Allogeneic Melanoma Research Articles

Long-Term Survival and Immune Response Dynamics in Melanoma Patients Undergoing TAPCells-Based Vaccination Therapy.

Cancer vaccines present a promising avenue for treating immune checkpoint blockers (ICBs)-refractory patients, fostering immune responses to modulate the tumor microenvironment. We revisit a phase I/II trial using Tumor Antigen-Presenting Cells (TAPCells) (NCT06152367), an autologous antigen-presenting cell vaccine loaded with heat-shocked allogeneic melanoma cell lysates. Initial findings showcased TAPCells inducing lysate-specific delayed-type hypersensitivity (DTH) reactions, correlating with prolonged survival. Here, we extend our analysis over 15 years, categorizing patients into short-term (<36 months) and long-term (≥36 months) survivors, exploring novel associations between clinical outcomes and demographic, genetic, and immunologic parameters. Notably, DTHpos patients exhibit a 53.1% three-year survival compared to 16.1% in DTHneg patients. Extended remissions are observed in long-term survivors, particularly DTHpos/M1cneg patients. Younger age, stage III disease, and moderate immune events also benefit short-term survivors. Immunomarkers like increased C-type lectin domain family 2 member D on CD4+ T cells and elevated interleukin-17A were detected in long-term survivors. In contrast, toll-like receptor-4 D229G polymorphism and reduced CD32 on B cells are associated with reduced survival. TAPCells achieved stable long remissions in 35.2% of patients, especially M1cneg/DTHpos cases. Conclusions: Our study underscores the potential of vaccine-induced immune responses in melanoma, emphasizing the identification of emerging biological markers and clinical parameters for predicting long-term remission.

Efficacy and safety of guttiferone E in melanoma-bearing mice.

Melanoma, an aggressive and potentially fatal skin cancer, is constrained by immunosuppression, resistance, and high toxicity in its treatment. Consequently, there is an urgent need for innovative antineoplastic agents. Therefore, this study investigated the antimelanoma potential of guttiferone E (GE). In an allogeneic murine B16 melanoma model, GE was administered subcutaneously and intraperitoneally. Antitumor evaluation included tumor volume/weight measurements and histopathological and immunohistochemical analysis. Furthermore, the toxicity of the treatments was evaluated through body/organ weights, biochemical parameters, and genotoxicity. Subcutaneous administration of 20 mg/kg of GE resulted in a significant reduction in both tumor volume and weight, effectively suppressing melanoma cell proliferation as evidenced by a decrease in mitotic figures. The tumor growth inhibition rate was equivalent to 54%. This treatment upregulated cleaved caspase-3, indicating apoptosis induction. On the other hand, intraperitoneal administration of GE showed no antimelanoma effect. Remarkably, GE treatments exhibited no toxicity, evidenced by non-significant differences in body weight gain, as well as organ weight, biochemical parameters of nephrotoxicity and hepatotoxicity, and genotoxic damage. This study revealed, for the first time, the efficacy of subcutaneous administration of GE in reducing melanoma, in the absence of toxicity. Furthermore, it was observed that the apoptotic signaling pathway is involved in the antimelanoma property of GE. These findings offer valuable insights for further exploring GE's therapeutic applications in melanoma treatment.

Abstract 3192: Antitumor activity of ex vivo expanded tumor antigen priming cytotoxic T cells against malignant melanoma

Abstract Background: Dendritic cells have a unique ability to pick up antigen from tissues and migrate to the lymph nodes where they can cross-present cell-associated antigens with CD8+ T cells by MHC class I. Cytotoxic T lymphocytes remove malignant cells through recognition by T cell receptors of specific antigenic peptides that appear on the surface of cancer cells by the major histocompatibility complex class I/beta-2-microglobulin complex. In this study, we show that naive CD8+ T cells against the melan A antigens cells primed by DCs loaded with melan A antigens are able to kill malignant melanoma cell line (SK-MEL-28). Methods: Peripheral blood mononuclear cells and Co-culture dendritic cells-T cells were seeded and MART-1 (100 ng/ml) reacted for 20 hours. ELISPOT ASSAY analyzes the degree of secretion of IFN-γ induced by Melan-A/MART-1 peptide stimulation in DC-T (1st week). In addition, the subtype analysis of T cells through FACS and the cytotoxicity activity of T cells was measured by tumor cell impedance with AXION. Results: In ELISPOT ASSAY, it was observed that the number of positive spots increased significantly over time compared to 1st week. In FACS result analysis, CD4+ and CD8+ T cells increased, CD4+ T cell was observed as a dominant T cell subtype. As a result of cell impedance assay using Axion, it was observed that the number of tumor cells (SK-MEL-28) decreased according to the CTL:T ratio. In particular, cytotoxicity was observed immediately after co-culture and gradually showed cytotoxicity up to 72 hours. Conclusion: Our study demonstrates that DCs loaded with killed allogeneic melanoma cells can cross-prime cytotoxic T cell which is specific for melan A antigen. In the future, additional T-cell studies targeting neoantigen and clinical studies applied to solid cancer patients will be conducted. Keywords: Dendritic cell, CD4+/CD8+ T cell, Melan A antigen, Malignant melanoma Citation Format: Jonggwon Choi. Antitumor activity of ex vivo expanded tumor antigen priming cytotoxic T cells against malignant melanoma [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 3192.

