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Chimeric antigen receptor (CAR) is a synthetic receptor that induces T cell-mediated lysis of abnormal cells. As cancer driver proteins are present at low levels on the cell surface, they can cause weak CAR reactivity, resulting in antigen sensitivity defects and consequently limited therapeutic efficacy. Although affinity maturation enhances the efficacy of CAR-T cell therapy, it causes off-target cross-reactions resulting in adverse effects. Preferentially expressed antigen in melanoma (PRAME) is an intracellular oncoprotein that is overexpressed in various tumors and restricted in normal tissues, except the testis. Therefore, PRAME could be an ideal target for cancer immunotherapy. In this study, we developed an experimental CAR system comprising six single-chain variable fragments that specifically recognizes the PRAMEp301/HLA-A∗24:02 complex. Cell-mediated cytotoxicity was demonstrated using a panel of CARs with a wide range of affinities (KD = 10−10–10−7 M) and affinity modulation. CAR-T cells with fast on-rates enhance antigen sensitivity by accelerating the killing rates of these cells. Alanine scanning data demonstrated the potential of genetically engineered CARs to reduce the risk of cross-reactivity, even among CARs with high affinities. Given the correlation between on-rates and dwell time that occurs in rebinding and cell-mediated cytotoxicity, it is proposed that CAR-binding characteristics, including on-rate, play a pivotal role in the lytic capacity of peptide-major histocompatibility complex-targeting CAR-T cells, thus facilitating the development of strategies whereby genetically engineered CARs target intracellular antigens in cancer cells to lyse the cells. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors and adoptive transfer of gene-engineered T cells have emerged as novel therapeutic modalities for hard-to-treat solid tumors; however, many patients are refractory to these immunotherapies, and the mechanisms underlying tumor immune resistance have not been fully elucidated. By comparing the tumor microenvironment of checkpoint inhibition-sensitive and -resistant murine solid tumors, we observed that the resistant tumors had low immunogenicity. We identified antigen presentation by CD11b+F4/80+ tumor-associated macrophages (TAMs) as a key factor correlated with immune resistance. In the resistant tumors, TAMs remained inactive and did not exert antigen-presenting activity. Targeted delivery of a long peptide antigen to TAMs by using a nano-sized hydrogel (nanogel) in the presence of a TLR agonist activated TAMs, induced their antigen-presenting activity, and thereby transformed the resistant tumors into tumors sensitive to adaptive immune responses such as adoptive transfer of tumor-specific T cell receptor-engineered T cells. These results indicate that the status and function of TAMs have a significant impact on tumor immune sensitivity and that manipulation of TAM functions would be an effective approach for improving the efficacy of immunotherapies. [ABSTRACT FROM AUTHOR]

The recent success of chimeric antigen receptor (CAR)-T cell therapy for treatment of hematologic malignancies supports further development of treatments for both liquid and solid tumors. However, expansion of CAR-T cell therapy is limited by the availability of surface antigens specific for the tumor while sparing normal cells. There is a rich diversity of tumor antigens from intracellularly expressed proteins that current and conventional CAR-T cells are unable to target. Furthermore, adoptively transferred T cells often suffer from exhaustion and insufficient expansion, in part, because of the immunosuppressive mechanisms operating in tumor-bearing hosts. Therefore, it is necessary to develop means to further activate and expand those CAR-T cells in vivo. The Wilms tumor 1 (WT1) is an intracellular oncogenic transcription factor that is an attractive target for cancer immunotherapy because of its overexpression in a wide range of leukemias and solid tumors, and a low level of expression in normal adult tissues. In the present study, we developed CART cells consisting of a single chain variable fragment (scFv) specific to the WT1235-243/HLAA* 2402 complex. The therapeutic efficacy of our CAR-T cells was demonstrated in a xenograft model, which was further enhanced by vaccination with dendritic cells (DCs) loaded with the corresponding antigen. This enhanced efficacy was mediated, at least partly, by the expansion and activation of CAR-T cells. CAR-T cells shown in the present study not only demonstrate the potential to expand the range of targets available to CAR-T cells, but also provide a proof of concept that efficacy of CAR-T cells targeting peptide/major histocompatibility complex can be boosted by vaccination. [ABSTRACT FROM AUTHOR]

