Improvement Of Immunotherapy Research Articles

Abstract 7547: Enhancers as targets for improvements in immunotherapy

Abstract Role of epigenetic elements in mediating tumor immune interaction remains poorly understood. We previously identified enrichment of active enhancer states on a set of genomic loci in non-responders to immune checkpoint blockade (ICB) therapy. We noted that the activated non-responder enhancers marked a group of key regulators of in melanoma cells that are known to mediate resistance to ICB therapy and several checkpoint receptors in T cells. Through spatial epigenomics (Spatial ATAC and CUT&Tag) approaches, we identified the tumor-immune niches harboring unique gene regulatory elements and transcription factors that could mediate melanoma-T cell interaction. Further, we aimed to identify functional epigenetic elements through custom CRISPRi screening of enhancers in patient tumor-derived melanoma and T cells. CRISPRi approaches identified novel functional enhancers that mediate T cell mediated killing. For example, we identified distal c-MET enhancers as important contributors to T cell mediated killing. In T cells, we identified enhancer hubs that target multiple genes. These hubs could be targeted to activate interferon gamma, reduce T cell exhaustion, and improve T cell mediated killing of melanoma cells. Such enhancer hubs could serve as targets in the ex vivo setting to improve adoptive T cell therapy. Overall, our results suggest that enhancers with unique spatial patterns in melanoma tumors contribute to the response to immunotherapy and could be novel targets for therapy. Citation Format: Anand Singh, Veena Kochat, Emre Arslan, Suresh Satpati, Wentao Deng, Sandeep Yadav, Efthalia Bataki, Cassian Yee, Kunal Rai. Enhancers as targets for improvements in immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7547.

The brief introduction of CAR T-cell: From mechanism to application

Chimeric antigen receptor T-cell (CAR-T) gene therapy is a newly developed solution to fight tumor cells. The therapy develops from the combination of the immunity and cell bioengineering, taking advantage of the cell killing function of T-cells, scientists modify them with a bioengineering method to target specific cells. In recent days, this therapy shows great results in several trials especially in hematological malignancies. However, there are still many obstacles, such as toxicities, that need to be overcome. Taking the promising potentials of CAR T-cell gene therapy, it is still a revolutionary step in the improvement of immunotherapy. In this paper, we focus on the structure and target antigens of CAR T-cell, summarize the generations of CAR T-cell gene therapies, as well as list out the FDA approved treatments using CAR. Most importantly, we will discuss the past advancements, recent innovations, and the existing factors that still prevent CAR T-cells from becoming the first line treatment for cancers.

Opaganib (ABC294640) Induces Immunogenic Tumor Cell Death and Enhances Checkpoint Antibody Therapy.