Abstract LB212: Allogeneic, IL-2-independent tumor-infiltrating lymphocytes expressing membrane-bound IL-15 (cytoTIL15࣪) eradicate tumors in a melanoma PDX model through recognition of shared tumor antigens

Abstract Standard tumor-infiltrating lymphocyte (TIL) therapy requires IL-2 administration to support TIL expansion and survival, but this cytokine is associated with T cell exhaustion and can result in severe toxicities that limit patient eligibility (1). To this end, we genetically engineered TIL to express membrane-bound IL-15 (mbIL15) under the control of Obsidian’s cytoDRIVE® technology (cytoTIL15࣪), which allows regulation of protein expression via a drug-responsive domain upon acetazolamide (ACZ) administration. IL-15 is a preferred cytokine over IL-2 to mediate TIL activation and expansion, because it does not result in CD8 T cell exhaustion or stimulate regulatory CD4 T cells, and enhances development of a memory T-cell phenotype. We have previously demonstrated IL-2-independent, 3-6-fold increased cytoTIL15 persistence in an antigen-independent setting relative to unengineered TIL therapy with IL-2 (uTIL) (2). Due to the challenge of generating autologous tumor/TIL-matched pairs and most importantly, to assess cytoTIL15 cell’s functional impact on anti-tumor growth across multiple donors, we developed an allogeneic patient-derived xenograft (PDX) model. To establish the model, different melanoma tumor digests were co-incubated in vitro with select HLA-A*02-matched, allogeneic melanoma TIL donors to assess their reactivity. Tumors were screened for expression of shared antigens, such as gp100 and MART1, and TIL donor TCRs were screened with tetramers. Once established, serially passaged tumor fragments were grown, measured, and randomized into groups to receive intravenous transfer of TIL (n=8/cohort). Mice receiving uTIL were treated with four saturating doses of recombinant IL-2, and mice receiving cytoTIL15 cells received either vehicle or oral 200 mg/kg ACZ daily for the entire study, without any IL-2. Three of four cytoTIL15 cell preparations from different donors dosed with ACZ achieved significant tumor growth inhibition compared to uTIL. Four mice developed complete responses as early as 17 days post cytoTIL15 cell transfer. The level of anti-tumor response was associated with increased frequency of MART1-reactive cytoTIL15 cells. On day 20 after TIL transfer, tumors and secondary lymphoid organs were collected (n=4/cohort). Tumors treated with cytoTIL15 cells + ACZ showed an 8-10-fold increased TIL infiltration compared to uTIL or cytoTIL15 cells + vehicle. Moreover, enhanced cytoTIL15 cell infiltration and anti-tumor activity was associated with increases in pro-inflammatory cytokines (e.g., IFNγ). Taken together, these data clearly demonstrate the superiority of cytoTIL15 cells over uTIL for controlling or eradicating melanoma tumor outgrowth and the utility of an allogeneic PDX model for comparative evaluation of tumor-antigen specific TIL reactivity.

Qa-1b functions as an oncogenic factor in mouse melanoma cells

BackgroundMalignant melanoma is one kind of rare cancer in human and animals, which occurs not only in skin but also in the mucous membranes of the nose, mouth, anus, digestive tract, and in the uvea (choroid) of the eye. In order to develop therapies, a better understanding of the genetic landscape and signal pathways are needed. Numerous studies highlighted the importance of NKG2A-HLA-E in tumor immunotherapy, but the function and mechanism of HLA-E in tumor cells have seldom been studied alone. The statistical analyses from publicly available database Oncomine and The Cancer Genome Atlas (TCGA) showed that HLA-E is highly expressed in melanoma compared to non-transformed counterparts. In addition, melanoma patients with HLA-E high expression had a worse OS rate than the patients with low expression. These data indicate the importance of HLA-E in human melanoma. Qa-1b is the homolog of HLA-E in mouse. ObjectiveTo investigate the function and mechanism of Qa-1b in mouse melanoma. MethodsMouse melanoma cell line B16-F10 and allogenic melanoma model were used to investigate the function and mechanism of Qa-1b in melanoma. ResultsQa-1b was highly expressed in B16-F10 compared with normal mouse epidermal cells. Qa-1b knockdown inhibited B16-F10 cell proliferation and migration in vitro and allogenic melanoma process in vivo. Furthermore, Qa-1b knockdown promoted cell cycle arrest at G0/G1 phase and cell apoptosis through Ras-Raf-MAPK signal pathway. ConclusionQa-1b functions as an oncogenic factor and it can be used as a new therapeutic target in melanoma.

Abstract P109: Targeting BRD4 in T cells with self-delivering RNAi PH-894 for immunotherapy

