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OX40 stimulation supports the survival of CD4 T cells, which received vaccine

S1. Poly-IC is a weaker adjuvant than poly-ICLC but IL2Cx improves its activity. S2. BiVax/IL2Cx generates large T cell responses in BALB/c mice. S3. Human IL2Cx has similar adjuvant effect to mouse IL2Cx. S4. WT mice were vaccinated with BiVax prime and 5 days later mice were boosted with pamTrp1/poly-IC (BiVax), IL2Cx25 alone, pamTrp1/IL2Cx25 or BiVax/IL2Cx25 and on day 12 the numbers of Tregs (CD4+ CD25+ Foxp3+) were evaluated in spleens. (N= 3 mice/group). S5. Antitumor effects BiVax/IL2Cx in BALB/c mice. S6. A-B, WT B6 mice received a BiVax prime followed by BiVax/IL2Cx122 boost. S7. A, WT mice were vaccinated with pamTrp1/poly-IC (BiVax), prime and 5 days later mice were boosted with either BiVax, BiVax + HD-IL2 (600,000 I.U 2X/day for 3 consecutive days), or BiVax + PEG-IL2 and on day 12 the numbers of Trp1+ T cells were evaluated in spleens. B, CD45.1 WT mice were adoptively transferred with TgTR1 CD8 T cells followed with pamTrp1/poly-IC (BiVax) prime and 5 days later mice were boosted with either BiVax, BiVax + HD-IL2 or BiVax + PEG-IL2. One hour after the last dose of IL2, pSTAT5 expression was evaluated in TgTR1 T cells from the spleens. (N= 3 mice/group). S8. Tumor cells isolated from tumor bearing mice express MHC-I and can stimulate CD8 T cells.

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Peptide vaccines can be a successful and cost-effective way of generating T-cell responses against defined tumor antigens, especially when combined with immune adjuvants such as poly-IC. However, strong immune adjuvants can induce a collateral increase in numbers of irrelevant, nonspecific T cells, which limits the effectiveness of the peptide vaccines. Here, we report that providing prolonged IL2 signaling in the form of either IL2/anti-IL2 complexes or pegylated IL2 overcomes the competitive suppressive effect of irrelevant T cells, allowing the preferential expansion of antigen-specific T cells. In addition to increasing the number of tumor-reactive T cells, sustained IL2 enhanced the ability of T cells to resist PD-1–induced negative signals, increasing the therapeutic effectiveness of the vaccines against established tumors. This vaccination strategy using peptides and sustained IL2 could be taken into the clinic for the treatment of cancer. Cancer Immunol Res; 6(5); 617–27. ©2018 AACR.

TriVax with or without OX40 agonist is superior to a conventional adjuvant in inducing CD4 T cell responses

Substantial evidence indicates that immunotherapy is a feasible and effective approach for the treatment of numerous types of cancer. Among various immunotherapy options, peptide vaccines to generate antitumor T cells appear as promising candidates, because of their cost effectiveness and ease of implementation. Nevertheless, most peptide vaccines are notorious for being weekly immunogenic and, thus, optimization of the vaccination strategy is essential to achieve therapeutic effectiveness. In addition, effective peptide vaccines must stimulate both CD8 cytotoxic and CD4 helper T lymphocytes. Our group has been successful in designing effective peptide vaccination strategies for inducing CD8 T-cell responses in mouse tumor models. Here, we describe a somewhat similar, but distinct, peptide vaccination strategy capable of generating vast CD4 T-cell responses by combining synthetic peptides with toll-like receptor (TLR) agonists and OX40/CD40 costimulation. This vaccination strategy was efficient in overcoming immune tolerance to a self-tumor–associated antigen and generated significant antitumor effects in a mouse model of malignant melanoma. The optimized peptide vaccine also allowed the expansion of adoptively transferred CD4 T cells without the need for lymphodepletion and IL2 administration, generating effective antimelanoma responses through the enhancement of proliferative and antiapoptotic activities of CD4 T cells. These results have practical implications in the design of more effective T-cell–based immunotherapies. Cancer Immunol Res; 5(1); 72–83. ©2016 AACR.

TriVax/OX40 elicits endogenous CD4 T cell responses against a non-immunogenic tumor-associated antigen

MHC Class II expression on B16F10 melanoma cells

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A major challenge for developing effective therapeutic vaccines against cancer is overcoming immunologic tolerance to tumor-associated antigens that are expressed on both malignant cells and normal tissues. Herein, we describe a novel vaccination approach, TriVax, that uses synthetic peptides representing CD8 T-cell epitopes, Toll-like receptor agonists that function as potent immunologic adjuvants and costimulatory anti-CD40 antibodies to generate large numbers of high-avidity antigen-reactive T cells capable of recognizing and killing tumor cells. Our results show that TriVax induced huge numbers of long-lasting antigen-specific CD8 T cells that displayed significant antitumor effects in vivo. The administration of a TriVax formulation containing a CD8 T-cell epitope derived from a melanosomal antigen (Trp2180-188) elicited antigen-specific CD8 T cells that induced systemic autoimmunity (vitiligo). More important, TriVax immunization was effective in eliciting potent protective antitumor immunity as well as remarkable therapeutic effects against established B16 melanoma. This therapeutic effect was mediated by CD8 T cells via perforin-mediated lysis and required the participation of type-I IFN but not IFNγ. These results suggest that similar strategies would be applicable for the design of effective vaccination for conducting clinical studies in cancer patients. [Cancer Res 2009;69(23):9012–9]

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PDF file - 86K, The effect of chemotherapy on expression of MPR in vivo

