Immunological analysis of hybrid neoantigen peptide encompassing class I/II neoepitope-pulsed dendritic cell vaccine.

Neoantigens/ are tumor-specific antigens that evade central immune tolerance mechanisms in the thymus. Long-term tumor-specific cytotoxic T lymphocyte activity maintenance requires class II antigen-reactive CD4+ T cells. We had previously shown that intranodal vaccination with class I neoantigen peptide-pulsed dendritic cells (DCs) induced a robust immune response in a subset of patients with metastatic cancer. The present study aimed to perform a detailed ex vivo analysis of immune responses in four patients receiving an intranodal hybrid human leukocyte antigen class II neoantigen peptide encompassing a class I neoantigen epitope (hybrid neoantigen)-pulsed DC vaccine. After vaccination, strong T-cell reactions against the hybrid class II peptide and the class I-binding neoantigen peptide were observed in all four patients. We found that hybrid class II neoantigen peptide-pulsed DCs stimulated CD4+ T cells via direct antigen presentation and CD8+ T cells via cross-presentation. Further, we demonstrated that hybrid class II peptides encompassing multiple class I neoantigen epitope-pulsed DCs could present multiple class I peptides to CD8+ T cells via cross-presentation. Our findings provide insight into the mechanisms underlying hybrid neoantigen-pulsed DC vaccine therapy and suggest future neoantigen vaccine design.

Contribution of pre-existing neoantigen-specific T cells to a durable complete response after tumor-pulsed dendritic cell vaccine plus nivolumab therapy in a patient with metastatic salivary duct carcinoma.

Although immune checkpoint inhibitors (ICIs) have emerged as new therapeutic options for refractory cancer, they are only effective in select patients. Tumor antigen-pulsed dendritic cell (DC) vaccine therapy activates tumor-specific cytotoxic T lymphocytes, making it an important immunotherapeutic strategy. Salivary ductal carcinoma (SDC) carries a poor prognosis, including poor long-term survival after metastasis or recurrence. In this study, we reported a case of refractory metastatic SDC that was treated with a tumor lysate-pulsed DC vaccine followed by a single injection of low-dose nivolumab, and a durable complete response was achieved. We retrospectively analyzed the immunological factors that contributed to these long-lasting clinical effects. First, we performed neoantigen analysis using resected metastatic tumor specimens obtained before treatment. We found that the tumor had 256 non-synonymous mutations and 669 class I high-affinity binding neoantigen peptides. Using synthetic neoantigen peptides and ELISpot analysis, we found that peripheral blood mononuclear leukocytes cryopreserved before treatment contained pre-existing neoantigen-specific T cells, and the cells obtained after treatment exhibited greater reactivity to neoantigens than those obtained before treatment. Our results collectively suggest that the rapid and long-lasting effect of this combination therapy in our patient may have resulted from the presence of pre-existing neoantigen-specific T cells and stimulation and expansion of those cells following tumor lysate-pulsed DC vaccine and ICI therapy.

Efficacy of Intranodal Neoantigen Peptide-pulsed Dendritic Cell Vaccine Monotherapy in Patients With Advanced Solid Tumors: A Retrospective Analysis.

BACKGROUND/AIM: Neoantigens are tumor-specific antigens that emerge due to gene mutations in tumor cells, and are highly antigenic epitopes that escape central immune tolerance in the thymus, making cancer vaccine therapy a desirable option. PATIENTS AND METHODS: Tumor neoantigens were predicted in 17 patients with advanced cancer. They were resistant to the standard treatment regime, and their synthetic peptides were pulsed to the patient's monocyte-derived dendritic cells (DCs), and administered to the patient's lymph nodes via ultrasound. RESULTS: Some patients showed sustained tumor shrinkage after this treatment, while some did not respond, showing no ELISpot reaction. Although the number of mutations and the predicted neoantigen epitopes differed between patients, the clinical effect depended more on the presence or absence of an immune response after vaccination rather than the number of neoantigens. CONCLUSION: Intranodal neoantigen peptide-pulsed DC vaccine administration therapy has clinical and immunological efficacy and safety.