Abstract BRD4 is a member of the family of BET proteins that act as epigenetic readers and regulators of gene transcription. BRD4 is a known driver of tumor growth via enhancement of transcription of oncogenes such as Myc. More recent evidence also suggests a role of BRD4 in T cells. T cell differentiation and T cell exhaustion is epigenetically regulated and BRD4 has been associated with T cell differentiation. Available small molecule inhibitors of BET proteins do not target BRD4 specifically, but also inhibit other members of the BET protein family. We therefore investigated the effects of BRD4 silencing on T cells with the INTASYL™ compound PH-894, a self-delivering RNAi specifically targeting BRD4. INTASYL self-delivering RNAi molecules consist of an asymmetric duplex structure, a small duplex region (≤ 15 base pairs), a single-stranded phosphorothioate tail, and chemical modifications to confer stability, hydrophobicity, and efficient cellular uptake. Primary human T cells from healthy donors, either unstimulated or activated by OKT3 or anti-CD3/CD28 beads, were treated with PH-894. Additional studies were performed with CD8+ T cells magnetically sorted from primary human T cells. Silencing of BRD4 mRNA was assessed by RT-qPCR, and protein by flow cytometry. Phenotypic markers of T cell activation, differentiation and function were assessed by flow cytometry and cytokine secretion by cytometric bead array. Expanded and PH-894-treated CD8+ T cells were studied for IFNγ production upon coculture with allogeneic melanoma cell lines. Silencing of BRD4 mRNA and protein by PH-894 was concentration dependent and durable for up to 10 days in activated and naïve T cells. Silencing was specific for BRD4 with no effect on BRD2 or BRD3 expression. In activated T cells there was a suppression of IL-10 secretion and no effect on inflammatory cytokine levels. Markers of T cell activation were elevated on naïve PH-894-treated T cells with a shift to an increased CD8+/CD4+ ratio. In addition, BRD4 silencing in activated CD8+ T cells coincided with a decreased expression of transcription factors typically induced immediately upon TCR activation and are known drivers of T cell differentiation towards an effector phenotype. Moreover, CD8+ T cells expanded in the presence of PH-894 produced increased levels of IFNγ when cocultured with two allogeneic melanoma cell lines. These data translated in vivo with PH-894 treatment eliciting a dose associated inhibition of tumor growth in a CT26 murine colon carcinoma model. Ex vivo analysis of tumors found PH-894 efficacy was associated with decreased expression of BRD4 in tumor infiltrating CD3 T cells, and a reduction of Foxp3hi CD25hi CD4+ CD3+ Tregs, compared with control tumors. Specific silencing of BRD4 with PH-894 resulted in phenotypic changes on T cells associated with T cell activation and reduced immunosuppression. These data suggest that BRD4 not only plays a role on tumor cells but can also regulate T cell function and that PH-894 can reprogram T cells to provide enhanced immunotherapeutic activity. Citation Format: Melissa Maxwell, Daisuke Ujiie, Benjamin Cuiffo, Brianna Rivest, Dingxue Yan, James Cardia, Jeroen Melief, Rolf Kiessling, Simon P. Fricker. Targeting BRD4 in T cells with self-delivering RNAi PH-894 for immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P109.

Immunization with alloantibodies-covered melanoma cells induces regional antitumor effects that become systemic when combined with 5-FU treatment

Alloantibodies, in particular immunoglobulin G (allo-IgG), confer a rejection advantage to tumors sharing the same major histocompatibility complex (MHC) in mice. However, when administrated intratumorally, this effect can only be achieved in combination with dendritic cells (DCs) activation. Here, we developed high titer allo-IgG by multiple rounds of immunization with allogenic B16 melanoma cells, which allows for the strong binding with B16cells. We demonstrate that B16cells incubated with these allo-IgG (referred to as allo-IgG-B16) become highly immunogenic, which release tumor antigens that are efficiently presented by classic DCs in lymph nodes (LNs). Injection of allo-IgG-B16 turns the tumor into an immune hot one and even elicits a systemic antitumor response when used together with 5-fluorouracil (5-FU). This systemic response is tumor-specific and relies on the critical site - LNs. Our findings provide a rationale for the use of allo-IgG in cancer treatment.

A Perspective on Cell Therapy and Cancer Vaccine in Biliary Tract Cancers (BTCs).

Simple SummaryIn this review, we discuss treatment strategies in biliary tract cancers (gallbladder cancer, intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma). In particular, we will describe advances in cellular therapies and cancer vaccines for biliary tract cancers, followed by our local experience with combining a melanoma-associated antigen (MAGE)-positive cell lysate-based autologous dendritic cell vaccine and anti-angiogenic therapy (bevacizumab) in a case of stage IV gallbladder cancer.Biliary tract cancer (BTC) is a rare, but aggressive, disease that comprises of gallbladder carcinoma, intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma, with heterogeneous molecular profiles. Advanced disease has limited therapeutic options beyond first-line platinum-based chemotherapy. Immunotherapy has emerged as a viable option for many cancers with a similar unmet need. Therefore, we reviewed current understanding of the tumor immune microenvironment and recent advances in cellular immunotherapy and therapeutic cancer vaccines against BTC. We illustrated the efficacy of dendritic cell vaccination in one patient with advanced, chemorefractory, melanoma-associated antigen (MAGE)-positive gallbladder carcinoma, who was given multiple injections of an allogenic MAGE antigen-positive melanoma cell lysate (MCL)-based autologous dendritic cell vaccine combined with sequential anti-angiogenic therapy. This resulted in good radiological and tumor marker response and an overall survival of 3 years from diagnosis. We postulate the potential synergism of adding anti-angiogenic therapy, such as bevacizumab, to immunotherapy in BTC, as a rational scientific principle to positively modulate the tumor microenvironment to augment antitumor immunity.

