Fully Automated Workflow for Integrated Sample Digestion and Evotip Loading Enabling High-Throughput Clinical Proteomics.

Protein identification and quantification is an important tool for biomarker discovery. With the increased sensitivity and speed of modern mass spectrometers, sample preparation remains a bottleneck for studying large cohorts. To address this issue, we prepared and evaluated a simple and efficient workflow on the Opentrons OT-2 robot that combines sample digestion, cleanup, and loading on Evotips in a fully automated manner, allowing the processing of up to 192 samples in 6 h. Analysis of 192 automated HeLa cell sample preparations consistently identified ∼8000 protein groups and ∼130,000 peptide precursors with an 11.5 min active liquid chromatography gradient with the Evosep One and narrow-window data-independent acquisition (nDIA) with the Orbitrap Astral mass spectrometer providing a throughput of 100 samples per day. Our results demonstrate a highly sensitive workflow yielding both reproducibility and stability at low sample inputs. The workflow is optimized for minimal sample starting amount to reduce the costs for reagents needed for sample preparation, which is critical when analyzing large biological cohorts. Building on the digesting workflow, we incorporated an automated phosphopeptide enrichment step using magnetic titanium-immobilized metal ion affinity chromatography beads. This allows for a fully automated proteome and phosphoproteome sample preparation in a single step with high sensitivity. Using the integrated digestion and Evotip loading workflow, we evaluated the effects of cancer immune therapy on the plasma proteome in metastatic melanoma patients.

A systematic review of cardiovascular toxicities induced by cancer immune therapies: underlying mechanisms, clinical manifestations and therapeutic approaches.

Immunotherapy has revolutionized the management of various types of cancers, even those previously deemed untreatable. Nonetheless, these medications have been associated with inflammation and damage across various organs. These challenges are exemplified by the adverse cardiovascular impacts of cancer immunotherapy, which need comprehensive understanding, clarification, and management integrated into the overall care of cancer patients. Numerous anticancer immunotherapies have been linked to the prevalence and severity of cardiovascular toxicity. These challenges emphasize the importance of conducting fundamental and applied research to elucidate disease causes, discover prognostic indicators, enhance diagnostic methods, and create successful therapies. Despite the acknowledged importance of T cells, there remains a knowledge gap regarding the inciting antigens, the reasons for their recognition, and the mechanisms of how they contribute to cardiac cell injury. In this review, we summarize the molecular mechanism, epidemiology, diagnosis, pathophysiology and corresponding treatment of cardiovascular toxicity induced by immunotherapy, including immune checkpoint inhibitors (ICIs), adoptive cell therapies (ACT), and bi-specific T-cell engagers (BiTEs) among others. By elucidating these aspects, we aim to provide a better understanding of immunotherapies in cancer treatment and offer guidance for their clinical application.

Differential ultracentrifugation enables deep plasma proteomics through enrichment of extracellular vesicles.

Human plasma is a rich source of biomedical information and biomarkers. However, the enormous dynamic range of plasma proteins limits its accessibility to mass spectrometric (MS) analysis. Here, we show that enrichment of extracellular vesicles (EVs) by ultracentrifugation increases plasma proteome depth by an order of magnitude. With this approach, more than two thousand proteins are routinely and reproducibly quantified by label-free quantification and data independent acquisition (DIA) in single-shot liquid chromatography tandem mass spectrometry runs of less than one hour. We present an optimized plasma proteomics workflow that enables high-throughput with very short chromatographic gradients analyzing hundred samples per day with deep proteome coverage, especially when including a study-specific spectral library generated by repeated injection and gas-phase fractionation of pooled samples. Finally, we test the workflow on clinical biobank samples from malignant melanoma patients in immunotherapy to demonstrate the improved proteome coverage supporting the potential for future biomarker discovery.

Effect of concomitant antibiotics use on patient outcomes and adverse effects in patients treated with ICIs.

