Antigen-specific Cytotoxic T Lymphocytes Research Articles

Toll-like receptor 9 is required for the maintenance of CD25+FoxP3+CD4+ Treg cells during Listeria monocytogenes infection

Abstract It has long been appreciated, but not understood, that the CD8+ cytotoxic T lymphocyte (CTL) dependence on CD4+T cell help (Th) is conditional; needed for some immunogens but not others. One explanation for this phenomenon envisions Th requirement as an intrinsic property of the pathogen itself rather than its introduction to the immune system. Here we show that dependence of the optimal CD8+ T cell response to Listeria monocytogenes (Lm) on CD4+ T cells is a function of the immunogen dose used for priming, with low dose Lm (LD; 50 or 103 CFU WT or ΔActA, respectively) inducing a primary antigen-specific CTL response profoundly dependent on CD4+ Th cells while that induced by high dose Lm (HD; 4×103 or 106 CFU WT or ΔActA, respectively) is significantly inhibited by CD4+CD25+FoxP3+regulatory T cells (Treg). The Th-independence of HD immunization is not overcome by additional antigen but instead involves the inflammatory response to more bacteria. Evaluation of various TLR pathways as the relevant sensing mechanism showed that TLR2 is required for CTL responses to LD immunization, and that HD immunization in the absence of TLR9 results in a simultaneous loss of CD25+FoxP3+CD4+ Treg cells and increase in conventional CD4+ Th cells and CTLs. Our data thus reveal that the CTL response to the same pathogen is determined by distinct roles for CD4+ T cells as helpers versus regulators based on immunogen dose and demonstrate a previously undescribed role for TLR9 in the regulation of CD4+ Th and Treg cells.

A TLR3-Specific Adjuvant Relieves Innate Resistance to PD-L1 Blockade without Cytokine Toxicity in Tumor Vaccine Immunotherapy

Cancer patients having anti-programmed cell death-1 (PD-1)/PD ligand 1 (L1)-unresponsive tumors may benefit from advanced immunotherapy. Double-stranded RNA triggers dendritic cell (DC) maturation to cross-prime antigen-specific cytotoxic T lymphocytes (CTLs) via Toll-like receptor 3 (TLR3). The TLR3-specific RNA agonist, ARNAX, can induce anti-tumor CTLs without systemic cytokine/interferon (IFN) production. Here, we have developed a safe vaccine adjuvant for cancer that effectively implements anti-PD-L1 therapy. Co-administration of ARNAX with a tumor-associated antigen facilitated tumor regression in mouse models, and in combination with anti-PD-L1 antibody, activated tumor-specific CTLs in lymphoid tissues, enhanced CTL infiltration, and overcame anti-PD-1 resistance without cytokinemia. The TLR3-TICAM-1-interferon regulatory factor (IRF)3-IFN-β axis in DCs exclusively participated in CD8+ Tcell cross-priming. ARNAX therapy established Th1 immunity in the tumor microenvironment, upregulating genes involved in DC/T cell/natural killer (NK) cell recruitment and functionality. Human exvivo studies disclosed that ARNAX+antigen induced antigen-specific CTL priming and proliferation in peripheral blood mononuclear cells (PBMCs), supporting the feasibility of ARNAX for potentiating anti-PD-1/PD-L1 therapy in human vaccine immunotherapy.

Engineering of a self-adjuvanted iTEP-delivered CTL vaccine.