PTPN3 expressed in activated T lymphocytes is a candidate for a non-antibody-type immune checkpoint inhibitor.

It has been shown that protein tyrosine phosphatase non-receptor type (PTPN) 3 inhibits T-cell activation. However, there is no definitive conclusion about how the inhibition of PTPN3 in lymphocytes affects immune functions in human lymphocytes. In the present study, we showed that PTPN3 inhibition significantly contributes to the enhanced activation of activated human lymphocytes. The PTPN3 expression of lymphocytes was significantly increased through the activation process using IL-2 and anti-CD3 mAb. Interestingly, inhibiting the PTPN3 expression in activated lymphocytes significantly augmented the proliferation, migration, and cytotoxicity through the phosphorylation of zeta-chain-associated protein kinase 70 (ZAP-70), lymphocyte-specific protein tyrosine kinase (LCK), and extracellular signal-regulated kinases (ERK). Lymphocyte activation by PTPN3 inhibition was observed only in activated CD3+ T cells and not in NK cells or resting T cells. In therapy experiments using autologous tumors and lymphocytes, PTPN3 inhibition significantly augmented the number of tumor-infiltrated lymphocytes and the cytotoxicity of activated lymphocytes. Our results strongly imply that PTPN3 acts as an immune checkpoint in activated lymphocytes and that PTPN3 inhibitor may be a new non-antibody-type immune checkpoint inhibitor for cancer therapy.

Usefulness of the nCounter Analysis System to Monitor Immune-related Biomarkers in PBMCs During Anti-PD-1 Therapy.

BACKGROUND/AIM: Immune checkpoint inhibitors (ICIs) have dramatically changed the clinical outcomes of advanced tumours. However, biomarkers for monitoring immunological features during immunotherapy remain unclear, especially those in the peripheral blood, which are easily available. This study evaluated the usefulness of nCounter Analysis System in identifying immunological biomarkers in peripheral blood mononuclear cells (PBMCs) during ICI therapy. PATIENTS AND METHODS: PBMCs from two patients who responded well to ICI therapy were used, and the expression levels of immune-related mRNA and extracellular proteins were analyzed. RESULTS: Changes in the expression levels of 55 genes from pre-treatment to on-treatment were bioinformatically similar between the two cases. The expression levels of PD-1 were consistent with those by flow cytometry analysis, a reliable tool for monitoring various markers. CONCLUSION: The nCounter Analysis System may be a potent tool to simultaneously investigate genes and proteins on PBMCs as biomarkers during immunotherapy using a small amount of sample.

Long-term survival of a patient with recurrent gallbladder carcinoma, treated with chemotherapy, immunotherapy, and surgery: a case report.

BACKGROUND: Gallbladder cancer (GBC) is one of the refractory diseases. Multidisciplinary approach including immunotherapy for such cancers has received much attention in recent years. CASE PRESENTATION: A 59-year-old man underwent an extended cholecystectomy for GBC (pathological stage II, T2 N0 M0, [per UICC 7th edition]) that was incidentally found during cholelithiasis surgery, and was then treated with adjuvant gemcitabine (GEM). Three months later, when a recurrence-suspected lesion was detected in segment 5 (S5) of his liver, we started adoptive immunotherapies with cytokine-activated killer (CAK) cell infusions, combined with chemotherapy. After a year of adjuvant immunochemotherapy, the S5 lesion disappeared on imaging, but lesions suspected metastatic recurrence again appeared in S7 and S8 at 4 years and 6 months post-surgery, for which GEM and cisplatin (CDDP) were administered as second-line chemotherapy. Immunochemotherapy produced stable disease (per RECIST) for 9 months, when tumor growth was detected; open microwave coagulo-necrotic therapy (MCN) was performed for these lesions. Three years after MCN, a solitary liver metastasis was detected in S4. MCN was conducted again, and peritoneal dissemination was found intraoperatively. A month after the second MCN, the patient's carcinoembryonic antigen (CEA) level had increased. Therefore, GEM and tegafur-gimeracil-oteracil potassium (TS-1) were administered as third-line chemotherapy. We also switched the adoptive immunotherapy for tumor-associated antigen-pulsed dendritic cell-activated killer (DAK) cell immunotherapy. After nine courses of GEM and TS-1 administration, CEA had decreased to a normal level. At the time of reporting, 9 years and 6 months have passed since the initial surgery, and 18 months have passed since the peritoneal metastasis was detected. GEM and CDDP are currently administered as fourth-line chemotherapy because of re-increased CEA. Although an undeniable metastasis was found in his para-aortic lymph node, this patient visits our clinic regularly for immunotherapy. CONCLUSION: We here report a rare case of long-term survival of recurrent GBC well controlled by multidisciplinary therapy. Immunotherapy may be a promising modality among multidisciplinary methods for advanced cancer.