A phase I+II clinical trial of vaccination with MAGE-A4 protein complexed with cholesteryl pullulan melanoma antigen gene-A4 nanogel (CHP-MAGE-A4) is currently underway in patients with MAGE-A4-expressing cancer. In the present study, the primary phase I endpoint was to test the safety of the administration of 300 µg CHP-MAGE-A4 with and without OK-432. Another aim of the study was to clarify the details of the specific humoral immune response to vaccination. The 9 patients enrolled for phase I were vaccinated 6 times, once every 2 weeks: 3 patients with 100 µg and 3 patients with 300 µg CHP-MAGE-A4, and 3 patients with 300 µg CHP-MAGE-A4 plus 0.5 clinical units of OK-432. Toxicities were assessed using Common Terminology Criteria for Adverse Events v3.0. Clinical response was evaluated by modified Response Evaluation Criteria in Solid Tumours. Immunological monitoring of anti-MAGE-A4-specific antibodies was performed by ELISA of pre- and post-vaccination patient sera. The 6 vaccinations produced no severe adverse events. Stable disease was assessed in 4/9 patients. Anti-MAGE-A4 total immunoglobulin (Ig)G titers increased in 7/9 patients. Efficacious anti-MAGE-A4 IgG1, 2 and 3 antibody responses were observed in 7/9 patients. Among them, positive conversions to T helper 2 (Th2)-type antibody responses (IgG4 and IgE) were observed after frequent vaccination in 4/7 patients. The Th2 conversion was possibly associated with undesirable clinical observations, including progressive disease and the appearance of a new relapse lesion. The present study suggested that frequent vaccinations activated a Th2-dominant status in the cancer patients. The identification of a time-dependent IgG subclass and IgE antibody production during vaccination protocols may be a useful surrogate marker indicating a potentially undesirable change of the immunological environment for an effective antitumor immune response in cancer patients. [ABSTRACT FROM AUTHOR]

The Fas/FasL (CD95/CD178) system is required for immune regulation; however, it is unclear in which cells, when, and where Fas/FasL molecules act in the immune system. We found that CD8+CD122+ cells, which are mostly composed of memory T cells in comparison with naïve cells in the CD8+CD122- population, were previously shown to include cells with regulatory activity and could be separated into CD49dlow cells and CD49dhigh cells. We established in vitro and in vivo experimental systems to evaluate the regulatory activity of CD122+ cells. Regulatory activity was observed in CD8+CD122+CD49dlow but not in CD8+CD122+CD49dhigh cells, indicating that the regulatory cells in the CD8+CD122+ population could be narrowed down to CD49dlow cells. CD8+CD122- cells taken from lymphoproliferation (lpr) mice were resistant to regulation by normal CD122+ Tregs. CD122+ Tregs taken from generalized lymphoproliferative disease (gld) mice did not regulate wild-type CD8+CD122- cells, indicating that the regulation by CD122+ Tregs is Fas/FasL-dependent. CD122+ Tregs taken from IL-10-deficient mice could regulate CD8+CD122- cells as equally as wild-type CD122+ Tregs both in vitro and in vivo. MHC class I-missing T cells were not regulated by CD122+ Tregs in vitro. CD122+ Tregs also regulated CD4+ cells in a Fas/FasL-dependent manner in vitro. These results suggest an essential role of Fas/FasL as a terminal effector of the CD122+ Tregs that kill activated T cells to maintain immune homeostasis. [ABSTRACT FROM AUTHOR]

Tumor antigen-specific CD4+ T cells generally orchestrate and regulate immune cells to provide immune surveillance against malignancy. However, activation of antigen-specific CD4+ T cells is restricted at local tumor sites where antigen-presenting cells (APCs) are frequently dysfunctional, which can cause rapid exhaustion of anti-tumor immune responses. Herein, we characterize antitumor effects of a unique human CD4+ helper T-cell subset that directly recognizes the cytoplasmic tumor antigen, NY-ESO-1, presented by MHC class II on cancer cells. Upon direct recognition of cancer cells, tumor-recognizing CD4+ T cells (TR-CD4) potently induced IFN-γ-dependent growth arrest in cancer cells. In addition, direct recognition of cancer cells triggers TR-CD4 to provide help to NY-ESO-1-specific CD8+ T cells by enhancing cytotoxic activity, and improving viability and proliferation in the absence of APCs. Notably, the TR-CD4 either alone or in collaboration with CD8+ T cells significantly inhibited tumor growth in vivo in a xenograft model. Finally, retroviral gene-engineering with T cell receptor (TCR) derived from TR-CD4 produced large numbers of functional TRCD4. These observations provide mechanistic insights into the role of TR-CD4 in tumor immunity, and suggest that approaches to utilize TR-CD4 will augment anti-tumor immune responses for durable therapeutic efficacy in cancer patients. [ABSTRACT FROM AUTHOR]