Abstract CT194: Low-dose Interferon-alpha pre-conditioning and adoptive cell therapy in metastatic melanoma patients refractory to standard (immune) therapies - a phase 1/2 study

Abstract Background: Adoptive cell therapy (ACT) with tumor-reactive T cells has shown consistent clinical efficacy. We evaluated the response to ACT in combination with interferon alpha (IFNa) preconditioning in stage IV metastatic melanoma patients, most of which were progressive on CTLA-4 and/or PD-1 checkpoint blockade therapy. Methods: Thirty-four patients were treated with ex vivo expanded tumor reactive T cells, derived from mixed-lymphocyte-autologous tumor cultures, or with autologous tumor-infiltrating lymphocytes and evaluated for clinical response. Clinical and immunological parameters associated with response were also evaluated. Results: Best overall response defined as clinical benefit, comprising either complete response, partial response or stable disease for more than 6 months, was observed in 29% of the patients. Six of the 14 (43%) immunotherapy naïve patients and 4 of the 20 (20%) patients progressive on prior immunotherapy benefited from ACT. The overall survival (OS) was 90% versus 28.6% at 1 year and 46.7% versus 0% at 3 years follow-up, of clinical responder and non-responder patients, respectively. Median OS was 36 versus 7 months, respectively. IFNa pre-treatment resulted in leuko-, neutro- and lymphopenia, which was sustained during the treatment in clinical responders and associated with response. Differences in antigen-specificity, but not in phenotype, cytokine profile, or CD8+ T cell number of the ACT products correlated with clinical response. Cross-reactivity of the ACT products to one or more allogeneic HLA-matched melanoma cell lines was associated with short OS after treatment while the ACT products of very long-term survivors showed solely recognition of patient-specific neo-antigens. Conclusion: This study demonstrates that ACT in combination with a mild IFNa preconditioning regimen can induce clinical benefit even in immunotherapy pre-treated patients, albeit with lower success than in immunotherapy naïve patients. ACT products comprising neo-antigen reactivity may be more effective. Future Perspectives: As a substantial percentage of the infused T cells expressed one or more inhibitory checkpoint molecules (CTLA-4, PD-1, or TIM-3), we started a new trial combining ACT and anti-PD-1 (ACTME trial; NCT03638375). The phase Ia of this trial was just completed with nine immunotherapy pre-treated patients showing that the combined treatment is feasible and safe. Updated and accumulated disease response data will be presented. Citation Format: Monique K. van der Kooij, Els M. Verdegaal, Marten Visser, Carolien E. van der Minne, Linda de Bruin, Pauline Meij, Anton G. Terwisscha van Scheltinga, Marij J. Welters, Saskia J. Santegoets, Noel F. de Miranda, Inge C. Roozen, Gerrit-Jan Liefers, Ellen Kapiteijn, Sjoerd H. van der Burg. Low-dose Interferon-alpha pre-conditioning and adoptive cell therapy in metastatic melanoma patients refractory to standard (immune) therapies - a phase 1/2 study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT194.

Low-dose interferon-alpha preconditioning and adoptive cell therapy in patients with metastatic melanoma refractory to standard (immune) therapies: a phase I/II study

BackgroundAdoptive cell therapy (ACT) with tumor-reactive T cells has shown consistent clinical efficacy. We evaluated the response to ACT in combination with interferon alpha (IFNa) preconditioning in patients with stage...

Cancer/testis antigens expression during cultivation of melanoma and soft tissue sarcoma cells

BackgroundAutologous dendritic cells (DC) loaded with tumor-associated antigens (TAAs) are a promising approach for anticancer immunotherapy. Polyantigen lysates appear to be an excellent source of TAAs for loading onto the patient’s dendritic cells. Cancer/testis antigens (CTA) are expressed by a wide range of tumors, but are minimally expressed on normal tissues, and could serve as a universal target for immunotherapy. However, CTA expression levels can vary significantly in patients with the same tumor type. We proposed that patients who do not respond to DC-based therapy may have distinct features of the CTA expression profile on tumor cells.Patients and methodsWe compared the gene expression of the principal families CTA in 22 melanoma and 27 soft tissue and bone sarcomas cell lines (STBS), received from patients and used for DC vaccine preparation.ResultsThe majority (47 of 49, 95.9%) cell lines showed CTA gene activity. The incidence of gene expression of GAGE, NYESO1, MAGEA1, PRAME’s was significantly different (adj. p < 0.05) between melanoma and sarcoma cell lines. The expression of the SCP1 gene was detected neither in melanoma cells nor in the STBS cells. Clustering by the gene expression profile revealed four different expression patterns. We found three main patterns types: hyperexpression of multiple CTA, hyperexpression of one CTA with almost no expression of others, and no expression of CTA. All clusters types exist in melanoma and sarcoma cell lines. We observed dependence of killing efficacy from the PRAME (rho = 0.940, adj. p < 0.01) expression during real-time monitoring with the xCELLigence system of the interaction between melanoma or sarcoma cells with the T-lymphocytes activated by the lysate of selected allogenous melanoma cell lines with high expression of CTA.ConclusionOur results demonstrate that one can use lysates from allogeneic melanoma cell lines as a source of CTA for DC load during the production of anticancer vaccines for the STBS treatment. Patterns of CTA expression should be evaluated as biomarkers of response in prospective clinical trials.