Combined Gemcitabine and Metronidazole Is a Promising Therapeutic Strategy for Cancer Stem-like Cholangiocarcinoma.

BACKGROUND/AIM: Metronidazole (MNZ) is a common antibiotic that exerts disulfiram-like effects when taken together with alcohol. However, the relationship between MNZ and aldehyde dehydrogenase (ALDH) activity remains unclear. This study investigated whether MNZ reduces cancer stemness by suppressing ALDH activity and accordingly reducing the malignancy of cholangiocarcinoma (CCA). MATERIALS AND METHODS: We developed gemcitabine (GEM)-resistant TFK-1 cells and originally established CCA cell line from a patient with GEM-resistant CCA. Using these cell lines, we analyzed the impacts of MNZ for cancer stem cell markers, invasiveness, and chemosensitivity. RESULTS: MNZ reduced ALDH activity in GEM-resistant CCA cells, leading to decreased invasiveness and enhanced chemosensitivity. MNZ diminished the invasiveness by inducing mesenchymal-epithelial transition and enhancing chemosensitivity by increasing ENT1 (equilibrative nucleoside transporter 1) and reducing RRM1 (ribonucleotide reductase M1). CONCLUSION: MNZ reduced cancer stemness in GEM-resistant CCA cells. Combined GEM and MNZ would be a promising therapeutic strategy for cancer stem-like CAA.

Stage IV gastric cancer successfully treated by multidisciplinary therapy including chemotherapy, immunotherapy, and surgery: a case report.

BACKGROUND: The prognosis of stage IV gastric cancer (GC) still remains unfavorable. Multidisciplinary approaches should therefore be considered to improve the survival of patients with stage IV GC. We report here a case of primary GC with potentially unresectable metastasis, successfully treated by a multidisciplinary approach including chemotherapy, immunotherapy, and surgery. CASE PRESENTATION: A 74-year-old man presented with multiple left neck masses. Abdominal computed tomography showed a thickened gastric wall and multiple lymphadenopathies including left supraclavicular lymph node. Gastroenterological endoscopy revealed tumor lesions in the gastric cardia. Tumor biopsy indicated a pathological diagnosis of poorly differentiated adenocarcinoma. Open left cervical lymph node biopsy showed histological features identical with the gastric tumor, indicating left clavicle lymph node metastasis of GC. After 2 years of chemo-immunotherapy with S-1/CDDP, paclitaxel, and cytokine-activated killer cells, lesions other than the stomach lesion had regressed to undetectable on imaging studies. The patient then underwent laparoscopy-assisted total gastrectomy with Roux-en-Y reconstruction followed by adjuvant chemo-immunotherapy with paclitaxel and S-1 for 1 year, and immunotherapy with tumor lysate-pulsed dendritic cell-activated killer cells for 5 years. The patient remained well after 5 years and 6 months of follow-up, with no signs of recurrence. CONCLUSION: Therapeutic combinations including immunotherapy may thus allow surgery to be performed in patients previously considered unsuitable for surgical intervention, potentially leading to a clinical cure, as in the current case.

Random migration contributes to cytotoxicity of activated CD8+ T-lymphocytes but not NK cells.