Antibody-based cancer drugs that target the checkpoint proteins CTLA-4, PD-1 and PD-L1 provide marked improvement in some patients with deadly diseases such as lung cancer and melanoma. However, most patients are either unresponsive or relapse following an initial response, underscoring the need for further improvement in immunotherapy. Certain drugs induce immunogenic cell death (ICD) in tumor cells in which the dying cells promote immunologic responses in the host that may enhance the in vivo activity of checkpoint antibodies. Sphingolipid metabolism is a key pathway in cancer biology, in which ceramides and sphingosine 1-phosphate (S1P) regulate tumor cell death, proliferation and drug resistance, as well as host inflammation and immunity. In particular, sphingosine kinases are key sites for manipulation of the ceramide/S1P balance that regulates tumor cell proliferation and sensitivity to radiation and chemotherapy. We and others have demonstrated that inhibition of sphingosine kinase-2 by the small-molecule investigational drug opaganib (formerly ABC294640) kills tumor cells and increases their sensitivities to other drugs and radiation. Because sphingolipids have been shown to regulate ICD, opaganib may induce ICD and improve the efficacy of checkpoint antibodies for cancer therapy. This was demonstrated by showing that in vitro treatment with opaganib increases the surface expression of the ICD marker calreticulin on a variety of tumor cell types. In vivo confirmation was achieved using the gold standard immunization assay in which B16 melanoma, Lewis lung carcinoma (LLC) or Neuro-2a neuroblastoma cells were treated with opaganib in vitro and then injected subcutaneously into syngeneic mice, followed by implantation of untreated tumor cells 7 days later. In all cases, immunization with opaganib-treated cells strongly suppressed the growth of subsequently injected tumor cells. Interestingly, opaganib treatment induced crossover immunity in that opaganib-treated B16 cells suppressed the growth of both untreated B16 and LLC cells and opaganib-treated LLC cells inhibited the growth of both untreated LLC and B16 cells. Next, the effects of opaganib in combination with a checkpoint antibody on tumor growth in vivo were assessed. Opaganib and anti-PD-1 antibody each slowed the growth of B16 tumors and improved mouse survival, while the combination of opaganib plus anti-PD-1 strongly suppressed tumor growth and improved survival (p < 0.0001). Individually, opaganib and anti-CTLA-4 antibody had modest effects on the growth of LLC tumors and mouse survival, whereas the combination of opaganib with anti-CTLA-4 substantially inhibited tumor growth and increased survival (p < 0.001). Finally, the survival of mice bearing B16 tumors was only marginally improved by opaganib or anti-PD-L1 antibody alone but was nearly doubled by the drugs in combination (p < 0.005). Overall, these studies demonstrate the ability of opaganib to induce ICD in tumor cells, which improves the antitumor activity of checkpoint antibodies.

IMMU-31. B-CELL THERAPY (BVAX) PROMOTES CD8 T CELLS' PERSISTENCE AND FUNCTION IN THE GLIOBLASTOMA MICROENVIRONMENT BY MAINTAINING THEIR STEM-LIKE FEATURES

Abstract The last decade witnessed tremendous improvement in immunotherapy to treat cancers. Its application in GBM has been particularly challenging due to the immunosuppressive tumor microenvironment, which greatly challenges T-cell infiltration and function. We developed a B-cell therapy with potent antigen-presenting functions that promote CD8+ tumor tropism and persistence in the tumor microenvironment. The present study investigates how BVax activates CD8+ T cells differently from other professional APCs like dendritic cells (DC) that could explain the persistence of CD8+ T cells in the tumor microenvironment. Upon in vitro activation by coculturing with BVax, most CD8+ T cells proliferated into TCF+ PD1- intermediate state, with CD44+ CD62L+ memory phenotype. Less than 10% of CD8+ were in the terminally-proliferation state. In contrast, DCs preferentially expanded TCF- PD1+ terminally-proliferating CD8+ T cells that were exhibiting CD44+ CD62L- effector phenotype. In vivo, activation of CD8+ T cells by co-injecting BVax or DC with CD8+ T cells to CT2A-bearing mice indicated that BVax promoted the proliferation of tumor-reactive CD8 T cells while maintaining the low PD1+ expression. Re-exposing the ex vivo-activated CD8+ T cells to tumor by adoptive transferring them to CT2A tumor-bearing mice revealed that BVax elicited immunological memory and potentiated GzmB+ production of CD8+ T cells. This study suggests that BVax and DC have distinctive interactions with CD8+ T cells, with the former poise CD8+ T cells at a more stem-like state, allowing them to differentiate upon tumor encounter.

Understanding the role of Toll-like receptors in lung cancer immunity and immunotherapy

Lung cancer is currently the leading cause of cancer-related deaths worldwide. Significant improvements in lung cancer therapeutics have relied on a better understanding of lung cancer immunity and the development of novel immunotherapies, as best exemplified by the introduction of PD-1/PD-L1-based therapies. However, this improvement is limited to lung cancer patients who respond to anti-PD-1 immunotherapy. Further improvements in immunotherapy may benefit from a better understanding of innate immune response mechanisms in the lung. Toll-like receptors (TLRs) are a key component of the innate immune response and mediate the early recognition of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). TLR signaling modulates the tumor microenvironment from "cold" to "hot" leading to immune sensitization of tumor cells to treatments and improved patient prognosis. In addition, TLR signaling activates the adaptive immune response to improve the response to cancer immunotherapy through the regulation of anti-tumor T cell activity. This review will highlight recent progress in our understanding of the role of TLRs in lung cancer immunity and immunotherapy.

