Source Of Tumor Antigens Research Articles

Discovery of high-expressing lncRNA-derived sORFs as potential tumor-associated antigens in hepatocellular carcinoma.

This study is based on deep mining of Ribo-seq data for the identification of lncRNAs that have highly expressed sORFs in HCC. In this paper, dynamic prospects associated with sORFs acting as newly defined tumor-specific epitopes are discussed with possible improvement in strategies for tumor immunotherapy. Using ribosome profiling to identify and characterize sORFs within lncRNAs in HCC, identify potential therapeutic targets and tumor-specific epitopes applicable for immunotherapy. MetamORF performed the identification of sORFs with deep analysis of the data of ribosome profiling in lncRNAs associated with HCC. The translation efficiency in these molecules was estimated, and epitope prediction was done by pVACbind. Peptide search was done to check the presence of micropeptides translated from these identified sORFs. validated translational activity and identified potential epitopes. Higher translation efficiency was noted in the case of lncRNAs associated with HCC compared to normal tissues. Of particular note is ORF3418981, which results in the highest expression and has supporting experimental evidence at the protein level. Epitope prediction identified a putative epitope at the C-terminus of ORF3418981. This study uncovers the as-yet-unknown potential of lncRNA-derived sORFs as sources of tumor antigens, shifting the research focus from protein-coding genes to non-coding RNAs also in the HCC context. Moreover, this study highlights the contribution of a subset of lncRNAs, especially LINC00152, to the development of tumors and modulation of the immune response by its sORFs.

Knowledge mapping of image-guided tumor ablation and immunity: A bibliometric analysis.

Various ablation techniques have been successfully applied in tumor therapy by locally destroying tumor. In the process of tumor ablation, a large number of tumor cell debris is released, which can be used as a source of tumor antigens and trigger a series of immune responses. With the deepening of the research on the immune microenvironment and immunotherapy, researches exploring tumor ablation and immunity are continuously published. However, no research has systematically analyzed the intellectual landscape and emerging trends for tumor ablation and immunity using scientometric analysis. Therefore, this study aimed to conduct a bibliometric analysis to quantify and identify the status quo and trend of tumor ablation and immunity. Data of publications were downloaded from the Web of Science Core Collection database. CiteSpace and VOSviewer were used to conduct bibliometric analysis to evaluate the contribution and co-occurrence relationship of different countries/regions, institutions and authors in the field, and to determine the research hotspots in this field. By searching in the database, a total of 3531 English articles published between 2012 and 2021 were obtained. We observed rapid growth in the number of publications since 2012. The two most active countries were China and the United States, with more than 1,000 articles. Chinese Academy of Sciences contributed the most publications (n = 153). Jibing Chen and Xianzheng Zhang might have a keen interest in tumor ablation and immunity, with more publications (n = 14; n = 13). Among the top 10 co-cited authors, Castano AP (284 citations) was ranked first, followed by Agostinis P (270 citations) and Chen Qian (246 citations). According to the co-occurrence and cluster analysis, the results indicated that the focus of research was "photothermal therapy" and "immune checkpoint blockade". In the past decade, the neighborhood of tumor ablation domain immunity has been paid more and more attention. Nowadays, the research hotspots in this field are mainly focused on exploring the immunological mechanism in photothermal therapy to improve its efficacy, and the combination of ablation therapy and immune checkpoint inhibitor therapy.

Comparison of tumor-derived total RNA and cell lysate on antitumor immune activity

Tumor-derived total RNA (TdRNA) and cell lysate (TCL), with almost all the relevant tumor antigens, represent attractive alternative sources of antigens in antitumor immunotherapy. However, the comparison of their capacity to elicit immune responses against breast cancer is still lacking. In this study, the antitumor immune effects of TdRNA and TCL were systematically compared. We isolated TdRNA and TCL from 4T1 mouse breast cancer cells, and found that both sources of antigens could stimulate the maturation of dendritic cells (DCs) at the cellular and in vivo levels, and induce robust cellular immune responses, as evidenced by the increased percentages of both CD4+ and CD8+ T cells in the inguinal lymph nodes and spleen. But TdRNA performed stronger immunoactivities than TCL on the increase of T cell population through DCs activation. Additionally, the synergistic antitumor efficacy of paclitaxel (PTX) with TdRNA and TCL respectively was further evaluated in the murine 4T1 tumor model. Compared with TCL, TdRNA could inhibit tumor growth more effectively with low systemic toxicity when combined with PTX, which was, at least in part, attributable to the improvement of systemic immune function and tumor immune infiltration. Overall, TdRNA outperforms TCL in antitumor immunity, and is expected to be a promising candidate for application as the source of tumor antigens.