Activated lymphocytes have the ability to undergo non-directional cell movement known as random migration, although the biological role for this remains unclear. Herein, we investigated how random migration affects cytotoxicity of activated lymphocytes using time-lapse imaging analysis. The kinetics of random migration paralleled cytotoxicity in activated lymphocytes. Sphingosine-1-phosphate (S1P) and its receptor-1 (S1PR1) play an important role in lymphocyte migration. Phosphorylated FTY720 (FTYP), a structural analog of S1P, significantly inhibited random migration and cytotoxicity of activated CD3(+)NKG2D(+)CD8(+) T-lymphocytes but not CD3(-)NKG2D(+)CD56(+) natural killer (NK) cells. In a mouse xenograft model, FTYP-treated activated lymphocytes exhibited lower cytotoxicity and less tumor infiltration for activated CD3(+)NKG2D(+) T-lymphocytes but not CD3(-)NKG2D(+) NK cells. These results suggest that random migration contributes to the cytotoxicity of activated CD8(+) T-cells but not of NK cells.

An epithelial cell adhesion molecule- and CD3-bispecific antibody plus activated T-cells can eradicate chemoresistant cancer stem-like pancreatic carcinoma cells in vitro.

Cancer stem-like properties of various types of cancer, including pancreatic cancer, one of the most aggressive types, correlate with metastasis, invasion, and therapeutic resistance. More importantly, chemoresistance in cancer stem-like cells (CSLCs) is a critical problem for eradication of pancreatic cancer. Several cell surface markers, such as CD44 and epithelial cell adhesion molecule (EpCAM), are molecular targets on CSLCs of pancreatic carcinoma. In this study, we investigated whether catumaxomab, a clinical-grade bi-specific antibody that binds to both EpCAM on tumor cells and CD3 on T-cells, combined with activated T-cells can eliminate chemoresistant pancreatic CSLCs in vitro. Firstly, we established a CSLC line (MU-PK1) from human pancreatic carcinoma cells derived from a patient with chemoresistant and disseminated pancreatic cancer. These CSLCs were almost completely resistant to gemcitabine-mediated cytotoxicity up to a concentration of 10 µg/ml. The cells expressed high levels of CSLC markers (CD44 and EpCAM) and had significantly higher capacities for sphere formation, invasion, and aldehyde dehydrogenase-1 expression, which are associated with cancer stemness properties. We found that pre-treatment with catumaxomab and subsequent addition of interleukin-2/OKT3 activated autologous T-cells eliminated CSLCs during a short incubation period. Moreover, when MU-PK1 cells were cultured under hypoxic conditions, the CSLCs became more aggressive. However, the combination of cytokine-activated killer T-cells with catumaxomab successfully lysed almost all these cells. In conclusion, catumaxomab combined with activated T-cells may be a potent therapeutic modality to eradicate chemoresistant pancreatic CSLCs.

NKG2D-directed cytokine-activated killer lymphocyte therapy combined with gemcitabine for patients with chemoresistant metastatic solid tumors.

Natural-killer group 2, member D (NKG2D) is an activating receptor found on activated natural killer cells and on activated T-cells, here termed cytokine-activated killer (CAK) cells. NKG2D ligands are expressed on various human cancer types. Gemcitabine is an anticancer drug which is a less immune-destructive agent than others. Herein, we investigated the clinical efficacy and the underlying mechanisms of a combination of CAK cell infusion therapy and gemcitabine. Twenty-three patients with disseminated carcinomas were treated with chemo-immunotherapy consisting of CAK cell infusion therapy following gemcitabine treatment. To investigate the underlying mechanisms by which CAK cells synergize with gemcitabine, we used enzyme-linked immunosorbent assay, Real-time reverse transcription polymerase chain reaction assay, calcein-release assay, and adherent target detachment assay. Using these assays we determined the NKG2D ligands such as major histocompatibility complex-class I-related chain (MIC)A/B expression in carcinoma cells and the level of cellular cytotoxicity generated by treatment with gemcitabine with/without CAK cells. The tumor responses differed among the patients (n=23). In vitro experiments revealed that MICA/B protein and mRNA expression were up-regulated in several carcinoma cell lines after gemcitabine treatment. Pre-treatment with gemcitabine and subsequent exposure to CAK cells induced greater cytotoxicity than either treatment alone. Inclusion of soluble MICB in CAK cell-mediated cytotoxicity assay significantly reduced cytotoxicity. Our clinical results of gemcitabine-CAK combinatorial therapy demonstrated long-term stable disease despite chemoresistance. In conclusion, the combination of gemcitabine and CAK cells may have clinical therapeutic significance for pancreatic, hepato-biliary tract, and urothelial tract cancer. Our study shows that combining CAK therapy with gemcitabine can lead to successful treatment of metastatic cancer.