Binding of DNA to Natural Sepiolite: Applications in Biotechnology and Perspectives

AbstractDNA manipulation is crucial for many biotechnological prospects and for medical applications such as gene therapy. This requires the amplification and extraction of DNA from bacteria and the transfer of these DNA molecules into cells, including bacterial and mammalian cells. The capacity of the natural magnesium silicate clay mineral sepiolite to bind to DNA makes it a potentially useful tool for biotechnological/medical strategies. In addition, sepiolite is inexpensive and classified as non-toxic and non-carcinogenic. This review will first describe the physicochemical interactions between sepiolite and DNA. Then, the leverage of sepiolite/DNA interactions for DNA extraction from bacteria, to optimize DNA transfer into bacteria and DNA transfection into mammalian cells, are presented. Finally, the putative toxicity of sepiolite and its advantages and perspectives for future prospects, such as the improvement of immunotherapy, are also discussed.

Influence of nNav1.5 on MHC class I expression in breast cancer

Cancer metastasis occurs due to the ability of the tumour to evade immune recognition and response by altering the major histocompatibility complex class I (MHC class I). A comprehensive understanding of the MHC class I alteration in breast cancer metastasis is vital for the rational design and improvement of immunotherapies for advanced cancer. In this study, we associate it with the elevation of 'neonatal' Nav1.5 (nNav1.5) mRNA expression in the aggressive type of human breast cancer cells. Using real-time PCR, lower expression of TAP1 (by - 4.83-fold), which is a part of the MHC class I antigen processing machinery (APM) pathway component, in contrast to the greater expression of nNav1.5 (by 3.76-fold), along with other metastatic markers, MMP1 (by 57.48-fold) and fibronectin (by 2.88-fold) in aggressive human breast cancer cell line, MDA-MB231 were obtained. Subsequent knockdown of nNav1.5 (by 52.6%) in MDA-MB-231 via siRNA-SCN5A resulted in downregulation of another metastatic marker, fibronectin (by 52.9%) but importantly, rescued MHC class I expression (by 347% or 3.47-fold). Furthermore, the siRNA-SCN5A transfected cells failed to form a unified spheroid and incapable of efficiently invading the surrounding invasion matrix. In summary, the influence of nNav1.5 in regulating MHC class I mRNA expression to allow for breast cancer invasion is demonstrated, supporting the potential of nNav1.5 as an immunotherapy target to overcome tumour immune evasion thus preventing metastasis.

Transforming growth factor-beta1 and myeloid-derived suppressor cells: A cancerous partnership.

Transforming growth factor-beta1 (TGF-β1) plays a crucial role in tumor progression. It can inhibit early cancer stages but promotes tumor growth and development at the late stages of tumorigenesis. TGF-β1 has a potent immunosuppressive function within the tumor microenvironment that largely contributes to tumor cells' immune escape and reduction in cancer immunotherapy responses. Likewise, myeloid-derived suppressor cells (MDSCs) have been postulated as leading tumor promoters and a hallmark of cancer immune evasion mechanisms. This review attempts to analyze the prominent roles of both TGF-β1 and MDSCs and their interplay in cancer immunity. Furthermore, therapies against either TGF-β1 or MDSCs, and their potential synergistic combination with immunotherapies are discussed. Simultaneous TGF-β1 and MDSCs inhibition suggest a potential improvement in immunotherapy or subverted tumor immune resistance.

SITC cancer immunotherapy resource document: a compass in the land of biomarker discovery

Since the publication of the Society for Immunotherapy of Cancer’s (SITC) original cancer immunotherapy biomarkers resource document, there have been remarkable breakthroughs in cancer immunotherapy, in particular the development and...