Soluble HLA peptidome of pleural effusions is a valuable source for tumor antigens

BackgroundSoluble human leucocyte antigen (sHLA) molecules, released into the plasma, carry their original peptide cargo and provide insight into the protein synthesis and degradation schemes of their source cells and...

Cytoplasmic Transduction Peptide-Fused Recombinant Tumor-Associated Antigens Elicit Potent Myeloma-Specific Cytotoxic T Lymphocytes By Loading Onto Dendritic Cells: Implications of Feasible Tumor Antigens for Clinical Application Against Multiple Myeloma

Dendritic cell (DC)-based vaccines are one of promising tools in patients with multiple myeloma (MM). However, the source of tumor antigens is still challenging in this field. Here, we evaluated the feasibility of tumor-associated antigens (TAAs) to be loaded onto DCs for improving the efficacy of immunotherapy against MM. Quantitative PCR was performed to evaluate the expression of TAAs from malignant plasma cells isolated from bone marrow mononuclear cells of MM patients. Based on quantitative PCR analysis, 4 cancer testis antigens (SSX2, SSX4, MAGE-A3 and MAGE-C2), hTERT, BCMA, and CD38 were selected to make CTP-fused recombinant TAAs. Then, we made cytoplasm transduction peptide (CTP)-fused recombinant proteins (CTP-SSX2, CTP-SSX4, CTP-MAGEA3, CTP-MAGEC2, CTP-hTERT, CTP-BCMA, and CTP-CD38) for the efficient delivery of TAAs to DCs. After determination of optimal protein dose for loading onto DCs, several functional studies were performed by the DCs. The CTP-fused recombinant TAAs did not deteriorate the function of DCs in terms of phenotype expressions and cytokine productions, including a Th1-polarizing capacity of naïve T cells. DCs loaded with each CTP-fused recombinant TAA had successfully generated a myeloma-specific cytotoxic T lymphocyte (CTL) to kill myeloma cell lines and primary myeloma cells. This study showed that CTP-fused recombinant myeloma-associated proteins were a useful tumor antigen source to be loaded onto DCs for generation of myeloma-specific CTLs in vitro . Therefore, CTP-fused recombinant TAAs will become a feasible tumor antigen for the clinical application of DC-based cancer immunotherapy in the field of MM. DisclosuresNo relevant conflicts of interest to declare.

Towards the rational design of a next-generation dendritic cell vaccine for cancer immunotherapy

As professional antigen presenting cells (APCs) capable of eliciting primary immune responses among naïve T cells, dendritic cells (DCs) offer an attractive target for immune intervention. While some strategies for vaccination have sought to deliver antigens direct to DCs&nbsp;<em>in vivo</em>, others have pulsed DCs with target antigens&nbsp;<em>ex vivo</em>&nbsp;prior to administration. Indeed, numerous clinical studies of cancer immunotherapy have been conducted over the past two decades based on this approach, most of them benefitting from the ease with which DCs may be differentiated&nbsp;<em>in vitro</em>&nbsp;from the peripheral blood monocytes of individual patients. Nevertheless, while therapies exploiting monocyte-derived DCs (moDCs) have been shown to be safe, clinical outcomes have been disappointing, efficacy having been limited by factors including the type of DCs used and the source of tumor antigens. Here we review recent developments in identifying DC subsets with more favorable properties for use in cancer vaccination, with particular emphasis on CD141⁺&nbsp;DCs capable of antigen cross-presentation and discuss alternative sources, such as induced pluripotent stem cells (iPSCs), amenable to manufacture at scale. Furthermore, we assess how different sources of tumor antigens may complement this approach for the design of next generation DC vaccines.

Comparison of Exosomes Derived from Non- and Gamma-Irradiated Melanoma Cancer Cells as a Potential Antigenic and Immunogenic Source for Dendritic Cell-Based Immunotherapeutic Vaccine.