Background: Immune checkpoint inhibitors (ICIs) such as PD-1/PD-L1/CTLA-4 inhibitors have brought new opportunities for the cure of cancer patients and have been widely used and which are the most successful cancer immunotherapy drug in recent years. Gut microbiome and metabolites exert a critical regulatory function in cancer immunotherapy of ICIs, which can be affected by antibiotics intervention. However, inflammatory infections caused by impaired immune function in tumor patients often require antibiotic treatment.Objective: In this review, we briefly discussed the correlation between antibiotics and ICIs treatment to evaluate the impact of antibiotics on cancer progression.Methods: By searches of PubMed, we collected the data such as progression-free survival time (PFS) and overall survival time (OS) in patients with non-small cell lung cancer (NSCLC), kidney cancer, Melanoma, colorectal cancer, and other tumors.Results: Antibiotics have a negative effect on the prolongation of survival in cancer patients treated with ICIs. This may depend on the patient's cancer type and the type of ICIs and antibiotics they have used.Conclusions: Antibiotics may reduce the effectiveness of immunotherapy by depleting the body's microbiome. Therefore, paying attention to the changes in the level of microorganisms in cancer patients, while making more individualized and precise improvements in treatment regimens, may bring new opportunities to improve the efficacy of immunotherapy.

Noncoding RNAs as potential mediators of resistance to cancer immunotherapy.

Substantial evolution in cancer therapy has been witnessed lately, steering mainly towards immunotherapeutic approaches, replacing or in combination with classical therapies. Whereas the use of various immunotherapy approaches, such as adoptive T cell therapy, genetically-modified T cells, or immune checkpoint inhibitors, has been a triumph for cancer immunotherapy, the great challenge is the ability of the immune system to sustain long lasting anti-tumor response. Additionally, epigenetic changes in a suppressive tumor microenvironment can pertain to T cell exhaustion, limiting their functionality. Noncoding RNAs (ncRNAs) have emerged over the last years as key players in epigenetic regulation. Among those, microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) have been studied extensively for their potential role in regulating tumor immunity through direct regulation of genes involved in immune activation or suppression. In this review, we will provide an overview of contemporary approaches for cancer immunotherapy and will present the current state of knowledge implicating miRNAs and lncRNAs in regulating immune response against human cancer and their potential implications in resistance to cancer immunotherapy, with main emphasis on immune checkpoints regulation.

The TNF/TNFR2 signaling pathway is a key regulatory factor in endothelial progenitor cell immunosuppressive effect.

BACKGROUND: Endothelial progenitor cells (EPCs) are non-differentiated endothelial cells (ECs) present in blood circulation that are involved in neo-vascularization and correction of damaged endothelial sites. Since EPCs from patients with vascular disorders are impaired and inefficient, allogenic sources from adult or cord blood are considered as good alternatives. However, due to the reaction of immune system against allogenic cells which usually lead to their elimination, we focused on the exact role of EPCs on immune cells, particularly, T cells which are the most important cells applied in immune rejection. TNFα is one of the main activators of EPCs that recognizes two distinct receptors. TNFR1 is expressed ubiquitously and its interaction with TNFα leads to differentiation and apoptosis, whereas, TNFR2 is expressed predominantly on ECs, immune cells and neural cells and is involved in cell survival and proliferation. Interestingly, it has been shown that different immunosuppressive cells express TNFR2 and this is directly related to their immunosuppressive efficiency. However, little is known about immunological profile and function of TNFR2 in EPCs. METHODS: Using different in-vitro combinations, we performed co-cultures of ECs and T cells to investigate the immunological effect of EPCs on T cells. We interrupted in the TNFα/TNFR2 axis either by blocking the receptor using TNFR2 antagonist or blocking the ligand using T cells derived from TNFα KO mice. RESULTS: We demonstrated that EPCs are able to suppress T cell proliferation and modulate them towards less pro-inflammatory and active phenotypes. Moreover, we showed that TNFα/TNFR2 immune-checkpoint pathway is critical in EPC immunomodulatory effect. CONCLUSIONS: Our results reveal for the first time a mechanism that EPCs use to suppress immune cells, therefore, enabling them to form new immunosuppressive vessels. Furthermore, we have shown the importance of TNFα/TNFR2 axis in EPCs as an immune checkpoint pathway. We believe that targeting TNFR2 is especially crucial in cancer immune therapy since it controls two crucial aspects of tumor microenvironment: 1) Immunosuppression and 2) Angiogenesis. Video Abstract. (MP4 46355 kb).

