers were primarily MR+ with few CCR7+ cells. Docetaxel treatment polarized MDSCs toward an M1-like phenotype, resulting in 40% of MDSCs expressing CCR7 in vivo and in vitro, and macrophage differentiation markers such as MHC class II, CD11c, and CD86 were upregulated. Interestingly, docetaxel induced cell death selectively in MR+ MDSCs while sparing the M1-like phenotype. Finally, inhibition of signal transducer and activator of transcription 3 may in part be responsible for the observed results. Conclusions: These findings suggest potential clinical benefit for the addition of docetaxel to current immunotherapeutic protocols. Clin Cancer Res; 16(18); 4583–94. ©2010 AACR.

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

PV-10 is a 10% solution of Rose Bengal that is currently being examined as a novel cancer therapeutic. In melanoma patients, intralesional injection (IL) of PV-10 has led to regression of injected lesions as well as distant metastases. In this study, we examined the efficacy and potential immune mechanism of PV-10 treatment in murine models of breast cancer and melanoma. In BALB/c mice bearing MT-901 breast cancer, injection of PV-10 led to regression of injected and untreated contralateral subcutaneous lesions (p Citation Format: Shari A. Pilon-Thomas, Amy Weber, Krithika Kodumudi, Lisa Kuhn, Paul Toomey, Amod Sarnaik. Intralesional injection with PV-10 induces a systemic anti-tumor immune response in murine models of breast cancer and 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 1248. doi:10.1158/1538-7445.AM2013-1248