Cancer cells can secrete exosomes under various stressful conditions, whose functions are involved in the delivery of various biologically active materials into host cells and/or modulation of host immune responses. Therefore, an improved understanding of the immunological interventions that stress-induced tumor exosomes have may provide novel therapeutic approaches and more effective vaccine designs. Here, we confirmed the phenotypical and functional alterations of dendritic cells (DCs), which act as a bridge between the innate and adaptive arms of immunity, following non-irradiated (N-exo) and gamma-irradiated melanoma cancer cell-derived exosome (G-exo) stimulation, and evaluated the N-exo- and G-exo-stimulated DCs as therapeutic cancer vaccine candidates. We demonstrated that G-exo-stimulated DCs result in DC maturation by the upregulation of surface molecule expression, pro-inflammatory cytokine release, and antigen-presenting ability, and the downregulation of endocytic capacity. In addition, these cells promoted T cell proliferation and the generation of T helper type 1 (Th1) and interferon (IFN)-γ-producing CD8+ T cells. However, N-exo-stimulated DCs induced semi-mature phenotypes and functions, eventually inhibiting T cell proliferation, decreasing IFN-γ, and increasing IL-10-producing CD4+ T cells. In addition, although N-exo and G-exo stimulations showed similar levels of antigen-specific IFN-γ production, which served as tumor antigen sources in melanoma-specific T cells, G-exo-stimulated DC vaccination conferred a stronger tumor growth inhibition than N-exo-stimulated DC vaccination; further, this was accompanied by a high frequency of tumor-specific, multifunctional effector T cells. These results suggest that gamma irradiation could provide important clues for designing and developing effective exosome vaccines that can induce strong immunogenicity, especially tumor-specific multifunctional T cell responses.

Development and Optimization of a GMP-Compliant Manufacturing Process for a Personalized Tumor Lysate Dendritic Cell Vaccine.

With the emergence of immune checkpoint inhibitors and adoptive T-cell therapies, there is a considerable interest in using personalized autologous dendritic cell (DC) vaccines in combination with T cell-targeting immunotherapies to potentially maximize the therapeutic impact of DC vaccines. Here, we describe the development and optimization of a Good Manufacturing Practice (GMP)-compliant manufacturing process based on tumor lysate as a tumor antigen source for the production of an oxidized tumor cell lysate loaded DC (OC-DC) vaccine. The manufacturing process required one day for lysate preparation and six days for OC-DC vaccine production. Tumor lysate production was standardized based on an optimal tumor digestion protocol and the immunogenicity was improved through oxidation using hypochloric acid prior to freeze-thaw cycles resulting in the oxidized tumor cell lysate (OC-L). Next, monocytes were selected using the CliniMACS prodigy closed system and were placed in culture in cell factories in the presence of IL-4 and GM-CSF. Immature DCs were loaded with OC-L and matured using MPLA-IFNγ. After assessing the functionality of the OC-DC cells (IL12p70 secretion and COSTIM assay), the OC-DC vaccine was cryopreserved in multiple doses for single use. Finally, the stability of the formulated doses was tested and validated. We believe this GMP-compliant DC vaccine manufacturing process will facilitate access of patients to personalized DC vaccines, and allow for multi-center clinical trials.

IMMU-32. RNA-NANOPARTICLE VACCINES MEDIATE T CELL TRAFFICKING NECESSARY FOR BBB PASSAGE AND ANTI-GLIOMA IMMUNE RESPONSE

Abstract BACKGROUND The blood-brain barrier (BBB) remains a potent obstacle for development of new therapies against glioblastoma (GBM). While activated T cells can cross the BBB, immunotherapy has yet to be fully unlocked for malignant brain tumors due their heterogeneity and immunosuppressive microenvironments. To overcome these challenges, our group has developed a novel treatment platform, which leverages the use of a clinically translatable nanoparticles (NPs) combined with personalized tumor derived mRNA to peripherally activate T cells against a heterogenous source of tumor antigens and reprogram the intratumoral milieu into an immune activated state. OBJECTIVE We sought to assess if RNA-NPs could activate systemic/intratumoral dendritic cells (DCs) and mediate a peripheral T cell response that could penetrate the GBM microenvironment. RESULTS We uncovered that RNA-NPs elicit potent innate immunomodulating effects through release of interferon-α (IFN-α) from plasmacytoid DCs (pDCs). After only a single RNA-NP vaccine, the bulk of systemic and intratumoral DCs in mice display an activated phenotype; DCs, harvested from intracranial tumors, elicit expansion of antigen specific T cell immunity. Tumor-specific RNA-NPs elicited enhanced survival outcomes in immunocompetent animals bearing NF-1/p53 mutant gliomas with increased intratumoral memory CD8+T-cells. Unlike immune checkpoint blockade (anti-PD-L1 mAbs), we found that RNA-NPs increase LFA-1 on peripheral CD8 splenocytes, which is necessary for activated T cell passage across the BBB. RNA-NPs also increased CCR2 on peripheral CD8 cells, which was dependent on IFN-α/β, as the percentage of CCR2+CD8+ splenocytes and anti-tumor activity (mediated by RNA-NPs) was abrogated in animals receiving concomitant type I IFN receptor (IFNAR1) mAbs. CONCLUSION Since LFA-1 is important for T cell trafficking across the BBB and CCR2 may promote chemotaxis to the brain (as GBMs are known to secrete CCL2), RNA-NPs may offer a new treatment modality and immunologic mechanism for unlocking peripheral T cell immunity against malignant gliomas.