Therapeutic melanoma vaccine with cancer stem cell phenotype represses exhaustion and maintains antigen-specific T cell stemness by up-regulating BCL6

ABSTRACT We developed a therapeutic, gene-modified, allogeneic melanoma vaccine (AGI-101H), which, upon genetic modification, acquired melanoma stem cell-like phenotype. Since its initial clinical trial in 1997, the vaccine has resulted in the long-term survival of a substantial fraction of immunized patients (up to 20 years). Here, we investigated the potential molecular mechanisms underlying the long-lasting effect of AGI-101H using transcriptome profiling of patients’ peripheral T lymphocytes. Magnetically-separated T lymphocytes from AGI-101H-immunized long-term survivors, untreated melanoma patients, and healthy controls were subjected to transcriptome profiling using the microarray analyses. Data were analyzed with a multitude of bioinformatics tools (WebGestalt, DAVID, GSEA) and the results were validated with RT-qPCR. We found substantial differences in the transcriptomes of healthy controls and melanoma patients (both untreated and AGI-101H-vaccinated). AGI-101H immunization induced similar profiles of peripheral T cells as tumor residing in untreated patients. This suggests that whole stem cells immunization mobilizes analogous peripheral T cells to the natural adaptive anti-melanoma response. Moreover, AGI-101H treatment activated the TNF-α and TGF-β signaling pathways and dampened IL2-STAT5 signaling in T cells, which finally resulted in the significant up-regulation of a BCL6 transcriptional repressor, a known amplifier of the proliferative capacity of central memory T cells and mediator of a progenitor fate in antigen-specific T cells. In the present study, high levels of BCL6 transcripts negatively correlated with the expression of several exhaustion markers (CTLA4, KLRG1, PTGER2, IKZF2, TIGIT). Therefore, Bcl6 seems to promote a progenitor fate for cancer-experienced T cells from AGI-101H-vaccinated patients by repressing the exhaustion markers.

Dendritic Cells Loaded with Heat Shock-Conditioned Ovarian Epithelial Carcinoma Cell Lysates Elicit T Cell-Dependent Antitumor Immune Responses In Vitro.

Ovarian epithelial carcinoma (OEC) is the most frequent ovarian tumor, characterized by a high mortality in advanced stages where conventional therapies are not effective. Based on the role of the immune system in the progression of this disease, immunotherapy using checkpoint blockade has been considered as a therapeutic alternative. Nevertheless, its results do not match up to the positive results in entities like melanoma and other malignancies, suggesting the need to find other therapies to be used alone or in combination. Dendritic cell- (DC-) based vaccines have shown promising results in several types of cancer, such as melanoma, prostate, and lung cancers, due to the essential role played by DCs in the activation of specific T cells, thus using other ways of activating the immune response than immune checkpoint blockade. During the last decade, we have used DC-based vaccines loaded with an allogeneic heat shock-conditioned melanoma cell lysate in the treatment of advanced stage patients in a series of clinical trials. In these studies, 60% of treated patients showed immunological responses which correlated positively with improved survival. Considering the relevance of ovarian cancer and the promising results of our DC-based vaccine, we show here that heat shock-conditioned cell lysates derived from ovarian epithelial carcinoma cell lines have the potential to induce the phenotypic and functional maturation of human DC, which in turn, is able to induce an efficient CD4+ and CD8+ T cell-mediated immune responses against ovarian cancer cell lines in vitro. In summary, OEC heat shock-conditioned cell lysate-loaded DCs may be considered for future combined immunotherapy approaches against ovarian tumors.

Syngeneic B16-F1 cells are more efficient than allogeneic Cloudman cells as antigen source in DC-based vaccination in the B16-F1 murine melanoma model

A major obstacle to obtaining relevant results in cancer vaccination has been the lack of identification of immunogenic antigens. Dendritic cell (DC)-based cancer vaccines used preventively may afford protection against tumor inoculation, but the effect of antigen choice on anti-tumor protection is not clear. When using irradiated syngeneic tumor cells to load DCs, tumor self-antigens are provided, including tumor-associated antigens (TAAs) and neoantigens generated by tumor mutations. On the other hand, allogeneic tumor cells could only supply shared TAAs. To assess the advantages of each source in protective vaccination, we analyzed in C57BL/6 mice the effect of loading DCs with irradiated syngeneic B16-F1 or allogeneic Cloudman melanoma cells; both cell lines were characterized by whole exome sequencing and RNAseq. Tumor cell components from the two irradiated cell lines were efficiently internalized by DCs, and transported to MHC-class II positive tubulovesicular compartments (MIICs). DCs loaded with allogeneic irradiated Cloudman cells (DC-ApoNecALLO) induced a partially effective anti-melanoma protection, although Cloudman and B16-F1 cells share the expression of melanocyte differentiation antigens (MDAs), cancer-testis antigens (CTAs) and other TAAs. DCs loaded with syngeneic B16-F1 cells (DC-ApoNecSYN) established a more potent and long-lasting protection and induced a humoral anti-B16F1 response, thus suggesting that neoepitopes are needed for inducing long-lasting protection.