Complex interactions between the microbiome and cancer immune therapy.

Immuno-oncology has rapidly grown in the last thirty years, and immunotherapeutic agents are now approved to treat many disparate cancers. Immune checkpoint inhibitors (ICIs) are employed to augment cytotoxic anti-cancer activity by inhibiting negative regulatory elements of the immune system. Modulating the immune system to target neoplasms has improved survivability of numerous cancers in many individuals, but forecasting outcomes post therapy is difficult due to insufficient predictive biomarkers. Recently, the tumor and gastrointestinal microbiome and immune milieu have been investigated as predictors and influencers of cancer immune therapy. In this review, we discuss: (1) ways to measure the microbiome including relevant bioinformatic analyses, (2) recent developments in animal studies and human clinical trials utilizing gut microbial composition and function as biomarkers of cancer immune therapy response and toxicity, and (3) using prebiotics, probiotics, postbiotics, antibiotics, and fecal microbiota transplant (FMT) to modulate immune therapy. We discuss the respective benefits of 16S ribosomal RNA (rRNA) gene and shotgun metagenomic sequencing including important considerations in obtaining samples and in designing and interpreting human and animal microbiome studies. We then focus on studies discussing the differences in response to ICIs in relation to the microbiome and inflammatory mediators. ICIs cause colitis in up to 25% of individuals, and colitis is often refractory to common immunosuppressive medications. Researchers have measured microbiota composition prior to ICI therapy and correlated baseline microbiota composition with efficacy and colitis. Certain bacterial taxa that appear to enhance therapeutic benefit are also implicated in increased susceptibility to colitis, alluding to a delicate balance between pro-inflammatory tumor killing and anti-inflammatory protection from colitis. Pre-clinical and clinical models have trialed probiotic administration, e.g. Bifidobacterium spp. or FMT, to treat colitis when immune suppressive agents fail. We are excited about the future of modulating the microbiome to predict and influence cancer outcomes. Furthermore, novel therapies employed for other illnesses including bacteriophage and genetically-engineered microbes can be adapted in the future to promote increased advancements in cancer treatment and side effect management.

Precision glycocalyx editing as a strategy for cancer immunotherapy.

Cell surface sialosides constitute a central axis of immune modulation that is exploited by tumors to evade both innate and adaptive immune destruction. Therapeutic strategies that target tumor-associated sialosides may therefore potentiate antitumor immunity. Here, we report the development of antibody-sialidase conjugates that enhance tumor cell susceptibility to antibody-dependent cell-mediated cytotoxicity (ADCC) by selective desialylation of the tumor cell glycocalyx. We chemically fused a recombinant sialidase to the human epidermal growth factor receptor 2 (HER2)-specific antibody trastuzumab through a C-terminal aldehyde tag. The antibody-sialidase conjugate desialylated tumor cells in a HER2-dependent manner, reduced binding by natural killer (NK) cell inhibitory sialic acid-binding Ig-like lectin (Siglec) receptors, and enhanced binding to the NK-activating receptor natural killer group 2D (NKG2D). Sialidase conjugation to trastuzumab enhanced ADCC against tumor cells expressing moderate levels of HER2, suggesting a therapeutic strategy for cancer patients with lower HER2 levels or inherent trastuzumab resistance. Precision glycocalyx editing with antibody-enzyme conjugates is therefore a promising avenue for cancer immune therapy.

Treg-specific demethylated region activity in isolated regulatory t lymphocytes is a surrogate for disease severity in hepatocellular carcinoma.