Abstract 3188: Transimmunization prevents recurrence and reprograms the immune milieu in a mouse model of ovarian cancer

Abstract Introduction: Targeting recurrent disease is vital in the improvement of survival in ovarian cancer patients. Transimmunization (TI) is a modification of extracorporeal photochemotherapy, which is a dendritic cell (DC)-based vaccination, currently used in cutaneous T cell lymphoma but has never been tested in gynecologic tumors. The objective of this study is to determine if TI can induce a tumor-specific immune response capable of preventing recurrence in ovarian cancer. Methods of study: Ovarian cancer cells expressing mCherry fluorescent protein were injected i.p. in C57bl/6 mice to mimic the establishment of recurrent disease. These cells (TKO cancer cells) were isolated from spontaneously formed ovarian tumors from p53LSL-R172H/+Dicerflox/floxPtenflox/flox Amhr2cre/+ mice. Tumor burden was quantified using mCherry fluorescence ROI area. TI vaccination was conducted as follows: (1) cancer cells were treated with 8-MOP and exposed to UVA to induce apoptosis; (2) PBMCs were collected from tumor-bearing mice, mixed with apoptotic cancer cells, and circulated through the TI chamber to induce functional DC from monocytes; (3) cells collected were incubated overnight to facilitate antigen uptake and presentation and injected i.p. Treatment groups were as follows: (1) PBS control, n = 20 ; (2) TI TKO, n =10 (i.e. using apoptotic TKO cells); and (3) TI YUMM, n = 10 (using apoptotic mouse melanoma YUMM1.7 cells as antigen-unrelated control). Immune milieu was characterized by IHC on tumors and FACS analysis using cells isolated from peritoneal lavage. Results: Eighty percent of mice in the TI TKO group were disease free compared to 35% in the PBS control group and 40% in the TI YUMM group. In addition, TI TKO vaccination (p &amp;lt; 0.0001), but not TI YUMM vaccination (p = 0.1079) induced a statistically significant decrease in tumor kinetics compared to PBS control. IHC analysis showed significantly higher levels of infiltrating CD8+ (p = 0.0037) and CD4+ T cells (p = 0.0079) in TI TKO group compared to PBS group. Similarly, mice in the TI TKO group have higher i.p. levels of CD8+/CD44+/CD62L+ central memory T cells (p = 0.004), higher levels of CD8+/CD44+/CD62L- effector memory T cells (p = 0.03), lower levels of CD8+/CD44-/CD62L+ naïve T cells (p = 0.02), and lower levels of CD11b+/Gr-1high myeloid-derived suppressor cells (p = 0.01) compared to PBS control group. Levels of these cells in TI YUMM group were not significantly different from the PBS control. Conclusion: We demonstrate for the first time that TI vaccination with autologous cancer cells can prevent recurrence and modify the immune phenotype in ovarian cancer. TI efficacy depends on the tumor antigen source, confirming TI as an antigen-specific DC-based immunotherapy. Our results highlight the value of TI in ovarian cancer and its potential application to other i.p. cancers. Citation Format: Ayesha B. Alvero, Douglas Hanlon, Mary Pitruzzello, Renata Filler, Eve Robinson, Olga Sobolev, Roslyn Tedja, Alessandra Ventura, Richard L. Edelson, Gil Mor. Transimmunization prevents recurrence and reprograms the immune milieu in a mouse model of ovarian 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 3188.

Biomaterial-Based Activation and Expansion of Tumor-Specific T Cells.

Traditional tumor vaccination approaches mostly focus on activating dendritic cells (DCs) by providing them with a source of tumor antigens and/or adjuvants, which in turn activate tumor-reactive T cells. Novel biomaterial-based cancer immunotherapeutic strategies focus on directly activating and stimulating T cells through molecular cues presented on synthetic constructs with the aim of improving T cell survival, more precisely steer T cell activation and direct T cell differentiation. Synthetic artificial antigen presenting cells (aAPCs) decorated with T cell-activating ligands are being developed to induce robust tumor-specific T cell responses, essentially bypassing DCs. In this perspective, we approach these promising new technologies from an immunological angle, first by identifying the CD4+ and CD8+ T cell subtypes that are imperative for robust anti-cancer immunity and subsequently discussing the molecular cues needed to induce these cells types. We will elaborate on how biomaterials can be applied to stimulate T cells in vitro and in vivo to improve their survival, activation and function. Scaffold-based methods can also be used as delivery vehicles for adoptive transfer of T cells, including tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor expressing (CAR) T cells, while simultaneously stimulating these cells. Finally, we provide suggestions on how these insights could advance the field of biomaterial-based activation and expansion of tumor-specific T cells in the future.