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Background aims Immunotherapeutic approaches using γδ T cells have emerged as the function of γδ T cells in tumor surveillance and clearance has been discovered. In vitro expansion methods of γ9δ2 T cells have been based on phosphoantigens and cytokines, but expansion methods using feeder cells to generate larger numbers of γδ T cells have also been studied recently. However, there are no studies that directly compare γδ T cells cultured with phosphoantigens with those cultured with feeder cells. Therefore, this study aimed to compare the expansion, characteristics and effector functions of γδ T cells stimulated with K562-based artificial antigen-presenting cells (aAPCs) (aAPC-γδ T cells) and γδ T cells stimulated with only zoledronic acid (ZA) (ZA-γδ T cells). Methods Peripheral blood mononuclear cells were stimulated with ZA for 7 days, and aAPC-γδ T cells were stimulated weekly with K562-based aAPCs expressing CD32, CD80, CD83, 4-1BBL, CD40L and CD70, whereas ZA-γδ T cells were stimulated with only IL-2. Cultured γδ T cells were analyzed by flow cytometry for the expression of co-stimulatory molecules, activating receptors and checkpoint inhibitors. Differentially expressed gene (DEG) analysis was also performed to determine the difference in gene expression between aAPC-γδ T cells and ZA-γδ T cells. In vitro cytotoxicity assay was performed with calcein AM release assay, and in vivo anti-tumor effect was compared using a U937 xenograft model. Results Fold expansion on day 21 was 690.7 ± 413.1 for ZA-γδ T cells and 1415.2 ± 1016.8 for aAPC- γδ T cells. Moreover, aAPC-γδ T cells showed continuous growth, whereas ZA-γδ T cells showed a decline in growth after day 21. The T-cell receptor Vγ9+δ2+ percentages (mean ± standard deviation) on day 21 were 90.0 ± 2.7% and 87.0 ± 4.5% for ZA-γδ T cells and aAPC-γδ T cells, respectively. CD25 and CD86 expression was significantly higher in aAPC-γδ T cells. In DEG analysis, aAPC-γδ T cells and ZA-γδ T cells formed distinct clusters, and aAPC-γδ T cells showed upregulation of genes associated with metabolism and cytokine pathways. In vitro cytotoxicity revealed superior anti-tumor effects of aAPC-γδ T cells compared with ZA-γδ T cells on Daudi, Raji and U937 cell lines. In addition, in the U937 xenograft model, aAPC-γδ T-cell treatment increased survival, and a higher frequency of aAPC-γδ T cells was shown in bone marrow compared with ZA-γδ T cells. Conclusions Overall, this study demonstrates that aAPC-γδ T cells show long-term proliferation, enhanced activation and anti-tumor effects compared with ZA-γδ T cells and provides a basis for using aAPC-γδ T cells in further studies, including clinical applications and genetic engineering of γδ T cells.

Background Vaccination with tumor peptide epitopes associated with major histocompatibility complex class I molecules is an attractive approach directed at inducing tumor-specific cytotoxic T lymphocytes (CTLs). However, challenges remain in improving the therapeutic efficacy of peptide epitope vaccines, including the low immunogenicity of peptide epitopes and insufficient stimulation of innate immune components in vivo. To overcome this, we aimed to develop and test an innovative strategy that elicits potent CTL responses against tumor epitopes. The essential feature of this strategy is vaccination using tumor epitope-loaded nanoparticles (NPs) in combination with polyinosinic-polycytidylic acid (poly-IC) and anti-PD1 monoclonal antibody (mAb). Methods Carboxylated NPs were prepared using poly(lactic-co-glycolic acid) and poly(ethylene/maleic anhydride), covalently conjugated with anti-H-2KbmAbs, and then attached to H-2Kb molecules isolated from the tumor mass (H-2b). Native peptides associated with the H-2Kbmolecules of H-2Kb-attached NPs were exchanged with tumor peptide epitopes. The tumor-specific CTL-inducing and anti-tumor activities of the tumor epitope-loaded NPs were examined in mice bearing EG7-OVA thymoma or B16-F10 melanoma. In addition, the anti-tumor therapeutic efficacy of the NPs was examined in combination with poly-IC, anti-PD1 mAb, or both. Results Tumor peptide epitope-loaded NPs efficiently induced tumor-specific CTLs when used to immunize tumor-bearing mice as well as normal mice. This activity of the NPs significantly was increased when co-administered with poly-IC. Accordingly, the NPs exerted significant anti-tumor effects in mice implanted with EG7-OVA thymoma or B16-F10 melanoma, and the anti-tumor activity of the NPs was significantly increased when applied in combination with poly-IC. The most potent anti-tumor activity was observed when the NPs were co-administered with both poly-IC and anti-PD1 mAb. Conclusions Immunization with tumor epitope-loaded NPs in combination with poly-IC and anti-PD1 mAb in tumor-bearing mice can be a powerful means to induce tumor-specific CTLs with therapeutic anti-tumor activity.

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression by suppressing translation or facilitating mRNA decay. Differential expression of miRNAs is involved in the pathogenesis of several diseases including cancer. Here, we investigated the role of-miR-24-3p as a downregulated miRNA in metastatic cancer. miR-24-3p was decreased in metastatic cancer and lower expression of miR-24-3p was related to poor survival of cancer patients. Consistently, ectopic expression of miR-24-3p suppressed the cell migration, invasion, and proliferation of MCF7, Hep3B, B16F10, SK-Hep1, and PC-3 cells by directly targeting p130Cas. Stable expression of p130Cas restored miR-24-3p-mediated inhibition of cell migration and invasion. These results suggest that miR-24-3p functions as a tumor suppressor and the miR-24-3p/p130Cas axis is a novel factor of cancer progression by regulating cell migration and invasion.