In certain unique conditions like viral infections of the liver like hepatitis B (HBV) and hepatitis C (HCV), activation of Tregs may be associated with chronicity of the viral infections and subsequent predisposition to development of hepatocellular carcinoma (HCC) by the integrated viral genome. In parallel, potential persistence of Tregs activity may lead to immune evasion of cancerous cells and thus persistence of the carcinomatous conditions. In this study, we hypothesized that although the relative proportions of Tregs may remain unaltered in HCC, persistence of activity of Tregs may lead to immune evasion in advanced stages of HCC. To examine the issue of activation of Treg in liver cancer pathogenesis, we obtained liver biopsy and peripheral blood samples from patients with advanced grades of HCC, isolated Tregs, and examined the methylation status of "Treg-specific demethylated region" (TSDR), a key region whose methylation suppresses Treg activity and demethylation stimulates its genomic activity. This study provides evidence of demethylation of TSDR, increased gene expression examined by luciferase assays, and nuclear translocation of key transcription factors that function as gene enhancers in CD4+CD25+FoxP3 regulatory T cells in advanced grades of HCC.

The Microbiome Modulates the Immune System to Influence Cancer Therapy.

The gut microbiota composition can affect the tumor microenvironment and its interaction with the immune system, thereby having implications for treatment predictions. This article reviews the studies available to better understand how the gut microbiome helps the immune system fight cancer. To describe this fact, different mechanisms and approaches utilizing probiotics to improve advancements in cancer treatment will be discussed. Moreover, not only calorie intake but also the variety and quality of diet can influence cancer patients' immunotherapy treatment because dietary patterns can impair immunological activities either by stimulating or suppressing innate and adaptive immunity. Therefore, it is interesting and critical to understand gut microbiome composition as a biomarker to predict cancer immunotherapy outcomes and responses. Here, more emphasis will be given to the recent development in immunotherapies utilizing microbiota to improve cancer therapies, which is beneficial for cancer patients.

A multicenter retrospective analysis: Factors influencing hepatic adverse events induced by immunotherapy in advanced liver cancer.

OBJECTIVES: To analyze the clinical characteristics and influencing factors of hepatotoxicity in patients with advanced hepatocellular carcinoma (HCC) treated with programmed cell death protein-1 (PD-1) inhibitors, and to provide a theoretical basis for the treatment of immune-related hepatotoxicity in patients with advanced HCC. METHODS: Retrospective analysis of clinical data of patients with advanced HCC from February 2021 to February 2023, in order to summarize and statistically analyze the influencing factors of immune-related liver adverse reactions. RESULTS: A total of 135 patients met the inclusion criteria, among whom 46 patients experienced varying degrees of immune-related liver adverse reactions, with an incidence rate of 34.1% (46/135). The time range of immune-related liver adverse reactions was 3-26 weeks, with a median time of 4 weeks. The age range of immune-related liver adverse reactions was 34-73 years, with a median age of 62 years. Statistical analysis of the influencing factors and liver adverse reactions showed that age, total bilirubin level, and Child-Pugh (C-P) grading were influencing factors for the occurrence of liver adverse reactions (p < .05), and among these three influencing factors, the proportion of males with ≥2 influencing factors was higher than that of females; liver function C-P B was an independent influencing factor for liver adverse reactions (p < .05). CONCLUSION: For male patients over 60 years old, with bilirubin levels ≥51 µmol/L and liver function C-P B, close observation of the occurrence of immune-related adverse reactions during treatment is recommended.

A novel dual mechanism-of-action bispecific PD-1-IL-2v armed by a "ßγ-only" interleukin-2 variant.

Introduction: Interleukin-2 (IL-2) is one of the first cytokines to be discovered as an immune agonist for cancer immunotherapy. Biased IL-2 variants had been discovered to eliminate Treg activation or enhance the tumor specific T cell cytotoxicity. However, all the biased IL-2 variants pose the risk to overstimulate immune response at a low-dose range. Here, we introduce a novel dual-MOA bispecific PD-1-IL-2v molecule with great anti-tumor efficacy in a high dosed manner. Methods: The novel IL-2 variant was designed by structural truncation and shuffling. The single armed bispecific PD-1-IL-2v molecule and IL-2v were studied by immune cell activations in vitro and in vivo and anti-tumor efficacy in mouse model. Results and discussion: The IL-2 variant in this bispecific antibody only binds to IL-2Rßγ complex in a fast-on/off manner without α, ß or γ single receptor binding. This IL-2v mildly activates T and NK cells without over stimulation, meanwhile it diminishes Treg activation compared to the wild type IL-2. This unique bispecific molecule with "ßγ-only" IL-2v can not only "in-cis" stimulate and expand CD8 T and NK cells moderately without Treg activation, but also block the PD-1/L1 interaction at a similar dose range with monoclonal antibody.