Radiofrequency ablation of liver metastasis: potential impact on immune checkpoint inhibitor therapy.

Percutaneous radiofrequency ablation (RFA), a generally accepted alternative therapy for patients with liver metastases, is a minimally invasive approach with a favorable safety profile and a lower rate of major complications. The use of RFA or combined RFA plus resection can produce total tumor clearance in patients with unresectable liver metastases. However, the relatively high rate of local tumor progression has prevented the widespread use of RFA. Furthermore, its efficacy is controversial because there have been no comparisons for its effect on overall survival compared with standard options such as systemic chemotherapy. Meanwhile, immunotherapy has become a major research focus for oncology based on the recent successes reported for immune checkpoint inhibitors for melanoma, non-small cell lung cancer, gastric cancer, and other cancers. Immune checkpoints negatively regulate T cell function, and inhibition prevents the blockade of the immune system by cancer cells to prevent their destruction. Unfortunately, only some patients (< 25%) respond to immuno-oncology drugs, whereas other patients acquire resistance. However, RFA can induce massive necrotic cell death which might activate immunity and the presentation of cryptic antigens to induce tumor-specific T cell response. Because RFA can induce the rapid release of large amounts of tumor antigens, it can potentially stimulate transient immune responses to much tumor antigens. Combination therapies have induced synergistic enhancement of anticancer immune response in preclinical studies, indicating great promise for the future of oncologic treatment.Key Points • Only some patients respond to immuno-oncology drugs. • RFA causes the release of large amounts of cellular debris, a source of tumor antigens that elicit immune responses against tumors. • Combination RFA for liver metastases and immune checkpoint inhibitor therapies might synergistically enhance antitumor immunity.

Dendritic cells loaded with the lysate of tumor cells infected with Newcastle Disease Virus trigger potent anti-tumor immunity by promoting the secretion of IFN-γ and IL-2 from T cells.

Dendritic cells (DCs) are professional antigen-presenting cells that are pivotal in the generation and sustainability of antitumor immune responses. Whole tumor cell lysates (TCLs) have been used as sources of tumor antigens for the development of DC vaccines. However, the clinical outcomes of the use of TCL-based DC vaccines have so far been unsatisfactory because of the weak immunogenicity of tumor cells. To improve the efficacy of TCL-based DC vaccines, viruses have been used to enhance the immunity of TCLs and to further enhance the antigen delivery and antigen-presenting ability of DCs. The aim of the present study was to improve the antigen-presenting ability of DCs and to use them to effectively activate T lymphocytes. The present study demonstrated that DCs loaded with the lysate of Newcastle Disease Virus (NDV)-infected tumor cells (NDV-TCL) have increased levels of cluster of differentiation 80 (CD80), CD86, CD83 and human leukocyte antigen-antigen D-associated expression, compared with those loaded with TCL alone. The DCs loaded with the NDV-TCL promoted T-cell proliferation and antitumor cytokine secretion from T cells. These results indicated that loading DCs with NDV-TCL could enhance the antigen-presenting ability of the DCs. On the basis of the results of the present study, we hypothesize that this method of loading DCs with NDV-TCL can be used to develop novel DC vaccines for tumor immunotherapy in the future.

Development of a vaccine based on bacteria-mimicking tumor cells coated with novel engineered toll-like receptor 2 ligands.

For a successful tumor vaccine, it is necessary to develop effective immuno‐adjuvants and identify specific tumor antigens. Tumor cells obtained from surgical or biopsy tissues are a good source of tumor antigens but, unlike bacteria, they do not induce strong immune responses. Here, we designed 2 novel lipopeptides that coat tumor cell surfaces and mimic bacterial components. Tumor cells coated with these lipopeptides (called bacteria‐mimicking tumor cells [BMTC]) were prepared and their efficacy as a tumor vaccine examined. Natural bacterial lipopeptides act as ligands for toll‐like receptor 2 (TLR2) and activate dendritic cells (DC). To increase the affinity of the developed lipopeptides for the negatively charged plasma membrane, a cationic polypeptide was connected to Pam2Cys (P2C), which is the basic structure of the TLR2 ligand. This increased the non‐specific binding affinity of the peptides for the cell surface. Two such lipopeptides, P2CSK11 (containing 1 serine and 11 lysine residues) and P2CSR11 (containing 1 serine and 11 arginine residues) bound to irradiated tumor cells via the long cationic polypeptides more efficiently than the natural lipopeptide MALP2 (P2C‐GNNDESNISFKEK) or a synthetic lipopeptide P2CSK4 (a short cationic polypeptide containing 1 serine and 4 lysines). BMTC coated with P2CSR11 or P2CSK11 were efficiently phagocytosed by DC and induced antigen cross‐presentation in vitro. They also induced effective tumor‐specific cytotoxic T cell responses and inhibited tumor growth in in vivo mouse models. P2CSR11 activated DC but induced less inflammation‐inducing cytokines/interferons than other lipopeptides. Thus, P2CSR11 is a strong candidate antigen‐specific immuno‐adjuvant, with few adverse effects.