The Combined Detection of Immune Genes for Predicting the Prognosis of Patients With Non-Small Cell Lung Cancer.

Lung cancer is one of the leading causes of cancer-related death. In recent years, there has been an increasing interest in the fields of tumor and immunity. This study focused on the possible prognostic value of immune genes in non-small cell lung cancer patients. We used The Cancer Genome Atlas (TCGA) to download gene expression data and clinical information of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). The immune gene list was downloaded from the Immport database. We then constructed immune gene prognostic models on the basis of Cox regression analysis. We further evaluated the clinical significance of the models via survival analysis, receiver operating characteristic (ROC) curves, and independent prognostic factor analysis. Moreover, we analyzed the associations of prognostic models with both mutation burdens and neoantigens. Using the Gene Expression Omnibus (GEO) and Kaplan–Meier plotter databases, we evaluated the validity of the prognostic models. The prognostic model of LUAD included 13 immune genes, and the prognostic model of LUSC contained 10 immune genes. High-risk patients based on prognostic models had a lower 5-year survival rate than did low-risk patients. The ROC curve analysis demonstrated the prediction accuracy of the prognostic models, as the area under the curve (AUC) was 0.742, 0.707, and 0.711 for LUAD, and 0.668, 0.703, and 0.668 for LUSC, when the predicted survival times were 1, 3, and 5 years, respectively. The mutation burden analysis showed that mutation level was associated with the risk score in patients with LUAD. The analysis based on GEO and Kaplan–Meier plotter demonstrated the prognostic validity of the models. Therefore, immune gene-related models of LUAD and LUSC can predict prognosis. Further study of these genes may enable us to better distinguish between LUAD and LUSC and lead to improvement in immunotherapy for lung cancer.

ULTRASOUND TUMOR ABLATION: IMMUNE EFFECTS AND PERSPECTIVES OF INTEGRATION IN THE MODERN TREATMENT OF ADVANCED CANCER

Despite significant progress in molecular oncology and immuno-oncology, only 20-30 % of patients with advanced cancer can be cured with modern treatments indicating that new approaches are needed. Further improvements in immunotherapy of cancer are associated with enhanced tumor immunogenicity, induction of inflammatory phenotype and inhibition of immune suppression at the tumor microenvironment level. In this context, high-intensity focused ultrasound (HIFU) ablation have several advantages, particularly it is able to elicits a rapid clinical and immune response, is non-invasive, have low local morbidity, allows repeated sonications, have relative low cost and does not require long hospitalization. In addition to cytoreduction and decreasing of systemic immune suppression, HIFU generates a tumor debris depot acting as vaccine in situ. Immunogenic cell death elicits a CD4+ and CD8+ cytotoxic T-cell response, but several regulatory mechanisms, particularly PD-1L expression, are promoted in response to enhanced immune cells infiltration of heated and distal tumors. This results in low rate of durable and clinically relevant abscopal effects. For these reasons HIFU is currently viewed as a part of strategies targeting multiple steps of cancer immune cycle (TLR agonists, GM-CSF, cytokines, CTLA-4, PD-1 / PD-1L inhibitors, T-cell co-stimulation agonists, adoptive cell therapy etc). Higher rate of abscopal effects and improved survival have been shown in some preclinical studies using thermal ablations in combination with immunotherapy. In this setting, there is an opportunity to use check-point inhibitors in reduced doses. In addition, tumor ablation after non-effective immunotherapy could induce a new cancer antigens spreading, T-cell repertoire changes and enhance tumor responsiveness to treatment. Based on encouraging preclinical data, this exiting approach is currently explored in some ongoing trials aiming to evaluate the optimal treatment sequences and its clinical efficacy.

A simple in vitro method for evaluating dendritic cell-based vaccinations.