Background Therapeutic cancer vaccines are an attractive approach for treating malignant tumours, and successful tumour eradication depends primarily on controlling tumour immunosuppression status as well as heterogeneity of tumour cells driven by epigenetic alterations. Methods Peptide-loaded dendritic cell (DC) prime and non-infectious peptide booster heterologous immunisations were assessed for the immunogenicity of polo-like kinase-1 (PLK1)-derived peptides. Heterologous vaccination regimen targeting multiple shared tumour antigens simultaneously with PD-L1 blockade was assessed against murine myeloid leukaemia. Results A synthetic PLK1122 (DSDFVFVVL)-based heterologous vaccination generated large numbers of long-lasting antigen-specific CD8 T-cells eliciting therapeutic effects against various established tumours. The therapeutic efficacy of single antigen-targeting PLK1122-based vaccine with sufficient endurance of PD-L1 blockade toward C1498 leukaemia relied on the heterogeneous clonal levels of MHC-I and PD-L1 expression. A novel multi-peptide-based vaccination targeting PLK1 and survivin simultaneously along with PD1 blockade led to complete tumour eradication and long-term survival in mice with clonally heterologous C1498 myeloid leukaemia. Conclusions Our findings suggest that PLK1 could be an attractive immunotherapeutic target antigen for cancer immunotherapy, and that similar strategies would be applicable for the optimisation of cancer vaccines for the treatment of numerous viral diseases and malignant tumours.

Chimeric antigen receptor (CAR)-engineered adoptive cell therapy has demonstrated a feasible, attractive “off-the-shelf” approach against various malignancies. Success of CAR-engineered cell therapy depends primarily on providing optimized costimulatory signals capable of achieving robust cell proliferation, persistence, and antitumor reactivity. We describe here the capability of a novel CD30-derived costimulatory signaling domain to generate effective CAR-redirected cells capable of eradicating hematological and solid cancers. After establishing CD19-redirecting second-generation CARs incorporating the CD30-derived intracellular signaling domain and third-generation CARs combining CD30 with either CD28 or 4-1BB, the effector functionality of CAR-transduced T cells was assessed in vitro and in vivo. Further, we evaluated the efficacy of epithelial cell adhesion molecule (EpCAM)-targeting third-generation CD30-encompassing CAR constructs for targeting solid tumors. The CD30-signaling CAR-T cells exhibited similar levels of cell proliferation, cytolytic efficacies, and cytokine secretion in vitro, and demonstrated improved tumor regression and prolonged survival in vivo with increased persistence of the CAR-T cells as those of other CARs with CD28 or 4-1BB costimulatory domain, which are currently in clinical use. Also, third-generation CARs including the CD30 costimulatory domain retained potent antitumor efficacies in vitro and in vivo independent of their position or type of partner costimulatory domain. More significantly, EpCAM-targeting CAR-T cells containing CD30-signaling domain revealed effective cytolytic activities correlating with EpCAM expression levels in the target cells. Our results demonstrate that the CD30-derived costimulatory domain could be an alternative for developing CAR-engineered therapeutics which may be applicable for various designs of CAR constructs, with an emphasis on effectiveness against solid tumors. Citation Format: Hyun-Il Cho, Chung-Hyo Kang, Sang-Eun Lee, Mi-So Kim, Bo-Ra Lim, Jung-Min Ha, Hyun-Jung Sohn, Tai-Gyu Kim. A novel costimulatory signaling domain for chimeric antigen receptor-engineering [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 1788.

Adoptive transfer of immune killer cells genetically engineered to express chimeric antigen receptors (CARs) has demonstrated a feasible and effective approach against numerous types of cancers. Among the killer cells, γδT cells possessing a wide range of antigen-independent tumor recognition and low risk of developing GVHD offer a chance to develop CAR-engineered allogenic ‘off-the-shelf’ cellular therapeutics. Successful tumor eradication primarily depends on providing optimized signals affecting the proliferation, persistence, antitumor activity and cytokine secretion of CAR-γδT cells. We describe here a novel CD30 costimulatory domain for CAR signaling, which is known to be involved in cell anti-apoptosis, activation and proliferation. Conventional αβT cells and innate γδT cells activated by a CD30-ζ signaling-CAR displayed similar levels of cytolytic efficacies and cytokine secretion against antigen-expressing cancer cell lines as those of other CAR containing commonly used costimulatory domains CD28, CD27, 4-1BB, OX40, and ICOS. Furthermore, the third-generation CARs including the CD30 costimulatory domain retained potent antitumor efficacies independent of their position or type of partner costimulatory domain. More significantly, CAR-γδT cells containing CD30-ζ signaling revealed effective cytolytic activities against several solid cancer cell lines based on its antigen-recognizing antibody fragments. Our findings suggest that the CD30 costimulatory domain could be an attractive candidate for developing CAR-engineered killer cell therapeutics and may be applicable for the design of numerous antigen-recognizing CAR constructs, with an emphasis on targeting solid tumors. Citation Format: Hyun-Il Cho, Chung-Hyo Kang, Sang-Eun Lee, In-Sil Song, Jung-Min Ha, Seon-Duk Lee, Hyun-Jung Sohn, Tai-Gyu Kim. CD30 co-stimulatory domain enhances the efficacy of chimeric antigen receptor-engineered γδT cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5506.