Cytotoxic T lymphocyte (CTL) epitope peptide-based vaccines are widely used in cancer and infectious disease therapy. We previously generated an immune-tolerant elastin-like polypeptides (iTEPs)-based carrier to deliver a peptide CTL vaccine and enhance the efficiency of the vaccine. To further optimize the vaccine carrier, we intended to potentiate its function by designing an iTEP-based carrier that was able to deliver adjuvant and a vaccine epitope as one molecule. Thus, we fused a 9-mer H100, a peptide derived from the high-mobility group box 1 protein (HMGB1) that could induce activation of dendritic cells (DCs), with an iTEP polymer to generate a new iTEP polymer named H100-iTEP. The H100-iTEP still kept the feature of reversible phase transition of iTEPs and should be able to be used as a polymer carrier to deliver peptide vaccines. The expression levels of CD80/CD86 on DCs were assessed using flow cytometry. The iTEP fusion-stimulated IL-6 secretion by DCs was measured with ELISA. Activation of antigen-specific CD8+ T cells induced by iTEP fusions was examined through a B3Z hybridoma cell activation assay. In vivo CTL activation promoted by iTEP fusions was detected by an IFN-γ-based ELISPOT assay. The iTEP fused with H100 could induce maturation of DCs in vitro as evidenced by increased CD80 and CD86 expression. The iTEP fusion also promoted activation of DCs by increasing secretion of a proinflammatory cytokine IL-6. The N-terminus or C-terminus fusion of H100 to iTEP had a similar effect and a reduced form of cysteine in iTEP fusions was required for DC stimulation. iTEP fusions potentiated a co-administrated CTL vaccine by increasing an antigen-specific CTL response in vitro and in vivo. When the H100-iTEP was fused to a CTL epitope to generate a one-molecule vaccine, this self-adjuvanted vaccine elicited a stronger antigen-specific CTL response than a vaccine adjuvanted by Incomplete Freund's Adjuvant. Thus, we have successfully generated a functional, one-molecule iTEP-based self-adjuvanted vaccine.

IFNγ enhances cytotoxic efficiency of the cytotoxic T lymphocytes against human glioma cells

Cytotoxic T lymphocytes (CTLs) are a key player in cancer immunotherapies, and MHC class I molecules on the cell surface are crucial for cellular recognition. However, the aberrant expression of MHC class I molecules is frequently found in various malignancies. IFNγ has dual functions in cancer progression, and its effect on tumor immunity is controversial. To investigate whether IFNγ can enhance cytotoxic efficiency of the tumor antigen-specific CTLs, we generated the CTLs using modified human dendritic cells as antigen presenting cells, then studied the activities of CTLs on human leukocyte antigen (HLA)-A2 positive glioma cells treated with, or without IFNγ. The results from both ELISpot and cytotoxicity assays demonstrated that the CTLs recognized and eliminated the HLA-A2 positive glioma cells treated with IFNγ more effectively when compared to the glioma cells deprived of IFNγ treatment. In addition, in vitro experiments showed that the levels of MHC class I molecules were upregulated in all of the HLA-A2 positive glioma cells. Using the publicly accessed TCGA data of low-grade glioma, we found significantly positive associations between IFNγ and both MHC class I molecules and CD8+ T cell activation score (p<0.0001). Furthermore, we found a significantly reduced risk of death in the glioma patients with high T cell activation score in comparison to those with low score (p=0.022). These findings suggest that a clinical application of IFNγ treatment may have potential benefits.

Exosomes from M1-Polarized Macrophages Potentiate the Cancer Vaccine by Creating a Pro-inflammatory Microenvironment in the Lymph Node

Exosomes are small membrane-bound vesicular particles generated by most cells for intercellular communication and regulation. During biogenesis, specific lipids, RNAs, proteins, and carbohydrates are enriched and packaged into the vesicles so that the exosomal contents reflect not only the source but also the physiological conditions of the parental cells. These exosomes transport materials or signals to the target cells for diverse physiological purposes. Our study focused on the exosomes derived from M1-polarized, proinflammatory macrophages for the possibility of using M1 exosomes as an immunopotentiator for a cancer vaccine. The M1 exosomes displayed a tropism toward lymph nodes after subcutaneous injection, primarily taken up by the local macrophages and dendritic cells, and they induced the release of a pool of Th1 cytokines. We found that M1, but not M2, exosomes enhanced activity of lipid calcium phosphate (LCP) nanoparticle-encapsulated Trp2 vaccine, and they induced a stronger antigen-specific cytotoxic Tcell response. The M1 exosomes proved to be a more potent immunopotentiator than CpG oligonucleotide when used with LCP nanoparticle vaccine in a melanoma growth inhibition study. Thus, our study indicated that exosomes derived from M1-polarized macrophages could be used as a vaccine adjuvant.

