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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.

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.

The acidic pH of tumors profoundly inhibits effector functions of activated CD8 + T-cells. We hypothesize that this is a physiological process in immune regulation, and that it occurs within lymph nodes (LNs), which are likely acidic because of low convective flow and high glucose metabolism. Here we show by in vivo fluorescence and MR imaging, that LN paracortical zones are profoundly acidic. These acidic niches are absent in athymic Nu/Nu and lymphodepleted mice, implicating T-cells in the acidifying process. T-cell glycolysis is inhibited at the low pH observed in LNs. We show that this is due to acid inhibition of monocarboxylate transporters (MCTs), resulting in a negative feedback on glycolytic rate. Importantly, we demonstrate that this acid pH does not hinder initial activation of naïve T-cells by dendritic cells. Thus, we describe an acidic niche within the immune system, and demonstrate its physiological role in regulating T-cell activation., T-cell activation primarily occurs in the lymph nodes, highly organized and specialized secondary lymphoid organs. Here the authors show that the acidic extracellular pH in lymph node paracortical zones limits cytokine production by effector T-cells, but does not alter their activation by antigen-presenting cells.

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.

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.

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.

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.

The discovery of human epidermal growth factor receptor 2 (HER2) and its role in breast cancer led to the development of the first targeted antibody treatment for HER2-positive breast cancer. This treatment breakthrough led to remarkable improvements in both early and late survival. Unfortunately, not all patients with HER2 breast cancer responded positively; some have innate resistance to treatment and others develop resistance over time. In this review, we discuss some research that is currently underway to understand HER2 resistance and strategies in overcoming it.

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.

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.

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.

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.

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