P3.07-008 Development of Immunomonitoring Assays for Dendritic Cell-Based Lung Cancer Immunotherapy

Allogeneic cancer cell lines might serve as a universal source of tumor antigens in the development of dendritic cell-based cancer vaccines. We showed that selected antigenic profile of lung cancer cell lines overlaps with antigenic profile of primary non/small cell lung cancer (NSCLC) tumors. However, it is unclear if T cells responses to all of these antigens can be detected in blood of NSCLC patients.

Neem leaf glycoprotein generates superior tumor specific central memory CD8+ T cells than cyclophosphamide that averts post-surgery solid sarcoma recurrence.

The success of cancer vaccines is limited as most of them induce corrupted CD8+ T cell memory populations. We reported earlier that a natural immunomodulator, neem leaf glycoprotein (NLGP), therapeutically restricts tumor growth in a CD8+ T cell-dependent manner. Here, our objective is to study whether memory CD8+ T cell population is generated in sarcoma hosts after therapeutic NLGP treatment and their role in prevention of post-surgery tumor recurrence, in comparison to the immunostimulatory metronomic cyclophosphamide (CTX) treatment. We found that therapeutic NLGP and CTX treatment generates central memory CD8+ T (TCM) cells with characteristic CD44+CD62LhighCCR7highIL-2high phenotypes. But these TCM cells are functionally impaired to prevent re-appearance of tumors along with compromised proliferative, IL-2 secretive and cytotoxic status. This might be due to the presence of tumor load, even a small one in the host, which serves as a persistent source of tumor antigens thereby corrupting the TCM cells so generated. Surgical removal of the persisting tumors from the host restored the functional characteristics of memory CD8+ T cells, preventing tumor recurrence after surgery till end of the experiment. Moreover, we observed that generation of superior TCM cells in NLGP treated surgically removed tumor hosts is related to the activation of Wnt signalling in memory CD8+ T cells with concomitant inhibition of GSK-3β and stabilisation of β-catenin, which ultimately activates transcription of Wnt target genes, like, eomesodermin, a signature molecule of CD8+ TCM cells.

Immunotherapeutic efficacy of a Lactobacillus casei lysate as an adjuvant combined with a heated-4T1 mammary carcinoma cell lysate in a murine model of breast cancer

Abstract Background Immunotherapy, during which the immune system of the patient is manipulated to act against tumors has been among the most successful methods in the treatment of breast cancer, a leading cause of mortality among women worldwide. Objectives To investigate the immunotherapeutic efficacy of Lactobacillus casei lysate as an adjuvant in combination with a heated-4T1 mammary carcinoma cell lysate in a model of breast cancer. Methods After ethics committee approval of all animal procedures, a murine model of breast cancer was induced in BALB/c mice using 4T1 cells. These mice were immunized with a combination of lysates of heated 4T1 cells and L. casei. Subsequent changes in tumor size and weight, and the production of TNF-α, IL-2, IL-12, IL-17, and IL13 were measured. Lung weights were measured as an indicator of metastasis to other organs. Results The tumor size and weight in mice immunized with the combined vaccine were significantly reduced compared with controls. The combined immunotherapy altered the pattern of cytokine production to the advantage of antitumor immunity, and was significantly more potent than immunization with heated-4T1-cell lysate or L. casei lysate alone. Conclusions Coadministration of L. casei lysate enhanced the immunotherapeutic efficacy of the heated-4T1-cell lysate as a source of tumor-associated antigens. L. casei can potentially be used as an adjuvant combined with sources of tumor antigens in the treatment of cancers, and as a safe alternative to the current adjuvants that cause greater irritation to hosts. Further studies are required to clarify the mechanisms underlying these effects.