Dexamethasone turns tumor antigen-presenting cells into tolerogenic dendritic cells with T cell inhibitory functions

BackgroundDendritic cells (DCs) are usually immunogenic, but they are also capable of inducing tolerance under anti-inflammatory conditions. Immunotherapy based on autologous DCs loaded with an allogeneic melanoma cell lysate (TRIMEL/DCs) induces immunological responses and increases melanoma patient survival. Glucocorticoids can suppress DC maturation and function, leading to a DC-mediated inhibition of T cell responses. MethodsThe effect of dexamethasone, a glucocorticoid extensively used in cancer therapies, on TRIMEL/DCs phenotype and immunogenicity was examined. ResultsDexamethasone induced a semi-mature phenotype on TRIMEL/DC with low maturation surface marker expressions, decreased pro-inflammatory cytokine induction (IL-1β and IL-12) and increased release of regulatory cytokines (IL-10 and TGF-β). Dexamethasone-treated TRIMEL/DCs inhibited allogeneic CD4+ T cell proliferation and cytokine release (IFNγ, TNF-α and IL-17). Co-culturing melanoma-specific memory tumor-infiltrating lymphocytes with dexamethasone-treated TRIMEL/DC inhibited proliferation and effector T cell activities, including cytokine secretion and anti-melanoma cytotoxicity. ConclusionsThese findings suggest that dexamethasone repressed melanoma cell lysate-mediated DC maturation, generating a potent tolerogenic-like DC phenotype that inhibited melanoma-specific effector T cell activities. These results suggest that dexamethasone-induced immunosuppression may interfere with the clinical efficacy of DC-based melanoma vaccines, and must be taken into account for optimal design of cellular therapy against cancer.

Abstract A144: The transcriptomic profile of peripheral T-cells that maintain dormant state of melanoma cells in patients treated with allogenic melanoma vaccine

Abstract Melanoma (MM) cells leave primary tumors early and evolve at different sites in parallel. However, timing in human MM is unknown. Lymphatic dissemination is fast shortly after the dermal invasion, but metastases may occur late. Such delay in metastases formation suggests possible host defense mechanism, or particular nature of non-actively proliferative (dormant) MM stem cell (MSC) population. T lymphocytes may also induce tumor dormancy. The proposed mechanism involves a cytostatic mode of tumor silencing resulting from the suppression of both neoplastic cell proliferation and cell cycle progression. Significant progress in the development of novel advanced MM therapies was made. However, not all patients benefit from the therapy, developing late metastases decades after treatment ends. Moreover, using the immunotherapy-based model of MM dormancy in mouse, vaccination against MM prevented tumor growth but failed to prevent tumor cell dissemination or elimination of all MM cells. We have developed therapeutic gene modified allogenic MM vaccine (AGI-101H) that has been tested since 1997 and resulted in long-term survival of a substantial fraction of patients. Genetic modification of vaccine cells to express designer cytokine cDNA encoding Hyper-IL-6 protein (comprising of IL-6 linked with a soluble IL-6 receptor) has altered AGI-101H cells phenotype towards MSC-like with high activity of aldehyde dehydrogenase isoenzyme (ALDH1A1). We have found significant induction of anti-MSCs response in immunized patients by a generation of functionally active cytotoxic CD8+T-cells and antibodies specific for ALDH1A1 in circulation. However, the AGI-101H mode of action is complex: besides inducing a direct anticancer response to MSC population, long-term vaccination may induce MM dormancy through T-cell mediated immunity. The above hypothesis is based on clinical observations of AHI-101H treated patient who was progression-free for many years, but injury induced metastases formation in the bruise. This patient developed skin MM in 1999. In 2002 in transit metastases appeared (resected). The patient was enrolled into AGI-101H program. In 2003 metastases in liver (resected), 2004 metastasis in ovary (resected). 11 years (2004-2015) no progression. In Nov. 2015 she had bicycle accident, trauma, and MM metastasis presenting with a bruise (resected). The treatment continues with no progression. The aim was to determine the molecular mechanisms of peripheral T-cell-mediated induction of MM dormancy induced by vaccination. Therefore, T-cell mRNA expression profiling of long-term surviving patients was carried out. PBMCs were isolated from treated patients (18), untreated MM patients (13) and healthy controls (8). Untouched T-cells were separated by magnetic beads and total RNA isolated. The transcriptome profiling was performed with Affymetrix HG U219 microarray (19,285 markers). Differential gene expression (DGE) analysis between all groups was conducted and validated with RT-qPCR and FACS. The transcriptomic results were further analyzed with Gene Set Enrichment Analysis (GSEA) tool. DGE analyses comparing AGI-101H vaccinated (AV) and nonimmunized (C) patients revealed 538 differentially expressed genes (DEGs), with 373 downregulated and 165 upregulated in AV (adj p &amp;lt; 0.05). Among these 538 markers, the expression of 14 (with |log2FC| &amp;gt; 1) is now being validated with RT-qPCR and FACS. Also, GSEA analysis revealed significant enrichment of “TNFa_signaling_via_NFkB”, “TGFb_signaling” and “G2/M_checkpoint” hallmark processes (MSigDB Hallmark gene sets) in AGI-101H vaccinated patients, that indicates considerable activation of antitumor response and prevention of conversion of MM dormanT-cells into proliferative metastases. Transcriptome profiling of peripheral T-cells in long-surviving MM patients immunized with AGI-101H suggests activation of the molecular mechanism that sustains MM cells in the non-actively proliferative dormant state. Citation Format: Andrzej A. Mackiewicz, Partycja Czerwinska, Marcin Rucinski, Katarzyna Gryska, Iga Grzadzielewska, Anna Jaworska, Jacek Mackiewicz. The transcriptomic profile of peripheral T-cells that maintain dormant state of melanoma cells in patients treated with allogenic melanoma vaccine [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A144.