IFN-γ is required for cytotoxic T cell-dependent cancer genome immunoediting

Genetic evolution that occurs during cancer progression enables tumour heterogeneity, thereby fostering tumour adaptation, therapeutic resistance and metastatic potential. Immune responses are known to select (immunoedit) tumour cells displaying immunoevasive properties. Here we address the role of IFN-γ in mediating the immunoediting process. We observe that, in several mouse tumour models such as HA-expressing 4T1 mammary carcinoma cells, OVA-expressing EG7 lymphoma cells and CMS5 MCA-induced fibrosarcoma cells naturally expressing mutated extracellular signal-regulated kinase (ERK) antigen, the action of antigen-specific cytotoxic T cell (CTL) in vivo results in the emergence of resistant cancer cell clones only in the presence of IFN-γ within the tumour microenvironment. Moreover, we show that exposure of tumours to IFN-γ-producing antigen-specific CTLs in vivo results in copy-number alterations (CNAs) associated with DNA damage response and modulation of DNA editing/repair gene expression. These results suggest that enhanced genetic instability might be one of the mechanisms by which CTLs and IFN-γ immunoedits tumours, altering their immune resistance as a result of genetic evolution.

Preclinical evaluation of an mRNA HIV vaccine combining rationally selected antigenic sequences and adjuvant signals (HTI-TriMix).

The development of a prophylactic vaccine against HIV-1 has so far not been successful. Therefore, attention has shifted more and more toward the development of novel therapeutic vaccines. Here, we evaluated a new mRNA-based therapeutic vaccine against HIV-1-encoding activation signals (TriMix: CD40L + CD70 + caTLR4) combined with rationally selected antigenic sequences [HIVACAT T-cell immunogen (HTI)] sequence: comprises 16 joined fragments from Gag, Pol, Vif, and Nef). For this purpose, peripheral blood mononuclear cells from HIV-1-infected individuals on cART, lymph node explants from noninfected humans, and splenocytes from immunized mice were collected and several immune functions were measured. Electroporation of immature monocyte-derived dendritic cells from HIV-infected patients with mRNA encoding HTI + TriMix potently activated dendritic cells which resulted in upregulation of maturation markers and cytokine production and T-cell stimulation, as evidenced by enhanced proliferation and cytokine secretion (IFN-γ). Responses were HIV specific and were predominantly targeted against the sequences included in HTI. These findings were confirmed in human lymph node explants exposed to HTI + TriMix mRNA. Intranodal immunizations with HTI mRNA in a mouse model increased antigen-specific cytotoxic T-lymphocyte responses. The addition of TriMix further enhanced cytotoxic responses. Our results suggest that uptake of mRNA, encoding strong activation signals and a potent HIV antigen, confers a T-cell stimulatory capacity to dendritic cells and enhances their ability to stimulate antigen-specific immunity. These findings may pave the way for therapeutic HIV vaccine strategies based on antigen-encoding RNA to specifically target antigen-presenting cells.

Repurposing ospemifene for potentiating an antigen-specific immune response

Ospemifene, an estrogen receptor agonist/antagonist approved for the treatment of dyspareunia and vaginal dryness in postmenopausal women, has potential new indications as an immune modulator. The overall objective of the present series of preclinical studies was to evaluate the immunomodulatory activity of ospemifene in combination with a peptide cancer vaccine. Immune regulating effects, mechanism of action and structure activity relationships of ospemifene and related compounds were evaluated by examining expression of T-cell activating cytokines in vitro, and antigen-specific immune response and cytotoxic T-lymphocyte activity in vivo. The effects of ospemifene (OSP) on the immune response to a peptide cancer vaccine (PV) were evaluated after chronic [control (n = 22); OSP 50 mg/kg (n = 16); PV (n = 6); OSP+PV (n = 11)], intermittent [control (n = 10); OSP 10 and 50 mg/kg (n = 11); PV (n = 11); combination treatment (n = 11 each dose)] and pretreatment [control; OSP 100 mg/kg; PV 100 μg; combination treatment (n = 8 all groups)] ospemifene oral dosing schedules in a total of 317 mixed-sex tumor-bearing and nontumor-bearing mice. The results showed that ospemifene induced expression of the key TH1 cytokines interferon gamma and interleukin-2 in vitro, which may be mediated by stimulating T-cells through phosphoinositide 3-kinase and calmodulin signaling pathways. In combination with an antigen-specific peptide cancer vaccine, ospemifene increased antigen-specific immune response and increased cytotoxic T-lymphocyte activity in tumor-bearing and nontumor-bearing mice. The pretreatment, intermittent, and chronic dosing schedules of ospemifene activate naive T-cells, modulate antigen-induced tolerance and reduce tumor-associated, pro-inflammatory cytokines, respectively. Taken together, ospemifene's dose response and schedule-dependent immune modulating activity offers a method of tailoring and augmenting the efficacy of previously failed antigen-specific cancer vaccines for a wide range of malignancies.