Within an individual, six different HLA class II heterodimers are expressed co-dominantly by two alleles of HLA-DR, -DQ, and -DP loci. However, it remained unclear which HLA allotypes were used in T cell responses to a given antigen. For the measurement of the CD4+ T cell responses restricted by a single HLA allotype, we established a panel of artificial antigen-presenting cells (aAPCs) expressing each single HLA allele of 20 HLA-DRB1, 16 HLA-DQ, and 13 HLA-DP alleles. CD4+ T cell responses to cytomegalovirus (CMV) pp65 restricted by single HLA class II allotype defined in 45 healthy donors. The average magnitude of CD4+ T cell responses by HLA-DR allotypes was higher than HLA-DQ and HLA-DP allotypes. CD4+ T cell responses by DRA*01:01/DRB1*04:06, DQA1*01:02/DQB1*06:02, DPA1*02:02/DPB1*05:01 were higher among the other alleles in each HLA-DR, -DQ, and -DP locus. Interestingly, the frequencies of HLA-DR alleles and the positivity of specific allotypes showed an inverse correlation. One allotype within individuals is dominantly used in CD4+ T cell response in 49% of donors, and two allotypes showed that in 7% of donors, and any positive response was detected in 44% of donors. Even if one individual had several dominant alleles, CD4+ T cell responses tended to be restricted by only one of them. Furthermore, CD8+ and CD4+ T cell responses by HLA class I and class II were correlated. Our results demonstrate that the CD4+ T cell preferentially use a few dominant HLA class II allotypes within individuals, similar to CD8+ T cell response to CMV pp65.

Within an individual, six different HLA class II heterodimers are expressed co-dominantly by two alleles of HLA-DR, -DQ, and -DP loci. However, it remained unclear which HLA allotypes were used in T cell responses to a given antigen. For the measurement of the CD4+ T cell responses restricted by a single HLA allotype, we established a panel of artificial antigen-presenting cells (aAPCs) expressing each single HLA allele of 20 HLA-DRB1, 16 HLA-DQ, and 13 HLA-DP alleles. CD4+ T cell responses to cytomegalovirus (CMV) pp65 restricted by single HLA class II allotype defined in 45 healthy donors. The average magnitude of CD4+ T cell responses by HLA-DR allotypes was higher than HLA-DQ and HLA-DP allotypes. CD4+ T cell responses by DRA*01:01/DRB1*04:06, DQA1*01:02/DQB1*06:02, DPA1*02:02/DPB1*05:01 were higher among the other alleles in each HLA-DR, -DQ, and -DP locus. Interestingly, the frequencies of HLA-DR alleles and the positivity of specific allotypes showed an inverse correlation. One allotype within individuals is dominantly used in CD4+ T cell response in 49% of donors, and two allotypes showed that in 7% of donors, and any positive response was detected in 44% of donors. Even if one individual had several dominant alleles, CD4+ T cell responses tended to be restricted by only one of them. Furthermore, CD8+ and CD4+ T cell responses by HLA class I and class II were correlated. Our results demonstrate that the CD4+ T cell preferentially use a few dominant HLA class II allotypes within individuals, similar to CD8+ T cell response to CMV pp65.

Background: Adoptive transfer of genetically engineered T-cells to express antigen-specific T-cell receptor (TCR) is a feasible and effective therapeutic approach for numerous types of cancers, including Epstein–Barr virus (EBV)-associated malignancies. Here, we describe a TCR gene transfer regimen to rapidly and reliably generate T-cells specific to EBV-encoded latent membrane protein-1 (LMP1), which is a potential target for T-cell-based immunotherapy. Methods: A novel TCR specific to LMP1 (LMP1-TCR) was isolated from HLA-A*0201 transgenic mice that were immunised with the minimal epitope LMP1166 (TLLVDLLWL), and LMP1-TCR-transduced peripheral blood lymphocytes were evaluated for functional specificities. Results: Both human CD8 and CD4 T-cells expressing the LMP1-TCR provoked high levels of cytokine secretion and cytolytic activity towards peptide-pulsed and LMP1-expressing tumour cells. Notably, recognition of these T-cells to peptide-pulsed cells was maintained at low concentration of peptide, implying that the LMP1-TCR has high avidity. Infusion of these engineered T-cells revealed remarkable therapeutic effects and inhibition of tumour growth in a preclinical xenogeneic model. We observed explosive ex vivo proliferation of functional TCR-transduced T-cells with artificial antigen-presenting cells that express co-stimulatory molecules CD80 and 4-1BBL. Conclusions: These data suggest that the novel TCR-targeting LMP1 might allow the potential design of T-cell-based immunotherapeutic strategies against EBV-positive malignancies.

Human leukocyte antigens (HLAs) are essential immune molecules that affect transplantation and adoptive immunotherapy. When hematopoietic stem cells or organs are transplanted with HLA-mismatched recipients, graft-versus-host disease or graft rejection can be induced by allogeneic immune responses. The function of each HLA allele has been studied using HLA-deficient cells generated from mutant cell lines or by RNA interference, zinc finger nuclease, and the CRISPR/Cas9 system. To improve HLA gene editing, we attempted to generate an HLA class I null cell line using the multiplex CRISPR/Cas9 system by targeting exons 2 and 3 of HLA-A, HLA-B, and HLA-C genes simultaneously. Multiplex HLA editing could induce the complete elimination of HLA class I genes by bi-allelic gene disruption on target sites which was defined by flow cytometry and target-specific polymerase chain reaction. Furthermore, artificial antigen-presenting cells were generated by transfer of a single HLA class I allele and co-stimulatory molecules into this novel HLA class I null cell line. Artificial antigen-presenting cells showed HLA-restricted antigen presentation following antigen processing and were successfully used for the efficient generation of tumor antigen-specific cytotoxic T cells in vitro. The efficient editing of HLA genes may provide a basis for universal cellular therapies and transplantation.