Tumor Lysate Loaded Dendritic Cells Induce a T cell Specific Antitumor Response Against Gallbladder Cancer

Abstract Introduction Although infrequent, Gallbladder cancer (GBC) has high incidence rates in South America. In fact, Chile has the highest GBC incidence and mortality worldwide. Currently, surgery is the only effective treatment, and the five-year survival rate is less than 10%. Previously, we demonstrated that allogeneic melanoma cell lysates-loaded dendritic cells (DCs) provide a standardized, applicable tumor-specific vaccine; capable to induce a cell mediated immune response in melanoma patients, improving survival. Here, we investigated the effect of tumor lysates derived from gallbladder cancer cell (GCC) lines on immature DCs and their capacity to induce a T cell mediated anti GBC responses in vitro. Material and Methods DCs were stimulated with GCC lysates and analyzed by flow cytometry for a panel of surface markers. Autologous CD3+ T cells were co-cultured with these DCs for 14 days. CD4+ and CD8+ T cells were analyzed by flow cytometry for activation and chemokine receptor expression. CD8+ T cells were isolated and co-cultured with GCC lines GBd1, TGBC-2TKB and melanoma cell line Mel1 for 16hrs. IFN-γ secretion was measured by ELISPOT. Results All GCC lysates induced DCs maturation, increasing surface expression of MHC-I, MHC-II, CD80, CD83, CD86, CCR7. DCs maturated with GCC lysate induced CD3+ T cells activation, increasing expression of CD25, CD69, CXCR3, CXCR4 in both CD4+ and CD8+ T cells compared with non-stimulated lymphocytes. Additionally, CD8+ T cells co-cultured with GBC-loaded DCs, released higher amounts of IFN-γ than controls. Discussion Based on these results, DCs maturated with GCC lysates are capable to induce specific T cells activation against GBC and can be considered for future immunotherapy approaches.

IL-10 is required for polarization of macrophages to M2-like phenotype by mycobacterial DnaK (heat shock protein 70)

Macrophages are key cells in the innate immune system. They phagocytose pathogens and cellular debris, promote inflammation, and have important roles in tumor immunity. Depending on the microenvironment, macrophages can polarize to M1 (inflammatory) or M2 (anti-inflammatory) phenotypes. Extracellular DnaK (the bacterial ortholog of the mammalian Hsp70) from Mycobacterium tuberculosis (Mtb) was described to exert immune modulatory roles in an IL-10 dependent manner. We have previously observed that endotoxin-free DnaK can polarize macrophages to an M2-like phenotype. However, the mechanisms that underlie this polarization need to be further investigated. IL-10 has been described to promote macrophage polarization, so we investigated the involvement of this cytokine in macrophages stimulated with extracellular DnaK. IL-10 was required to induce the expression of M2 markers - Ym1 and Fizz, when macrophages were treated with DnaK. Blockade of IL-10R also impaired DnaK induced polarization, demonstrating the requirement of the IL-10/IL-10R signaling pathway in this polarization. DnaK was able to induce TGF-β mRNA in treated macrophages in an IL-10 dependent manner. However, protein TGF-β could not be detected in culture supernatants. Finally, using an in vivo allogeneic melanoma model, we observed that DnaK-treated macrophages can promote tumor growth in an IL-10-dependent manner. Our results indicate that the IL-10/IL-10R axis is required for DnaK-induced M2-like polarization in murine macrophages.

Abstract A37: Immunization of cutaneous melanoma patients with the allogeneic cell vaccine CSF-470 enhances immune infiltration of metastatic lesions and would favor subsequent response to Vemurafenib

Abstract Therapeutic cancer vaccines are aimed at promoting tumor-specific immunity with long-term memory. We have developed the CSF-470 therapeutic vaccine, a mini-allograft of four irradiated allogeneic cutaneous melanoma cell lines, combined with BCG and rhGM-CSF as adjuvants; that is currently being assayed in adjuvancy against medium-dose IFN-alfa2b in the CASVAC-0401 phase II/III clinical trial (ClinicalTrials.gov identifier: NCT01729663). An update to September 2014 reveals that after inclusion of 31 patients, a significant benefit in the distant metastasis-free survival for the CSF-470 arm is obtained (p=0.034). Of the 20 patients assigned to the CSF-470 arm, with a mean follow-up of 26 months, six patients developed distant metastases, of whom two died, and fourteen patients are distant metastases-free (70%). Five patients of the vaccine arm were treated with Vemurafenib after progression; two of them achieved complete remission and two of them partial remission (&amp;gt;50%); only one developed severe dermatitis. Before Vemurafenib treatment, metastases biopsies from only three patients could be obtained; two of them later achieved complete responses. In this work, we present the analysis of the peripheral blood and in situ immune populations during CSF-470 immunization in the two patients that achieved complete responses with Vemurafenib treatment. To attain this task, histopathological characterization of tumor biopsies, along with immune populations determined by immunohistochemistry, was performed by microscopy and image analysis. Peripheral blood mononucleated cells were obtained from patients at different times during CSF-470 immunization and immune populations were assessed by flow cytometry. Patient #006 entered the CASVAC-0401 study after a primary and lymph node tumor resection. By the end of CSF-470 immunization, she developed simultaneously subcutaneous and lung metastases. Following a 3-month cycle of Vemurafenib 960mg bid, patient #006 achieved complete remission of lung tumor nodules. Analysis of immune response evolution during protocol revealed a marked increase in in situ immune infiltration at the cutaneous metastasis relative to the primary tumor, especially in CD8+, CD4+ and CD20+ cells. Several dying tumor cells could be distinguished in the cutaneous metastasis, with attached and surrounding CD8+, CD4+ and CD45Ro+ T lymphocytes and CD68+ cells; and with Granzyme B vesicles and DNA strand breaks. Patient #005 developed sequential in transit metastases; CD8+, CD4+ and CD20+ lymphocyte infiltration increased in situ in biopsies following CSF-470 immunization. Vemurafenib treatment allowed achieving a complete response characterized by brisk infiltration of CD68+ macrophages, CD11c+ dendritic cells, CD8+ and CD4+ lymphocytes. In both patients, analysis of peripheral immune populations revealed an increment of NK cells detected after 6-months treatment, followed by an increase in CD4+ and CD8+ cells by the end of the protocol (2 years). Also, serum reactivity to CSF-470 cutaneous melanoma cells increased during vaccination. We suggest that previous immunization of melanoma patients with CSF-470 vaccine increases tumor immune infiltration and subsequent response to Vemurafenib. These encouraging results may open new options for combined therapies in cutaneous melanoma. Citation Format: Mariana Aris, María Betina Pampena, Estrella M. Levy, Alicia I. Bravo, Florencia P. Madorsky-Rowdo, Ana Mordoh, Julio Kaplan, Antonela Baron, Mariela Urrutia, Paula A. Blanco, María Marcela Barrio, José Mordoh. Immunization of cutaneous melanoma patients with the allogeneic cell vaccine CSF-470 enhances immune infiltration of metastatic lesions and would favor subsequent response to Vemurafenib. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A37.