Abstract B53: T lymphocyte engineering with responsive cytokine nanogels for enhanced efficacy and safety of adoptive cell therapy for cancer

Abstract Adoptive cell transfer (ACT) with autologous tumor-reactive T-cells has recently shown great promise in the treatment of leukemias in clinical trials. However, effective control of solid tumors, in particular resistant, metastatic, or recurrent cancers, remain challenging. One of the major limitations is the rapid decline of T-cell function in the immunosuppressive tumor microenvironment. In order to maintain high numbers of viable antigen-specific cytotoxic T-cells in tumors, co-administration of supporting immunostimulant agents such as cytokines together with transferred cells is often employed. However, the high systemic doses of such agents needed to enhance T-cell functionality inside solid tumors can also result in serious side effects. Here, we developed a cell-responsive nanogel (NG) based on a novel carrier-free delivery strategy to effectively and specifically support adoptively transferred T-cells with recombinant cytokines such as interleukin-2 (IL-2) or interleukin-15 (IL-15). IL-2-Fc or an IL-15 superagonist (IL-15Sa) were chemically crosslinked with a disulfide linker to form these protein NGs, which were subsequently bound to and stably anchored on the plasma membrane of adoptively transferred T-cells ex vivo. These NGs had exceptionally high loading of cytokines (~70 wt%) and released native cytokine proteins in physiological condition in a sustained manner through breakdown of a degradable disulfide linker in response to the reduction activity at the plasma membrane surface of activated T-cells, and thus enabled continuous pseudo-autocrine stimulation of transferred CD8+ T-cells. In a mouse model of subcutaneous B16F10 melanoma tumors, adoptively transferred mouse pmel-1 CD8+ T-cells with optimized IL-15Sa-NGs showed markedly enhanced expansion and persistence in tumors and thus improved the anti-cancer efficacy. Quantification of transferred tumor-specific T-cells in tumors 7 days after transfer showed that surface-bound IL-15Sa-NGs resulted in ~16-fold more tumor-reactive T-cells compared to T-cells supported by an equivalent dose of soluble IL-15Sa administered systemically. Surface-bound IL-15Sa-NGs could be used to “backpack” T-cells with doses of cytokine that slowly released and maintained T-cell function over at least 2 weeks in vivo, while the same doses administered systemically as a bolus or repeated lower-dose injections were very toxic, eliciting systemic cytokine syndrome. These data demonstrate that T-cell surface-bound responsive cytokine-NGs provide autocrine support to the tumor-reactive T-cells to enhance efficacy of ACT therapy against solid tumors and safely administered potent adjuvant immunostimulants to donor T-cells. Citation Format: Li Tang, Yiran Zheng, Llian Mabardi, Darrell Irvine. T lymphocyte engineering with responsive cytokine nanogels for enhanced efficacy and safety of adoptive cell therapy for cancer. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B53.

Dissecting thyroid hormone transport and metabolism in dendritic cells.

We reported thyroid hormone (TH) receptor expression in murine dendritic cells (DCs) and 3,5,3'-triiodothyronine (T3)-dependent stimulation of DC maturation and ability to develop a Th1-type adaptive response. Moreover, an increased DC capacity to promote antigen-specific cytotoxic T-cell activity, exploited in a DC-based antitumor vaccination protocol, was revealed. However, putative effects of the main circulating TH, l-thyroxine (T4) and the mechanisms of TH transport and metabolism at DC level, crucial events for TH action at target cell level, were not known. Herein, we show that T4 did not reproduce those registered T3-dependent effects, finding that may reflect a homoeostatic control to prevent unspecific systemic activation of DCs. Besides, DCs express MCT10 and LAT2 TH transporters, and these cells mainly transport T3 with a favored involvement of MCT10 as its inhibition almost prevented T3 saturable uptake mechanism and reduced T3-induced IL-12 production. In turn, DCs express iodothyronine deiodonases type 2 and 3 (D2, D3) and exhibit both enzymatic activities with a prevalence towards TH inactivation. Moreover, T3 increased MCT10 and LAT2 expression and T3 efflux from DCs but not T3 uptake, whereas it induced a robust induction of D3 with a parallel slight reduction in D2. These findings disclose pivotal events involved in the mechanism of action of THs on DCs, providing valuable tools for manipulating the immunogenic potential of these cells. Furthermore, they broaden the knowledge of the TH mechanism of action at the immune system network.