Systemic delivery of complexes of melanoma RNA with mannosylated liposomes activates highly efficient murine melanoma-specific cytotoxic T cells in vivo

The efficiency of the antitumor immune response triggered by dendritic cell (DC)-based vaccines depends predominantly on the efficiency of delivering tumor antigen-coding nucleic acids into DCs. Mannosylated liposomes were used to deliver tumor total RNA into DCs both ex vivo and in vivo, and the cytotoxic T-lymphocyte (CTL) antitumor response was assayed. The liposomes contained the mannosylated lipid conjugate 3-[6-(α-D-mannopyranosyloxy)hexyl]amino-4-{6-[rac-2,3-di(tetradecyloxy)prop-1-yl oxycarbonylamino]hexyl}aminocyclobut-3-en-1,2-dione), the polycationic lipid 2X3 (1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride), and the zwitterionic lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) at a molar ratio of 1: 3: 6 and were used as a transfection agent. Total RNA isolated from B16-F10 mouse melanoma cells served as a source of tumor antigens. Systemic administration of mannosylated liposomes-tumor RNA complexes into circulation of melanoma-bearing mice induced an efficient CTL response, which reduced the melanoma cell index in vitro with the same efficiency (by a factor of 2.8) as CTLs activated via an inoculation of DCs loaded with complexes of the same composition ex vivo. Complexes of tumor RNA with control liposomes, which lacked the mannosylated lipid conjugate, or DCs transfected with these complexes ex vivo were less efficient and reduced the melanoma cell count by a factor of only 1.6-1.8.

Cross-Presentation Is a Source of Tumor Antigens for Multiple Myeloma Immunotherapy

IntroductionMultiple myeloma (MM) remains incurable and accounts for 20% of deaths from hematologic malignancies. Immune modulating drugs and immunotherapy have been shown to be beneficial in MM patients, highlighting the importance of the immune system in MM. The success of these interventions relies on the unique ability of the immune system to specifically distinguish between MM and normal cells. One example involves the use of cytotoxic T-lymphocytes (CTL) that eliminate cancer cells by recognizing tumor specific antigens (TSA) that are presented on human leukocyte antigen (HLA) class I molecules on the tumor cells. TSA are presented on HLA class I via the canonical antigen-processing pathway, which causes the tumor cells to become targets for killing by CTL. On the other hand, exogenous antigens are typically presented on HLA class II molecules. Through cross-presentation, exogenous antigens are taken up and presented on HLA class I molecules. We have recently identified cross-presentation as a mechanism by which tumor cells, including MM, present exogenous antigens, leading to an expanded repertoire of possible tumor antigens that can be targeted. Here we demonstrate that targeting cross-presented antigens is a feasible immunotherapeutic strategy in MM. To this end, we used the anti-PR1/HLA-A2 antibody, 8F4, which effectively eliminates PR1/HLA-A2-expressing tumor cells, and identified novel cross-presented antigens.Experimental designNSG mice were sublethally irradiated with 200 Rad. After 24 hours, 2x106 U266 cells transduced with luciferase/GFP were IV injected. Engraftment was confirmed by measuring human IgE in the mouse serum by ELISA and bioluminescent imaging (BLI). Mice were treated with 8F4 (10 mg/kg), IgG isotype control (10 mg/kg) or PBS. Mice were sacrificed on week 9, bone marrow was harvested and analyzed by flow cytometry.To identify novel cross-presented antigens, polymorphonuclear cells (PMN) were isolated from HLA-A2- healthy donors. PMN were lysed and cultured with U266 cells for 24 hours at a 2:1 PMN:U266 cell ratio. U266 cells were lysed, incubated with anti-HLA-ABC antibody and protein-A/G-sepharose beads. Immunoprecipitated proteins were recovered by mild acid elution and peptides were discovered using tandem mass spectrometry. HLA-A2 specific peptides with strong predicted in silico binding were analyzed using the HLArestrictor 1.2 algorithm.Antigen processing machinery (APM) of primary patient samples were analyzed using western blots.Results8F4 treated mice showed a 2 fold decrease in the percent of U266 in the bone marrow compared to isotype and untreated mice (P<0.05) (Figure 1). ELISA demonstrated approximately a 3-fold decrease in human IgE ng/mL in serum of mice treated with 8F4 (P<0.001). BLI quantification showed approximately a 0.5 fold decrease in total flux (p/s) in mice treated with 8F4 compared to isotype and untreated mice. In addition to illustrating the therapeutic potential of targeting PR1, we also sought to identify other cross-presented MM peptides. Initially, over 4x103 total peptides were discovered by mass spectrometry (Figure 2). Using our reality score indexing and bioinformatics we identified approximately 5x102 unique, high confidence, cross-presented peptides. Our top candidate peptides have predicted HLA-A2 binding affinities of <18 nM, ion scores >21, retention time >30.42 minutes, and are rank 1 for their spectral matches. Since we have demonstrated the capability of MM to cross-present antigens, we investigated whether the APM was dysregulated in MM patients. Patients show dysregulation in tapasin, LMP2/7, and calnexin by western blot analysis.ConclusionCollectively, our data demonstrate that the MM antigen repertoire is much larger than previously appreciated, and that there is a new catalogue of potential immunotherapeutic targets in MM derived from exogenous antigens. [Display omitted] [Display omitted] DisclosuresSergeeva:Astellas Pharma: Patents & Royalties. Molldrem:Astellas Pharma: Patents & Royalties.