// Hyun-Jung Sohn 1, 6, * , Ji Yoon Lee 2, 5, * , Hyun-Joo Lee 1, 6 , Dae-Hee Sohn 4, 6 , Hyun-Il Cho 1, 2 , Hee-Je Kim 2, 3 and Tai-Gyu Kim 1, 4 1 Catholic Hematopoietic Stem Cell Bank, The Catholic University of Korea, Seoul, Korea 2 Leukemia Research Institute, Seoul St. Mary`s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea 3 Department of Hematology, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary`s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea 4 Departments of Microbiology, College of Medicine, The Catholic University of Korea, Seoul, Korea 5 Department of Biomedical Laboratory Science, College of Health Sciences, Sangji University, Wonju, Korea 6 ViGenCell Inc., Seoul, Korea * These authors have contributed equally to this work Correspondence to: Hee-Je Kim, email: cumckim@catholic.ac.kr Tai-Gyu Kim, email: kimtg@catholic.ac.kr Keywords: Wilms’ tumor protein 1, survivin, telomerase reverse transcriptase, dendritic cells, adoptive T cell therapy Received: December 21, 2016 Accepted: March 03, 2017 Published: April 19, 2017 ABSTRACT Previously, we found that most patients with acute myeloid leukemia (AML) expressed at least one of the leukemic associated antigens (LAAs) WT1, survivin and TERT , and different combinations of the three LAAs predicted negative clinical outcomes. Multi-tumor antigen-specific T cells were generated to overcome antigenic variation and may be sufficient to maximize antitumoral effects. To generate triple antigen-specific (Tri)-T cells that recognize three LAAs, dendritic cells (DCs) were transfected with three tumor antigen-encoding RNAs. These DCs were used to stimulate both CD8 and CD4 T cells and to overcome the limitation of known human leukocyte antigen-restricted epitopes. The sum of the antigen-specific T cell frequencies was higher in the Tri-T cells than in the T cells that recognized a single antigen. Furthermore, the Tri-T cells were more effective against leukemic blasts that expressed all three LAAs compared with blasts that expressed one or two LAAs, suggesting a proportional correlation between IFN-γ secretion and LAA expression. Engrafted leukemic blasts in the bone marrow of mice significantly decreased in the presence of Tri-T cells. This technique represents an effective immunotherapeutic strategy in AML.

Dendritic cell-derived exosomes (DEX) comprise an efficient stimulator of T cells. However, the production of sufficient DEX remains a barrier to their broad applicability in immunotherapeutic approaches. In previous studies, genetically engineered K562 have been used to generate artificial antigen presenting cells (AAPC). Here, we isolated exosomes from K562 cells (referred to as CoEX-A2s) engineered to express human leukocyte antigen (HLA)-A2 and costimulatory molecules such as CD80, CD83, and 41BBL. CoEX-A2s were capable of stimulating antigen-specific CD8 T cells both directly and indirectly via CoEX-A2 cross-dressed cells. Notably, CoEX-A2s also generated similar levels of HCMV pp65-specific and MART1-specific CD8 T cells as DEX in vitro. The results suggest that these novel exosomes may provide a crucial reagent for generating antigen-specific CD8 T cells for adoptive cell therapies against viral infection and tumors.

// Chang-Ae Shin 1 , Hyun-Woo Cho 1 , A-Ri Shin 2, 3 , Hyun-Jung Sohn 2 , Hyun-Il Cho 2, 3 , Tai-Gyu Kim 1, 2, 3 1 Department of Microbiology and Immunology, College of Medicine, The Catholic University of Korea, Seoul 137-701, South Korea 2 Catholic Hematopoietic Stem Cell Bank, College of Medicine, The Catholic University of Korea, Seoul 137-701, South Korea 3 Catholic Cancer Research Institute, College of Medicine, The Catholic University of Korea, Seoul 137-701, South Korea Correspondence to: Hyun-Il Cho, email: hyun.cho@catholic.ac.kr Tai-Gyu Kim, email: kimtg@catholic.ac.kr Keywords: cancer vaccine, B-cells, costimulatory ligand, anti-apoptosis, tumor immunity Received: February 24, 2016 Accepted: May 29, 2016 Published: June 15, 2016 ABSTRACT Activated B-cells are a promising alternative source of antigen-presenting cells. They can generally be obtained in sufficient numbers for clinical use, but in most instances produce weak immune responses and therapeutic effects that are suboptimal for use in therapeutic cancer vaccines. To improve the immunogenic potency and therapeutic efficacy of B-cell-based vaccines, ex vivo -activated B-cells were transduced with recombinant lentiviruses in order to express additional costimulatory ligands—CD40L, CD70, OX40L, or 4-1BBL—either individually or in pairs (CD70/CD40L, OX40L/CD40L, or 4-1BBL/CD40L). We observed that the expression of CD40L molecules on B-cells was crucial for T-cell priming and activation. Administration of B-cells co-expressing CD40L with the other costimulatory ligands provided substantial antigen-specific CD8 T-cell responses capable of provoking in vivo proliferation and potent cytolytic activities. Notably, expression of CD40L augmented B-cell viability by inhibiting apoptosis through upregulated expression of the anti-apoptotic molecules BCL2, Bcl-xL and Bax. B-cells co-expressing CD40L with CD70, OX40L, or 4-1BBL induced potent therapeutic antitumor effects in a B16 melanoma model. Moreover, the combination of genetically-modified B-cell vaccines with programmed cell death-1 blockade potentiated the therapeutic efficacy. These results suggest that B-cells endowed with additional costimulatory ligands enable the design of effective vaccination strategies against cancer.