The Hedgehog inhibitor suppresses the function of monocyte-derived dendritic cells from patients with advanced cancer under hypoxia.

Immunotherapy using monocyte derived dendritic cells (Mo-DCs) from cancer patients has been developed; however, the Mo-DCs regularly studied have been derived from non-cancer bearing donors or mice, and evaluated in normoxic conditions. In the present study, we investigated the effects of Hedgehog (Hh) inhibitors which are being developed as molecular target drugs for cancer on the functions of Mo-DCs derived from patients with advanced cancer when cultured in a tumor-like hypoxic environment. Mo-DC induction, migration, chemotaxis, phagocytosis, maturation, IL-12 p40 or p70 secretion and the allogeneic lymphocyte stimulation activity of Mo-DCs from patients with advanced cancer were all significantly inhibited by the Hh inhibitor, cyclopamine under hypoxic conditions. Our results suggest that Hh signaling plays an important role in the maintenance and function of Mo-DCs derived from patients with advanced cancer when cultured under hypoxic conditions.

Combining celecoxib with sorafenib synergistically inhibits hepatocellular carcinoma cells in vitro.

AIM: Sorafenib is a promising treatment for hepatocellular carcinoma (HCC) but recent toxicity concerns suggest that new strategies for its use are needed. One approach for reducing toxicity is to use lower doses of sorafenib in combination with other complementary mechanisms. Celecoxib, a cyclooxygenase-2 inhibitor, has been shown to inhibit HCC, and we hypothesized that low-dose sorafenib co-administered with celecoxib could act synergistically in the inhibition of HCC. In this study, the effect of sorafenib was tested in combination with celecoxib on the growth of human HCC cells in vitro. MATERIALS AND METHODS: Two human HCC cell lines, HepG2 and Huh7, were treated with sorafenib and celecoxib, alone and in combination, and the effect of these treatments on growth, apoptosis, and expression of phospho-AKT was evaluated by WST-8, DNA content, and immunocytochemical assays, respectively. RESULTS: When compared to the actions of either agent alone, the combination of low concentrations of sorafenib (<5 µM) and celecoxib (<20 µM) resulted in enhanced inhibition of both cell growth and AKT activation, and increased the induction of apoptosis. Combination index (CI) analysis showed that the growth inhibition effect was synergistic. CONCLUSION: This study shows that celecoxib synergistically potentiates the sorafenib-mediated antitumor effect. This finding establishes the foundation for clinical trials evaluating the efficacy of co-administration of soerafenib and celecoxib as a treatment for HCC.

The Hedgehog inhibitor cyclopamine impairs the benefits of immunotherapy with activated T and NK lymphocytes derived from patients with advanced cancer.

Hedgehog (Hh) signaling is activated in various types of cancer and contributes to the progression, proliferation, and invasiveness of cancer cells. Many Hh inhibitors are undergoing clinical trial and show promise as anticancer drugs. Hh signaling is also induced in the activated T and NK (TNK) lymphocytes that are used in immunotherapy. Activated TNK lymphocyte therapy is anticipated to work well within a tumor's hypoxic environment. However, most studies on the immunobiological functions of activated TNK lymphocytes have been conducted on healthy donor samples, under normoxic conditions. In the present study, we evaluated the effects of Hh inhibition and oxygen concentrations on the function of activated TNK lymphocytes derived from patients with advanced cancer. Proliferation, migration, surface NKG2D expression, and cytotoxicity were all significantly inhibited, and IFN-γ secretion was significantly increased upon Hh inhibitor treatment of activated TNK lymphocytes under hypoxic conditions in vitro. Tumors from mice injected with cyclopamine-treated activated TNK lymphocytes showed a significant increase in tumor size and had fewer apoptotic cells compared with the tumors in mice injected with control activated TNK lymphocytes. These results suggest that Hh signaling plays a pivotal role in activated TNK lymphocyte cell function. Combination therapy using Hh inhibitors and activated TNK lymphocytes derived from patients with advanced cancer may not be advantageous.