Dendritic cell subsets and implications for cancer immunotherapy.

Dendritic cells (DCs) play a central role in the orchestration of effective T cell responses against tumors. However, their functional behavior is context-dependent. DC type, transcriptional program, location, intratumoral factors, and inflammatory milieu all impact DCs with regard to promoting or inhibiting tumor immunity. The following review introduces important facets of DC function, and how subset and phenotype can affect the interplay of DCs with other factors in the tumor microenvironment. It will also discuss how current cancer treatment relies on DC function, and survey the myriad ways with which immune therapy can more directly harness DCs to enact antitumor cytotoxicity.

AN0025, a novel antagonist of PGE2-receptor E-type 4 (EP4), in combination with total neoadjuvant treatment of advanced rectal cancer.

PURPOSE: To assess the safety and efficacy of AN0025 in combination with preoperative radiotherapy and chemotherapy in either short course (SCRT) or long course radiotherapy (LCRT) settings for those with locally advanced rectal cancer. PATIENTS AND METHODS: Twenty-eight subjects with locally advanced rectal cancer participated in this multicenter, open-label, Phase Ib trial. Enrolled subjects received either 250 mg or 500 mg of AN0025 once daily for 10 weeks with either LCRT or SCRT with chemotherapy (7 subjects/group). Participants were assessed for safety/efficacy starting from the first dose of study drug administration and were followed for 2 years. RESULTS: No treatment-emergent adverse or serious adverse events meeting dose-limiting criteria were observed, with only 3 subjects discontinuing AN0025 treatment due to adverse events. Twenty-five of 28 subjects completed 10 weeks of AN0025 and adjuvant therapy and were evaluated for efficacy. Overall, 36.0% of subjects (9/25 subjects) achieved a pathological complete response or a complete clinical response, including 26.7% of subjects (4/15 subjects who underwent surgery) who achieved a pathological complete response. A total of 65.4% of subjects had magnetic resonance imaging-confirmed down-staging ≤ stage 3 following completion of treatment. With a median follow-up of 30 months. The 12-month disease-free survival and overall survival were 77.5% (95% confidence interval [CI]: 56.6, 89.2) and 96.3% (95% CI: 76.5, 99.5), respectively. CONCLUSIONS: Treatment with AN0025 administered for 10 weeks along with preoperative SCRT or LCRT did not appear to worsen the toxicity in subjects with locally advanced rectal cancer, was well-tolerated and showed promise in inducing both a pathological and complete clinical response. These findings suggest its activity deserves further investigation in larger clinical trials.

An arginase1- and PD-L1-derived peptide-based vaccine for myeloproliferative neoplasms: A first-in-man clinical trial.