Zoledronic acid (ZA) is used for treating osteoporosis and for preventing skeletal fractures in cancer patients suffering from myeloma and prostate cancer. It is also reported to directly induce cancer cell apoptosis and indirectly modulate T-cell immune response as an antitumor agent. In this study, the effect of ZA following peptide/polyinosinic-polycytidylic acid (poly-IC) vaccination was investigated in a murine tumor model. The combination of ZA with peptide/poly-IC vaccine showed a synergistic effect on the induction of antigen-specific CD8 T-cell response. Three consecutive intravenous administrations of ZA was defined to induce the highest CD8 T-cell response. Further, total splenocyte counts and antigen-specific CD8 T-cell response gradually increased depending on the dose of ZA. In tumor-bearing mice, ZA showed a dose-dependent decrease of growth and prolonged survival. Treatment with ZA only decreased the number of CD11b(+)Gr1(+) myeloid cells in blood. Our results demonstrate that the use of ZA could improve antitumor immune responses induced by the peptide/poly-IC vaccine.

Therapeutic cancer vaccines are an attractive approach for treating malignant tumors, and successful tumor eradication depends primarily on controlling tumor immunosuppression status as well as heterogeneity of tumor cells driven by epigenetic alterations. Here we describe an effective peptide vaccination approach capable of circumventing antigenic heterogeneity by targeting multiple shared tumor antigens simultaneously in murine myeloid leukemia. Peptide-loaded dendritic cell (DC) prime and noninfectious peptide booster heterologous immunizations were assessed for the immunogenicity of polo-like kinase-1 (PLK1)-derived peptides, and targeting multiple shared tumor antigens simultaneously with PD-L1 blockade was evaluated for antitumor effects against murine myeloid leukemia. A synthetic PLK1122 (DSDFVFVVL)-based heterologous vaccination generated large numbers of long-lasting antigen-specific CD8 T-cells eliciting therapeutic effects against various established tumors. The therapeutic efficacy of single antigen-targeting PLK1122-based vaccine with sufficient endurance of PD-L1 blockade toward C1498 leukemia relied on the heterogeneous clonal levels of MHC-I and PD-L1 expression. A novel multi-peptide-based vaccination targeting PLK1 and survivin simultaneously along with PD1 blockade led to complete tumor eradication and long-term survival in mice with clonally heterologous C1498 myeloid leukemia. Our findings suggest that PLK1 could be an attractive immunotherapeutic target antigen for cancer immunotherapy, and that similar strategies would be applicable for the optimization of cancer vaccines for the treatment of numerous viral diseases and malignant tumors.

Human γδ T cells play important roles in the regulation of infection and cancer. To understand the roles of costimulatory signals in activation and expansion ex vivo, Vγ9Vδ2 T cells were grown with artificial APCs that express CD83, 4-1BB ligand, and/or CD32, which allowed a loading of αCD3 and αCD28 antibodies. The costimulatory signals through CD80, 4-1BB, and CD83 ligand in low levels of IL-2 triggered an explosive ex vivo proliferation of Vγ9Vδ2 T cells capable of secreting high levels of IL-2, IFN-γ, and TNF-α. Moreover, the triple-costimulatory signals cause augmented cell viabilities for long-term growth of Vγ9Vδ2 T cells, resulting in phenotypic changes to CD27−CD45RA+ effector memory-like cells. Notably, we observed that CD83 ligand signaling is crucial to promote ex vivo expansion, survival, and cytolytic effector functions of Vγ9Vδ2 T cells. In contrast, 4-1BB signaling is moderately important in up-regulating surface molecules on Vγ9Vδ2 T cells. Consequently, γδ T cells stimulated in the presence of triple-costimulatory signals have diverse cytolytic effector molecules, including perforin, granzyme A, granzyme B, and Fas ligand, eliciting potent cytolytic activities against tumor cells. Overall, our results provide insights into the roles of costimulatory signals in manufacturing long-lived and fully functional Vγ9Vδ2 T cells that could be useful against cancers.

Extranodal NK/T-cell lymphoma (ENKTCL) is associated with latent Epstein-Barr virus (EBV) infection and frequent relapse even after complete response (CR) to intensive chemotherapy and radiotherapy. The expression of EBV proteins in the tumor provides targets for adoptive immunotherapy with antigen-specific cytotoxic T cells (CTL). To evaluate the efficacy and safety of EBV latent membrane protein (LMP)-1 and LMP-2a-specific CTLs (LMP1/2a CTLs) stimulated with LMP1/2a RNA-transferred dendritic cells, we treated 10 ENKTCL patients who showed complete response to induction therapy. Patients who completed and responded to chemotherapy, radiotherapy, and/or high-dose therapy followed by stem cell transplantation (HDT/SCT) were eligible to receive eight doses of 2 × 10(7) LMP1/2a CTLs/m(2). Following infusion, there were no immediate or delayed toxicities. The 4-year overall survival (OS) and progression-free survival (PFS) were 100%, and 90% (95% CI: 71.4 to 100%) respectively with a median follow-up of 55·5 months. Circulating IFN-γ secreting LMP1 and LMP2a-specific T cells within the peripheral blood corresponded with decline in plasma EBV DNA levels in patients. Adoptive transfer of LMP1/2a CTLs in ENKTCL patients is a safe and effective postremission therapeutic approach. Further randomized studies will be needed to define the role of EBV-CTLs in preventing relapse of ENKTCL.