Because WT1 is expressed in leukemia cells, the development of cancer immunotherapy targeting WT1 has been an attractive translational research topic. However, concern of this therapy still remains, since WT1 is abundantly expressed in renal glomerular podocytes. In the present study, we clearly showed that WT1-specific cytotoxic T lymphocytes (CTLs) certainly exerted cytotoxicity against podocytes in vitro; however, they did not damage podocytes in vivo. This might be due to the anatomical localization of podocytes, being structurally separated from circulating CTLs in glomerular capillaries by an exceptionally thick basement membrane. [ABSTRACT FROM AUTHOR]

Transactivating factor PuF which interacts with a nuclease hypersensitive element locates upstream from the c-myc gene. PuF was recently identified as being encoded by nm23-H2/NDP kinase gene [Postel, E. H., Berberich, S. J., Flint, S. J., and Ferrone, C. A. (1993) Science 261, 428-429]. Here we have studied the correlation of expression between c-myc and nm23 genes in vitro. By a comparative study of the expression of 2 genes in cell lines, no direct correlation of kinetics was found. A plasmid containing the human c-myc fragment (c-myc CAT) was cloned upstream from the bacterial chloramphenicol acetyltransferase (CAT) gene. When the murine melanoma cell line was cotransfected with a nm23-M2/NDP kinase expression vector and c-myc CAT, CAT activity was elevated. In addition, cell cycle phases in the murine cell lines transfected with nm23/NDP kinase were estimated; an alteration of the cell cycle, prolonged S-phase was found in the cell lines transfected with nm23-M2/NDP kinase, whereas human nm23-H2/NDP kinase did not play a role in transactivating the c-myc gene or in S-phase prolongation in murine cell lines. From these results we conclude that the murine nm23-M2 gene transactivates the c-myc gene and controls the cell cycle, S-phase, indirectly via a cellular cofactor in the murine cell line, which does not work with the human nm23-H2 gene. [ABSTRACT FROM AUTHOR]

3-Methylcholanthrene (MCA)-induced sarcomas have been used as conventional tools for investigating immunosurveillance against tumor development. However, MCA-induced sarcoma is not always an ideal model for the study of the human cancer system because carcinomas and not sarcomas are the dominant types of human cancers. To resolve this problem, we established a novel and simple method to induce mouse squamous cell carcinomas (SCCs). As well known, the subcutaneous injection of MCA caused the formation of sarcomas at 100% incidence. However, we here first succeeded at inducing SCC at 60% of incidence within 2 months by a single intra-dermal injection of MCA. Using this primary SCC model, we demonstrated the critical role of interferon (IFN)-γ-dependent type 1 immunity in immunosurveillance against SCC from the following results: (i) The incidence of SCC was accelerated in IFN-γ-deficient mice compared with that in wild-type mice; (ii) In vivo injection of CpG-oligodeoxynucleotides (CpG-ODN) caused a marked reduction in the incidence of SCC in parallel with the activation of type 1-dependent antitumor immunity and (iii) The antitumor activity of CpG-ODN was significantly decreased in IFN-γ-deficient mice. Thus, our established MCA-induced mouse SCC model could be a powerful tool for evaluating immunosurveillance mechanisms during the development of SCC and might result in a novel strategy to address immunosurveillance mechanisms of human cancer. [ABSTRACT FROM AUTHOR]

It is well documented that UV radiation present in sunlight suppresses immune responses, especially Th1-driven cellular immune responses, resulting in the exacerbation of skin cancer and infectious diseases. However, the effects of UV irradiation on humoral immune responses remain less clearly defined. In addition, the majority of studies documenting immunosuppressive effects of UV irradiation has been demonstrated in animals exposed to UV radiation before immunization. In the present study, therefore, we examined the effects of UV irradiation on humoral immune responses in mice that had been immunized before UV irradiation. Both Th1- and Th2-associated Ig responses were significantly suppressed by UV irradiation given 7 days after immunization in an antigen-specific manner. Adoptive transfer experiments revealed that CD4+ T cells from UV-irradiated mice are responsible for the UV-induced suppression of antibody responses. These CD4+ regulatory T cells suppressed proliferation of conventional CD4+ T cells in vivo and in vitro and contained IL-10-producing cells that did not express Foxp3. Mice depleted of CD25+ cells also exhibited reduced antibody responses by UV irradiation. Finally, we showed that CD4+ T cells from UV-irradiated mice treated with anti-IL-10 mAb failed to suppress antibody responses upon transfer. These results indicate that UV irradiation after immunization suppresses Th1- and Th2-mediated humoral immunity via the generation of Tr1-like regulatory T cells, in the process of which IL-10 appears to be important. Possible detrimental effects of UV irradiation after vaccination are also discussed. [ABSTRACT FROM AUTHOR]