Introduction: Arginase-1 (ARG1) and Programed death ligand-1 (PD-L1) play a vital role in immunosuppression in myeloproliferative neoplasms (MPNs) and directly inhibit T-cell activation and proliferation. We previously identified spontaneous T-cell responses towards PD-L1 and ARG1 derived peptide epitopes in patients with MPNs. In the present First-in-Man study we tested dual vaccinations of ARG1- derived and PD-L1-derived peptides, combined with Montanide ISA-51 as adjuvant, in patients with Janus Kinase 2 (JAK2) V617F-mutated MPN. Methods: Safety and efficacy of vaccination with ARG1- derived and PD-L1-derived peptides with montanide as an adjuvant was tested in 9 patients with MPN The primary end point was safety and toxicity evaluation. The secondary end point was assessment of the immune response to the vaccination epitope (www.clinicaltrials.gov identifier NCT04051307). Results: The study included 9 patients with JAK2-mutant MPN of which 8 received all 24 planned vaccines within a 9-month treatment period. Patients reported only grade 1 and 2 vaccine related adverse events. No alterations in peripheral blood counts were identified, and serial measurements of the JAK2V617F allelic burden showed that none of the patients achieved a molecular response during the treatment period. The vaccines induced strong immune responses against both ARG1 and PD-L1- derived epitopes in the peripheral blood of all patients, and vaccine-specific skin-infiltrating lymphocytes from 5/6 patients could be expanded in vitro after a delayed-type hypersensitivity test. In two patients we also detected both ARG1- and PD-L1-specific T cells in bone marrow samples at the end of trial. Intracellular cytokine staining revealed IFNγ and TNFγ producing CD4+- and CD8+- T cells specific against both vaccine epitopes. Throughout the study, the peripheral CD8/CD4 ratio increased significantly, and the CD8+ TEMRA subpopulation was enlarged. We also identified a significant decrease in PD-L1 mRNA expression in CD14+ myeloid cells in the peripheral blood in all treated patients and a decrease in ARG1 mRNA expression in bone marrow of 6 out of 7 evaluated patients. Conclusion: Overall, the ARG1- and PD-L1-derived vaccines were safe and tolerable and induced strong T-cell responses in all patients. These results warrant further studies of the vaccine in other settings or in combination with additional immune-activating treatments.

Strategies to overcome myeloid cell induced immune suppression in the tumor microenvironment.

Cancer progression and metastasis due to tumor immune evasion and drug resistance is strongly associated with immune suppressive cellular responses, particularly in the case of metastatic tumors. The myeloid cell component plays a key role within the tumor microenvironment (TME) and disrupts both adaptive and innate immune cell responses leading to loss of tumor control. Therefore, strategies to eliminate or modulate the myeloid cell compartment of the TME are increasingly attractive to non-specifically increase anti-tumoral immunity and enhance existing immunotherapies. This review covers current strategies targeting myeloid suppressor cells in the TME to enhance anti-tumoral immunity, including strategies that target chemokine receptors to deplete selected immune suppressive myeloid cells and relieve the inhibition imposed on the effector arms of adaptive immunity. Remodeling the TME can in turn improve the activity of other immunotherapies such as checkpoint blockade and adoptive T cell therapies in immunologically "cold" tumors. When possible, in this review, we have provided evidence and outcomes from recent or current clinical trials evaluating the effectiveness of the specific strategies used to target myeloid cells in the TME. The review seeks to provide a broad overview of how myeloid cell targeting can become a key foundational approach to an overall strategy for improving tumor responses to immunotherapy.

Functional integration of protein A binding ability to antibody fragments for convenient and tag-free purification.

Although the development of small therapeutic antibodies is important, the affinity tags used for their purification often result in heterogeneous production and immunogenicity. In this study, we integrated Staphylococcus aureus protein A (SpA) binding ability into antibody fragments for convenient and tag-free purification. SpA affinity chromatography is used as a global standard purification method for conventional antibodies owing to its high binding affinity to the Fc region. SpA also has a binding affinity for some variable heavy domains (VH) classified in the VH3 subfamily. Through mutagenesis based on alignment and structural modeling results using the SpA-VH3 cocrystal structure, we integrated the SpA-binding ability into the anti-CD3 single-chain Fv. Furthermore, we applied this mutagenesis approach to more complicated small bispecific antibodies and successfully purified the antibodies using SpA affinity chromatography. The antibodies retained their biological function after purification. Integration of SpA-binding ability into conventional antibody fragments simplifies the purification and monitoring of the production processes and, thus, is an ideal strategy for accelerating the development of small therapeutic antibodies. Furthermore, because of its immunoactivity, the anti-CD3 variable region with SpA-binding ability is an effective building block for developing engineered cancer therapeutic antibodies without the Fc region.