BackgroundDendritic cell (DC) therapy is a promising therapy for cancer-targeting treatments. Recently, DCs have been used for treatment of some cancers. We aimed to develop an in vitro assay to evaluate DC therapy in cancer treatment using a breast cancer model.MethodsDCs were induced from murine bone marrow mononuclear cells in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with GM-CSF (20 ng/mL) and IL-4 (20 ng/mL). Immature DCs were primed with breast cancer stem cell (BCSC)-derived antigens. BCSCs were sorted from 4T1 cell lines based on aldehyde dehydrogenase expression. A mixture of DCs and cytotoxic T lymphocytes (CTLs) were used to evaluate the inhibitory effect of antigen-primed DCs on BCSCs. BCSC proliferation and doubling time were recorded based on impedance-based cell analysis using the xCELLigence system. The specification of inhibitory effects of DCs and CTLs was also evaluated using the same system.ResultsThe results showed that impedance-based analysis of BCSCs reflected cytotoxicity and inhibitory effects of DCs and CTLs at 72 hours. Differences in ratios of DC:CTL changed the cytotoxicity of DCs and CTLs.ConclusionThis study successfully used impedance-based cell analysis as a new in vitro assay to evaluate DC efficacy in cancer immunotherapy. We hope this technique will contribute to the development and improvement of immunotherapies in the near future.

Dendritic cell subsets in neoplastic tissue and peripheral blood of laryngeal cancer patients: relation with grade and stage of the disease

Despite improvements in immunotherapy, little is known about dendritic cell (DC) subpopulations naturally occurring in the laryngeal cancer tissue (LCT) and peripheral blood (PB) of untreated laryngeal cancer (LC) patients. The purpose of the present study was to evaluate mature, immature myeloid and plasmacytoid DCs in the LCT and PB of patients with various stages and grades of squamous cell carcinoma of the larynx (n=66) and PB of healthy donors (n=20), and to explore the correlation of the percentages of the DCs to clinical parameters. The percentage of DCs in LCT and PB was determined using monoclonal antibodies and flow cytometry. It has been revealed that DCs accumulate in LCT in comparison with their content in PB. The myeloid to lymphoid/plasmacytoid DC ratio was higher in LCT compared to PB. It was found that in cases of poorly-differentiated LC, there were higher percentages of lymphoid/plasmacytoid DCs in LCT in comparison to their content in the PB. Moreover, in the blood of patients with T4 cancers we found significantly lower percentages of myeloid DCs in comparison to individuals with T1 neoplasms. The percentage of myeloid DCs infiltrating cancer tissue positively correlated with the T stage. In patients with no metastases in the lymph nodes, PB contained less mature DCs but higher amount of myeloid DCs compared to LCT. Alteration of the DC proportion in LC patients may result in the development of immunotolerance.

Immunotherapy of autoimmunity and cancer: the penalty for success

Advances in our understanding of autoimmunity and tumour immunity have led to improvements in immunotherapy for these diseases. Ironically, effective tumour immunity requires the induction of the same responses that underlie autoimmunity, whereas autoimmunity is driven by dysregulation of the same mechanisms that are involved in host defence and immune surveillance. Therefore, as we manipulate the immune system to treat cancer or autoimmunity, we inevitably unbalance the vital mechanisms that regulate self tolerance and antimicrobial resistance. This Science and Society article aims to dissect the conundrum that is inherent to the concept of immunotherapy and highlights the need for new and more specific therapeutic approaches.