To define whether individual human leukocyte antigen (HLA) class I allotypes are used preferentially in human cytomegalovirus (CMV)-specific cytotoxic T lymphocyte responses, CD8+ T cell responses restricted by up to six HLA class I allotypes in an individual were measured in parallel using K562-based artificial antigen-presenting cells expressing both CMV pp65 antigen and one of 32 HLA class I allotypes (7 HLA-A, 14 HLA-B, and 11 HLA-C) present in 50 healthy Korean donors. The CD8+ T cell responses to pp65 in the HLA-C allotypes were lower than responses to those in HLA-A and -B allotypes and there was no difference between the HLA-A and HLA-B loci. HLA-A*02:01, -B*07:02, and -C*08:01 showed the highest magnitude and frequency of immune responses to pp65 at each HLA class I locus. However, HLA-A*02:07, -B*59:01, -B*58:01, -B*15:11, -C*03:02, and -C*02:02 did not show any immune responses. Although each individual has up to six different HLA allotypes, 46% of the donors showed one allotype, 24% showed two allotypes, and 2% showed three allotypes that responded to pp65. Interestingly, the frequencies of HLA-A alleles were significantly correlated with the positivity of specific allotypes. Our results demonstrate that specific HLA class I allotypes are preferentially used in the CD8+ T cell immune response to pp65 and that a hierarchy among HLA class I allotypes is present in an individual.

Conventional therapeutic approaches to post-transplant lymphoproliferative disorder (PTLD) occurring after solid-organ transplantation have shown only limited success in achieving durable response. Key factors driving the pathogenesis of PTLD include Epstein–Barr virus (EBV) reactivation and impaired immune surveillance due to prolonged immune suppression. Thus, EBV-specific cytotoxic T lymphocytes (EBV-CTLs) have emerged as an alternative therapeutic approach for the treatment of EBV-associated PTLD by enhancing EBV-specific immunity. We evaluated the safety and efficacy of EBV latent membrane proteins (LMP)-1- and 2-specific CTLs in two PTLD patients at high risk for relapse. Following diagnosis, patients were initially treated with a combination of chemotherapy and/or radiotherapy. Patients then received a total of eight doses of 2 × 107 EBV-CTLs/m2. Following initial therapy, both patients achieved complete remission confirmed by FDG-PET/CT imaging. Post-remission therapy using adoptive transfer of EBV-CTLs was safe without immediate or late toxicities. Infusion of EBV-CTLs led to an overall reduction in plasma EBV levels in the peripheral blood, which was associated with long-term remission of both patients during a follow-up of more than 65 months. Further prospective studies with larger number of patients will be needed to confirm the role of EBV-CTLs as post-remission therapy in high-risk PTLD.

Therapeutic cancer vaccines are an attractive alternative to conventional therapies to treat malignant tumors, and more importantly, to prevent recurrence after primary therapy. However, the availability of professional antigen-presenting cells (APCs) has been restricted by difficulties encountered in obtaining sufficient professional APCs for clinical use. We have prepared an alternative cellular vaccine with CD4 T-cells that can be expanded easily to yield a pure and homogeneous population in vitro. To enhance their potency as a therapeutic vaccine, in vitro expanded CD4 T-cells were transfected with RNAs encoding the costimulatory ligands CD80, 4-1BBL, or both (CD80-T, 4-1BBL-T, and CD80/4-1BBL-T-cells, respectively). We observed augmented cell vitality in CD80/4-1BBL-T-cells in vitro and in vivo. Significant CD8 T-cell responses eliciting in vivo proliferation and cytotoxicity were obtained with CD80/4-1BBL-T-cell vaccination compared to CD80-T and 4-1BBL-T-cell vaccinations. In contrast, β2m-deficient CD80/4-1BBL-T-cells were not as effective as wile-type CD80/4-1BBL-T-cells in priming CD8 T-cells. Furthermore, CD80/4-1BBL-T-cell immunization resulted in curing established EG7 tumors, resulting in the generation of memory CD8 T-cell responses, and elicited therapeutic antitumor responses against B16 melanoma. These results suggest that CD4 T-cells endowed with costimulatory ligands allow the design of effective vaccination strategies against cancer.

Bilayer CuGa/In precursors were deposited on low-alkali, high-strain-point glass with a thickness of 1.8 mm by in-line DC-magnetron sputtering of CuGa (24 Ga wt%) and pure In targets. Se layers of different thicknesses, e.g., 1, 3, 5, and 8 μm, were coated on the sputter-deposited glass/Mo/CuGa/In precursors with no external heating by evaporating Se in a high vacuum evaporator. Possible reaction pathways to Cu(InGa)Se 2 formation from glass/Mo/CuGa/In/(Se) precursors were suggested on the basis of the high-temperature X-ray diffraction analysis. It is also proposed that the high temperature ramp rates might be preferred for the improved Ga diffusion toward the sample surface and thus reduced phase separation between CuInSe 2 and Cu(InGa)Se 2 . In the rapid thermal process system used in this study, the Se thickness of 3 μm demonstrated the best Ga incorporation during the selenization of 600 nm-thick CuGa/In precursors at 550–570 °C.