Dendritic cells engineered to secrete anti-DcR3 antibody augment cytotoxic T lymphocyte response against pancreatic cancer in vitro.

AIMTo investigate the enhanced cytotoxic T lymphocyte responses against pancreatic cancer (PC) in vitro induced by dendritic cells (DCs) engineered to secrete anti-DcR3 monoclonal antibody (mAb).METHODSDCs, T lymphocytes and primary PC cells were obtained from PC patients. DCs were transfected with a designed humanized anti-DcR3 monoclonal antibody heavy and light chain mRNA and/or total tumor RNA (DC-tumor-anti-DcR3 RNA or DC-total tumor RNA) by using electroporation technology. The identification, concentration and function of anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA were determined by western blotting and enzyme-linked immunosorbent assay. After co-culturing of autologous isolated PC cells with target DCs, the effects of secreting anti-DcR3 mAb on RNA-DCs’ viability and apoptosis were assessed by MTT assay and flow cytometry. Analysis of enhanced antigen-specific immune response against PC induced by anti-DcR3 mAb secreting DCs was performed using a 51Cr releasing test. T cell responses induced by RNA-loaded DCs were analyzed by measuring cytokine levels, including IFN-γ, IL-10, IL4, TNF-α and IL-12.RESULTSThe anti-DcR3 mAb secreted by DCs reacted with recombinant human DcR3 protein and generated a band with 35 kDa molecular weight. The secreting mAb was transient, peaking at 24 h and becoming undetectable after 72 h. After co-incubation with DC-tumor-anti-DcR3 RNA for designated times, the DcR3 level in the supernatant of autologous PC cells was significantly down-regulated (P < 0.05). DCs secreting anti-DcR3 mAb could improve cell viability and slow down the apoptosis of RNA-loaded DCs, compared with DC-total tumor RNA (P < 0.01). The anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA could enhance the induction of cytotoxic T lymphocytes (CTLs) activity toward RNA-transfected DCs, primary tumor cells, and PC cell lines, compared with CTLs stimulated by DC-total tumor RNA or control group (P < 0.05). Meanwhile, the antigen-specific CTL responses were MHC class I-restricted. The CD4+ T cells and CD8+ T cells incubated with anti-DcR3 mAb secreting DCs could produce extremely higher level IFN-γ and lower level IL4 than those incubated with DC-total tumor RNA or controls (P < 0.01).CONCLUSIONDCs engineered to secrete anti-DcR3 antibody can augment CTL responses against PC in vitro, and the immune-enhancing effects may be partly due to their capability of down-regulating DC apoptosis and adjusting the Th1/Th2 cytokine network.

Differential Potency and Breadth of the Same Epitope-Specific CTLs to Inhibit HIV-1 Replication based on TCR Usage

Increasing evidence suggests that the immune control of viral replication by HIV-1-specific cytotoxic T lymphocytes (CTLs) is relevant to the selection of human leukocyte antigen (HLA) allele-restricted antigen-specific CTL repertoire. But the underlying factors accounting for CTL functional difference from T cell receptor (TCR) clonal diversity are currently unclear. Here we found that CTL repertoire specific for HLA-B*27-restricted HIV-1 Gag p24 epitope KK10 (KRWIILGLNK, residues 263-272) selected multiple TCR clonotypes. By dissection of the bulk KK10-specific CTL compartment, differential potency and breadth of KK10-specific CTL clones to inhibit HIV-1 replication was defined due to the distinct TCR usage. Superior control of viral replication of wild-type HIV-1 isolates was observed by the TCR-clonotypic CTLs characteristic of higher ability to produce MIP-1β. A unique TCR-equipped KK10- specific CTLs efficiently controlled wide-type HIV-1 isolates and broadly cross-recognized viral variants. These data suggest that clonally diverse CTL responses to a viral epitope increase the likelihood that multiple divergent strains of viruses may be recognized by cross-reactive CTLs through immunization with only a single viral epitope sequence.

Effective Respiratory CD8 T-Cell Immunity to Influenza Virus Induced by Intranasal Carbomer-Lecithin-Adjuvanted Non-replicating Vaccines.