The combined actions of NK and T lymphocytes are necessary to reject an EGFP+ mesenchymal tumor through mechanisms dependent on NKG2D and IFNγ

Better understanding of the mechanisms that mediate spontaneous immune rejections ought to be important in the quest for improvements in immunotherapy of cancer. A set of intraperitoneal tumors of mesenchymal origin that had been chemically induced in ubiquitously expressing EGFP transgenic mice provided a model in which both T and NK cells were absolutely required for tumor rejection. Tumor cells were traceable because of being fluorescent and readily grafted in RAG1(-/-) immunodeficient mice, whereas they were rejected in a majority of syngeneic C57BL/6 and EGFP-transgenic mice. Tumor-cell clones with the highest EGFP expression tended to be rejected, but a direct involvement of EGFP as the antigen recognized for the immune rejections was ruled out. Rejections were absolutely dependent on NK cells as well as on CD4(+) and CD8(+) T lymphocytes according to selective depletion studies. Furthermore, CD8(+) and CD4(+) T lymphocytes as well as NK cells were detected in the inflammatory infiltrate that mediates tumor rejection along with some DC. The effects of IFN gamma, produced at the tumor site by T and NK lymphocytes, were only required at the malignant cell level and were necessary for tumor eradication. NK recognition of tumor cells was mediated by the NKG2D-activating receptor and blocking its function in vivo partially interfered with rejection. Therefore, complete rejection of these mesenchymal tumors requires a concerted set of activities including direct tumor-cell destruction and IFN gamma production that are mediated by both NK and T cells.

T cell immune responses against melanoma and melanocytes in cancer and autoimmunity.

T cell responses specific for melanoma cells and melanocytes appear to be involved in the rejection of melanoma tumors, as well as in the development of autoimmune reactions in patients with Vogt-Koyanagi-Harada disease (VKH), sympathetic ophthalmia, or autoimmune vitiligo. Some of the target antigens for those T cells have been isolated using cDNA expression cloning with melanoma reactive T cells derived from lymphocytes tumor infiltrating (TIL) of patients with melanoma. These include melanocyte specific proteins, such as tyrosinase, TRP1, TRP2, gp100, and MART-1, cancer-testis antigens, and mutated peptides derived from genetic alterations in melanoma cells. Some of the melanoma reactive T cells appear to respond to cryptic or subdominant self epitopes in melanosomal proteins. Modification of those epitopes to increase their immunogenicity by replacement of amino acids at primary anchor residues for peptide/MHC binding, allowed an improvement in immunotherapy for patients with melanoma. Targets for autoreactive T cells against melanocytes in those autoimmune disorders remain to be identified. Isolation of novel target antigens is important for understanding these pathological T cell responses, as well as for developing new diagnostic and treatment methods for these diseases. A variety of techniques, including cDNA expression cloning with T cells, serological analysis of recombinant cDNA expression libraries (SEREX), cDNA subtraction with representational differential analysis (RDA), and serial analysis of gene expression (SAGE) are now being applied to identify novel melanoma/melanocyte antigens recognized by T cells and antibodies.

Pharmacokinetics of the toxic fraction of Centruroides limpidus limpidus venom in experimentally envenomed rabbits and effects of immunotherapy with specific F(ab ′) 2

Envenomations after scorpion stings are a major health problem throughout the world. Their specific treatment is immunotherapy which consists of the injection of specific antibody. In this article, we studied the pharmacokinetics of the toxic fraction of Centruroides limpidus limpidus venom (fraction II) in experimentally envenomed rabbits. After an intravenous injection, fraction II (FII) was rapidly distributed and eliminated from the body (terminal half-life of 1.9 h). When injected subcutaneously, high concentrations of FII were measured in the vascular space rapidly after the injection ( T max=1 h) and FII was eliminated with a terminal half-life of 1.8 h, close to that determined after intravenous injection. These observations go along with the rapid onset of clinical symptoms observed after accidental envenomations. To investigate the mechanism of action of antivenom, we examined the effects of the intravenous administration of antivenom (horse F(ab′) 2 directed against Centruroides venoms) on the pharmacokinetics of FII. Immunotherapy performed 2 h after the experimental envenomation largely increased the area under the concentration time curve of FII compared to that calculated in absence of immunotherapy (13,000 versus 170 ng h ml −1, respectively). These observations agree with previous findings which showed that specific antibody fragments are able to remove drugs from their site of action and sequester them in the vascular space. These studies provide a powerful tool to determine an excellent procedure for further improvement of immunotherapy.