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression by suppressing translation or facilitating mRNA decay. Differential expression of miRNAs is involved in the pathogenesis of several diseases including cancer. Here, we investigated the role of-miR-24-3p as a downregulated miRNA in metastatic cancer. miR-24-3p was decreased in metastatic cancer and lower expression of miR-24-3p was related to poor survival of cancer patients. Consistently, ectopic expression of miR-24-3p suppressed the cell migration, invasion, and proliferation of MCF7, Hep3B, B16F10, SK-Hep1, and PC-3 cells by directly targeting p130Cas. Stable expression of p130Cas restored miR-24-3p-mediated inhibition of cell migration and invasion. These results suggest that miR-24-3p functions as a tumor suppressor and the miR-24-3p/p130Cas axis is a novel factor of cancer progression by regulating cell migration and invasion.

Substantial evidence indicates that immunotherapy is a feasible and effective approach for the treatment of numerous types of cancer. Among various immunotherapy options, peptide vaccines to generate antitumor T cells appear as promising candidates, because of their cost effectiveness and ease of implementation. Nevertheless, most peptide vaccines are notorious for being weekly immunogenic and, thus, optimization of the vaccination strategy is essential to achieve therapeutic effectiveness. In addition, effective peptide vaccines must stimulate both CD8 cytotoxic and CD4 helper T lymphocytes. Our group has been successful in designing effective peptide vaccination strategies for inducing CD8 T-cell responses in mouse tumor models. Here, we describe a somewhat similar, but distinct, peptide vaccination strategy capable of generating vast CD4 T-cell responses by combining synthetic peptides with toll-like receptor (TLR) agonists and OX40/CD40 costimulation. This vaccination strategy was efficient in overcoming immune tolerance to a self-tumor–associated antigen and generated significant antitumor effects in a mouse model of malignant melanoma. The optimized peptide vaccine also allowed the expansion of adoptively transferred CD4 T cells without the need for lymphodepletion and IL2 administration, generating effective antimelanoma responses through the enhancement of proliferative and antiapoptotic activities of CD4 T cells. These results have practical implications in the design of more effective T-cell–based immunotherapies. Cancer Immunol Res; 5(1); 72–83. ©2016 AACR.

Therapeutic vaccines for the treatment of cancer are an attractive alternative to some of the conventional therapies that are currently used. More importantly, vaccines could be very useful to prevent recurrences when applied after primary therapy. Unfortunately, most therapeutic vaccines for cancer have performed poorly due to the low level of immune responses that they induce. Previous work done in our laboratory in cancer mouse models demonstrated that vaccines consisting of synthetic peptides representing minimal CD8 T-cell epitopes administered i.v. mixed with poly-IC and anti-CD40 antibodies (TriVax) were capable of inducing massive T cell responses similar to those found during acute infections. We now report that some peptides are capable of inducing similarly large T cell responses after vaccination with poly-IC alone (BiVax). The results show that amphiphilic peptides are more likely to function as strong immunogens in BiVax and that systemic immunizations (i.v. or i.m.) were more effective than local (s.c.) vaccine administration. The immune responses induced by BiVax were found to be effective against established tumors in two mouse cancer models. The roles of various immune-related pathways such as type-I IFN, CD40 costimulation, CD4 T cells, TLRs and the MDA5 RNA helicase were examined. The present findings could facilitate the development of simple and effective subunit vaccines for diseases where CD8 T cells provide a therapeutic benefit.

Toll-like receptor 2 (TLR2) is a target for immune system stimulation during cancer immunotherapy and a cell-surface marker for pancreatic cancer. To develop targeted agents for cancer imaging and therapy, we designed, synthesized, and characterized 13 novel, fully synthetic high affinity TLR2 agonists. Analogue 10 had the highest agonist activity (NF-κB functional assay, EC(50) = 20 nM) and binding affinity (competitive binding assay, K(i) = 25 nM). As an immune adjuvant, compound 10 stimulated the immune system in vivo by generation and persistence of antigen-specific CD8+ T cells indicating its potential use in cancer immunotherapy. After conjugation of near-infrared dye to 10, agonist activity (EC(50) = 34 nM) and binding affinity (K(i) = 11 nM) were retained in 13. Fluorescence signal was present in TLR2 expressing pancreatic tumor xenografts 24 h after injection of 13, while an excess of unlabeled ligand blocked 13 from binding to the tumor, resulting in significantly decreased signal (p < 0.001) demonstrating in vivo selectivity.

Autophagy attenuates the efficacy of conventional chemotherapy but its effects on immunotherapy have been little studied. Here, we report that chemotherapy renders tumor cells more susceptible to lysis by CTL in vivo. Moreover, bystander tumor cells that did not express antigen were killed by CTL. This effect was mediated by transient but dramatic upregulation of the mannose-6-phosphate receptor (MPR) on the tumor cell surface. Antitumor effects of combined treatment related to the kinetics of MPR upregulation and abrogation of this event abolished the combined effect of immunotherapy and chemotherapy. MPR accumulation on the tumor cell surface during chemotherapy was observed in different mouse tumor models and in patients with multiple myeloma. Notably, this effect was the result of redistribution of the receptor caused by chemotherapy-inducible autophagy. Together, our findings reveal one molecular mechanism through which the antitumor effects of conventional cancer chemotherapy and immunotherapy are realized. Cancer Res; 72(21); 5483–93. ©2012 AACR.

An experimental study on the failure of aluminum sandwich joints under shear loading was conducted. A total of 60 specimens including three different insert types and two different potting types were fabricated and tested. The test results showed that the through-clearance type of joint fails at the highest load among insert type joints. The failure load of the potted joints with the dimple washer increased by 10% compared to the simple potted joints. As expected, the shear failure load became higher in accordance with the face thickness increase. It was also found that the upper face in contact with the loading tool is more dominant over the failure load.