A Phase I/II Study of Vaccination By Autologous Leukemic Apoptotic Corpse Pulsed Dendritic Cells for Elderly Acute Myeloid Leukemia Patients in First or Second Complete Remission (LAM DC trial)

Introduction: A number of approaches have been explored to prevent relapse in AML setting, including immune-strategies such as dendritic cells (DC) vaccination. There is no report so far of the use of autologous apoptotic leukemic cells as a source of tumor antigen for DC vaccine.Methods: The main objective of this prospective monocentric Phase I/II study was to explore the feasibility to produce autologous leukemic apoptotic corpse-pulsed DC for elderly AML patients in first or second complete remission (CR) and to report the toxicity of such a vaccine. Inclusion criteria were AML (except promyelocytic) patients older than 59 years with a good performans status (ECOG <=2), not eligible for allogeneic transplantation or another trial, with >=50% of leukemic blasts in bone marrow (BM) or peripheral blood, and with no contra-indications to apheresis. Vaccines were produced by Nantes UTCG according to good manufacturing practices for cell and gene based therapies in order to comply to the French AFSSAPS (currently ANSM) agency guidelines. Patients had to be pre-included (refractory or not) at diagnosis or at time of first relapse in order to collect sufficient leukemic cells (>2.4 108) prior to chemotherapy after 1 or 2 days of collection. After blasts collection, the choice of chemotherapy regimen was at the discretion of the investigator. Few courses of chemotherapy were allowed before vaccine production but not after. Patients were definitively included only in case of CR to allow collecting autologous non-leukemic peripheral monocytes by apheresis to generate the DC vaccine. Patients were programmed to receive up to 5 doses of vaccine (days +1 +7 +14 +21 and +35 +2) which consisted of 10 millions pulsed DC, including 9 millions administered subcutaneously (1 mL) and 1 million administered intradermally (0.1mL). Minimal residual disease (MRD) was studied after vaccines using flow cytometry.Results: Between November 2009 and March 2015, 23 patients were pre-included but 2 patients were excluded for analyses because blast collection was finally not performed. Thus, overall, 21 elderly AML patients (male n=14; median age: 74 years (range: 65-84), secondary AML n=8) were considered either at time of diagnosis or at time of first relapse. The median % of BM blasts was 63% (range: 20-92), including 3 and 4 cases with less than 40% and between 40-49%, respectively (protocol deviation). Although it was not the case for one patient with >50% of BM blasts, all patients between 40-49% of BM blasts reached the threshold of 2.4x108blasts required for the study. Two patients out of 3 with less than 40% BM blasts had insufficient blast collection to pursue the protocol. After blast collection, the majority of patients (n=19) received non-intensive chemotherapy. 5/21 (24%) cases achieved CR, a rate that was expected for this very old population. All of CR patients could proceed to apheresis after 2 (n=4) or 4 (n=1) courses of non-intensive consolidation. Production of the 5 vaccines was possible for all of them and first infusion was made at a median of 25 days (range: 20-28) from the apheresis. However, a median of 27 vaccines (range: 8-85) could have been theoretically produced in CR patients, suggesting the possibility to realize a longer maintenance therapy to prevent relapse in the future.All patients received as expected the 5 vaccines and no adverse events were documented. Durations of response from CR were: +8.5, +8, +4.5, +4, +12 months and from first vaccine: +5.5, +4.5, +1.8, +1.8, and +9 months. Two patients had relapsed before day+55. At this time, the 3 other patients were documented with negative MRD. In July 2016, 2 patients are still alive, 1 at +30 months from CR in relapse and 1 at +13 months in CR. The 3 other cases died of relapse at +15.5, +8 and +5.5 months from CR. The median OS from pre-inclusion was significantly higher for vaccinated CR patients (13 months (9-41) vs 4.75 months (1-24), p=0.009).Conclusion: Our strategy seems promising for elderly AML patients achieving CR in terms of relapse prevention. Vaccine production is reproducible and compliant for clinical use. Larger Phase 2 studies are required to confirm our results in younger and older AML population. The trial is registered at Clinicaltrials.gov NCT01146262. This study was supported by a grant from the French National Cancer Institute. DisclosuresMoreau:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Janssen: Honoraria, Speakers Bureau; Novartis: Honoraria; Takeda: Honoraria; Amgen: Honoraria.