Combining cetuximab with killer lymphocytes synergistically inhibits human cholangiocarcinoma cells in vitro.

AIM: We explored the possibility of combining adoptive immunotherapy with cytokine-activated killer (CAK) cells and the epidermal growth factor receptor monoclonal antibody, cetuximab, as a treatment for cholangiocarcinoma. MATERIALS AND METHODS: CAK cells were cultured with a high-dose of interleukin-2 and anti-CD3 monoclonal antibodies. This cell population contained both activated CD16+/CD56+ (NK) cells and CD3+/NKG2D(high+) T-cells. The effect of CAK cells and cetuximab, alone and in combination, on the viability of human cholangiocarcinoma cells was evaluated. RESULTS: Culture of CAK cells alone, but not cetuximab alone, exhibited modest cytotoxicity toward cholangiocarcinoma cells. However, combining CAK cells with cetuximab significantly enhanced cytotoxicity. This enhancement was inhibited by the addition of excess human immunoglobulins, suggesting that antibody-dependent cytotoxicity, mediated by activated NK cells in the CAK cell culture was involved in this mechanism. CONCLUSION: Cetuximab may be used to enhance CAK cell therapeutic activity in patients with cholangiocarcinoma, by potentiating antibody-dependent cellular cytotoxicity.

A new method for rapid cytotoxic T-lymphocyte induction using a multiple cytokine cocktail.

Immunotherapy using cytotoxic T-lymphocytes (CTLs) still has limited success. An increase in the frequency of CTL administration is one method to improve immunotherapy using CTLs. The conventional method (C-method) that generates CTLs after the induction of dendritic cells requires a long time period. If CTLs can be more rapidly and simply induced, the frequency of immunotherapy could be increased and unexpected contamination could be avoided. In this study, in order to more rapidly induce functional CTLs, we investigated a new method (N-method) that uses a cytokine cocktail, including interleukin (IL)-2, IL-4, granulocyte macrophage colony-stimulating factor, tumour necrosis factor-α and interferon-α, together with a tumour lysate. CTLs induced by the N-method had equivalent functions, such as proliferation, surface antigen expression and cytotoxicity, compared with those induced by the C-method. These results suggest that the N-method can substitute the C-method in order to improve the effect of immunotherapy using CTLs.

Combinatorial cytotoxicity of gemcitabine and cytokine-activated killer cells in hepatocellular carcinoma via the NKG2D-MICA/B system.

AIM: Natural-killer group 2, member D (NKG2D) is an activating receptor on natural killer cells and activated T-cells, designated cytokine-activated killer (CAK) cells here. The MHC class I chain-related A and B (MICA and MICB, respectively) are ligands of NKG2D and are expressed on various human tumor cells, including hepatocellular carcinoma (HCC) cells. Here, we investigate whether gemcitabine, a chemotherapeutic agent, affects MICA/B expression in HCC. MATERIALS AND METHODS: We used ELISA, RT-PCR and adherent target detachment assays to determine expression of MICA/B in HepG2 HCC cells and the level of cellular cytotoxicity generated by treatment with gemcitabine and/or CAK cells. RESULTS: Surface expression of MICA/B was evident after gemcitabine treatment, and MICB-specific mRNA was up-regulated. Pre-treatment with gemcitabine and subsequent exposure to CAK cells induced greater cytotoxicity than either treatment alone. Inclusion of soluble MICB significantly reduced cytotoxicity. CONCLUSION: Gemcitabine induced MICA/B expression in HepG2 cells, resulting in synergistic enhancement of the cytotoxic effects of NKG2D-high CAK cells. The combination of gemcitabine and CAK cells may have clinical therapeutic significance for HCC.