Engineering Multienzyme-Mimicking Covalent Organic Frameworks as Pyroptosis Inducers for Boosting Antitumor Immunity.

The engineering of a series of multienzyme-mimicking covalent organic frameworks (COFs), COF-909-Cu, COF-909-Fe, and COF-909-Ni, as pyroptosis inducers, remodeling the tumor microenvironment to boost cancer immunotherapy, is reported. Mechanistic studies reveal that these COFs can serve as hydrogen peroxide (H2 O2 ) homeostasis disruptors to elevate intracellular H2 O2 levels, and they not only exhibit excellent superoxide dismutase (SOD)-mimicking activity and convert superoxide radicals (O2 •- ) to H2 O2 to facilitate H2 O2 generation, but also possess outstanding glutathione peroxidase (GPx)-mimicking activity and deplete glutathione (GSH) to alleviate the scavenging of H2 O2 . Meanwhile, the outstanding photothermal therapy properties of these COFs can accelerate the Fenton-like ionization process to facilitate their chemodynamic therapy efficiency. One member, COF-909-Cu, can robustly induce gasdermin E (GSDME)-dependent pyroptosis and remodel the tumor microenvironment to trigger durable antitumor immunity, thus promoting the response rate of αPD-1 checkpoint blockade and successfully restraining tumor metastasis and recurrence.

Therapeutic Cancer Vaccination With a Peptide Derived From the Calreticulin Exon 9 Mutations Induces Strong Cellular Immune Responses in Patients With CALR-Mutant Chronic Myeloproliferative Neoplasms.

BACKGROUND: The calreticulin (CALR) exon 9 mutations that are identified in 20% of patients with Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPN) generate immunogenic antigens. Thus, therapeutic cancer vaccination against mutant CALR could be a new treatment modality in CALR-mutant MPN. METHODS: The safety and efficacy of vaccination with the peptide CALRLong36 derived from the CALR exon 9 mutations was tested in a phase I clinical vaccination trial with montanide as adjuvant. Ten patients with CALRmut MPN were included in the trial and received 15 vaccines over the course of one year. The primary end point was evaluation of safety and toxicity of the vaccine. Secondary endpoint was assessment of the immune response to the vaccination epitope (www.clinicaltrials.gov identifier NCT03566446). RESULTS: Patients had a median age of 59.5 years and a median disease duration of 6.5 years. All patients received the intended 15 vaccines, and the vaccines were deemed safe and tolerable as only two grade three AE were detected, and none of these were considered to be related to the vaccine. A decline in platelet counts relative to the platelets counts at baseline was detected during the first 100 days, however this did not translate into neither a clinical nor a molecular response in any of the patients. Immunomonitoring revealed that four of 10 patients had an in vitro interferon (IFN)-γ ELISPOT response to the CALRLong36 peptide at baseline, and four additional patients displayed a response in ELISPOT upon receiving three or more vaccines. The amplitude of the immune response increased during the entire vaccination schedule for patients with essential thrombocythemia. In contrast, the immune response in patients with primary myelofibrosis did not increase after three vaccines. CONCLUSION: Therapeutic cancer vaccination with peptide vaccines derived from mutant CALR with montanide as an adjuvant, is safe and tolerable. The vaccines did not induce any clinical responses. However, the majority of patients displayed a marked T-cell response to the vaccine upon completion of the trial. This suggests that vaccines directed against mutant CALR may be used with other cancer therapeutic modalities to enhance the anti-tumor immune response.

Reprogramming Immune Cells for Enhanced Cancer Immunotherapy: Targets and Strategies.

Cancer is one of the leading causes of death and a major public health problem all over the world. Immunotherapy is becoming a revolutionary clinical management for various cancer types. Restoration of aberrant immune surveillance on cancers has achieved markable progress in the past years by either in vivo or ex vivo engineering of the immune cells. Here, we summarized the central roles of immune cells in tumor progression and regression, and the existing and emerging strategies for different immune cell-based immunotherapies. In addition, the current challenges and the potential solutions in translating the immunotherapies into the clinic are also discussed.