Abstract A50: Immunomonitoring in a phase II/III trial of therapeutic vaccination with CSF-470 plus BCG plus GM-CSF versus IFN-alfa in patients with cutaneous melanoma

Abstract Melanoma is the deadliest form of skin cancer. Once it has metastasized, treatment options are limited and prognosis is bleak. Immunotherapy is one of the current promising treatments for patients (pts) after surgery, but markers for prediction of immune response are still lacking. In a phase II/III randomized clinical trial (CASVAC-0401, NCT01729663), cutaneous melanoma pts with Stages IIB, IIC and III were randomized to receive CSF-470 versus medium-dose IFN-alpha in a 2:1 ratio. The duration of the study is two years, during which the vaccinated pts would receive 13 vaccinations. CSF-470 vaccine is a mini-allograft consisting of four lethally-irradiated allogeneic cutaneous melanoma cell lines, combined with BCG and rhGM-CSF as adjuvants with the goal of stimulating adaptive and innate immunity. Thirty one pts have been included so far in the study; 20 pts have been randomized to the vaccine arm and 11 to the IFN-alfa arm. The DMFS (distant-metastasis-free-survival) is significantly longer for pts in the CSF-470 arm than in those of the IFN-alfa arm (p=0.034, Gehan-Breslow-Wilcoxon Test); besides, quality of life is significantly better in the CSF-470 arm. Looking for parameters involved in the immune system stimulation in vaccinated pts, DTH (Delayed-Type Hypersensitivity) and lymphocyte subsets were periodically evaluated. A significant increase in the DTH response between NED (non evident disease) and PRO (progressed) was observed in vaccinated pts (p=0.0362, unpaired t test with Welch's correction). In order to investigate if the increase in DTH was accompanied by changes in lymphocyte peripheral populations, we investigated changes between pre-and post-treatment samples from 24 enrolled pts, 16 from the vaccine arm and 8 from the IFN-alfa arm. We did not observe significant differences between PRE and POST1 samples (6 months; five vaccinations) in T CD4+, CD8+ or regulatory cells, neither in vaccinated nor in IFN-alfa treated pts. Furthermore, we did not detect any changes in the frequencies of T CD8+ specific clones against MART-1 or gp100 Ags after treatment in HLA*0201 pts. Instead, we observed that only vaccinated pts increased the percentage of Natural Killer (NK) in the POST1 sample (PRE 16.6±8.0% vs POST1 22±12.4% p=0.0106, paired t test). Given these observations, we investigated, using a degranulation assay after challenge with live melanoma cell lines or K562 cells, if NK cells functionality was also augmented. Nevertheless, we did not see significant differences in NK cell activity after vaccination or IFN-alfa treatment. In contrast, we observed significant increases in antibody (Ab) responses against melanoma cells in vaccinated pts sera, that was not observed in IFN-alfa treated pts, as determined by ELISA (vaccine arm: PRE 0.3±0.2 vs POST1 0.8±0.3; p&amp;lt;0.0001, paired t test; IFN-alfa arm: PRE 0.4±0,2 vs POST1 0.4±0,3; p&amp;gt;0.05, paired t test). Anti-melanoma Abs increased both in NED and in PRO vaccinated pts. In conclusion, we suggest that the increase in circulating NK cells that coexist with anti-melanoma Abs could contribute to eliminate circulating tumor cells, thus hampering further metastatic dissemination. Also, since DTH was clearly increased in responding vaccinated pts, we suggest that lymphocyte subsets located in peripheral tissues, which give rise to DTH reactivity, would more closely reflect clinical responses and should receive closer examination than circulating lymphocytes populations. Efforts to understand the impact of CSF-470 in the pts´ immune system are needed to identify the effectors involved in clinical responses. Citation Format: María Betina Pampena, Estrella Mariel Levy, María Paula Blanco, María Marcela Barrio, José Mordoh. Immunomonitoring in a phase II/III trial of therapeutic vaccination with CSF-470 plus BCG plus GM-CSF versus IFN-alfa in patients with cutaneous melanoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A50.