CD8+ cytotoxic T lymphocytes (CTLs) are critical for clearing many viral infections, and protective CTL memory can be induced by vaccination with attenuated viruses and vectors. Non-replicating vaccines are typically potentiated by the addition of adjuvants that enhance humoral responses, however few are capable of generating CTL responses. Adjuplex is a carbomer-lecithin-based adjuvant demonstrated to elicit robust humoral immunity to non-replicating antigens. We report that mice immunized with non-replicating Adjuplex-adjuvanted vaccines generated robust antigen-specific CTL responses. Vaccination by the subcutaneous or the intranasal route stimulated systemic and mucosal CTL memory respectively. However, only CTL memory induced by intranasal vaccination was protective against influenza viral challenge, and correlated with an enhancement of memory CTLs in the airways and CD103+ CD69+ CXCR3+ resident memory-like CTLs in the lungs. Mechanistically, Myd88-deficient mice mounted primary CTL responses to Adjuplex vaccines that were similar in magnitude to wild-type mice, but exhibited altered differentiation of effector cell subsets. Immune potentiating effects of Adjuplex entailed alterations in the frequency of antigen-presenting-cell subsets in vaccine draining lymph nodes, and in the lungs and airways following intranasal vaccination. Further, Adjuplex enhanced the ability of dendritic cells to promote antigen-induced proliferation of naïve CD8 T cells by modulating antigen uptake, its intracellular localization, and rate of processing. Taken together, we have identified an adjuvant that elicits both systemic and mucosal CTL memory to non-replicating antigens, and engenders protective CTL-based heterosubtypic immunity to influenza A virus in the respiratory tract. Further, findings presented in this manuscript have provided key insights into the mechanisms and factors that govern the induction and programming of systemic and protective memory CTLs in the respiratory tract.

Anti-Tumor Activities of XBP1 Antigen-Specific Cytotoxic T Lymphocytes Are Enhanced By HDAC6 Inhibitor ACY241

The effects of histone deacetylase (HDAC) inhibition on immune effector cells may have significant clinical implications; however, this has not yet been elucidated. The goal of this study was to investigate the immunomodulatory potential of the selective HDAC6 inhibitor ACY241 in combination with a cancer vaccine to enhance the efficacy of antigen-specific cytotoxic T lymphocytes (CTL) and the specific activities against tumor cells. Here, we report the effects of ACY241 treatment on antigen expression, immune activation, proliferation, and functional activities of XBP1 antigen-specific cytotoxic T lymphocytes (XBP1-CTL). The antigen-specific CTL were generated in vitro by repeated stimulation with novel immunogenic heteroclitic HLA-A2 XBP1 peptides (YISPWILAV, YLFPQLISV), as described previously by our group (Bae et al. Leukemia 2011; Bae et al. Oncoimmunology 2014l; Bae et al. Leukemia 2016). We found that treatment with ACY241 up-regulated key co-stimulatory (CD28, CD40L) and activation (CD38, CD69, CD137) molecules on XBP1-CTL, without inducing expression of co-inhibitory checkpoints (PD1, LAG3, CTLA4, VISTA). In addition, ACY241 increased the frequency of memory CTL subsets and enhanced their anti-tumor activities (cytotoxic activity, Th1-type cytokine production, CTL proliferation) against HLA-A2+ and XBP1+ multiple myeloma, breast cancer, and colon cancer cells. The XBP1-CTL responses were dramatically increased in combination with ACY241, including higher levels of tumor-specific CD107a up-regulation, perforin release, IFN-g/IL-2/TNF-a cytokine production and proliferation of the CD3+CD8+ T cells expressing CD28/CD38 in response to the specific XBP1 peptides. ACY241 also enhanced the expression of various tumor-associated antigens (XBP1, CD138, CS1, BCMA, CD44), MHC class I/II molecules, along with co-stimulatory B7 molecules (CD80, CD86) on HLA-A2+ myeloma (U266), breast cancer (MDA-MB231) and colon cancer (SW480) cell lines. Furthermore, in vitro ACY241 treatment consistently decreased the frequency of immune suppressor cells including myeloid-derived suppressor cells (CD14- CD15+/CD11b+ CD33+/HLA-DRlow) and regulatory T cells (CD25+ FOXP3+/CD3+ CD4+) in peripheral blood or bone marrow mononuclear cells from multiple myeloma patients in a dose-dependent manner. In conclusion, our data demonstrates the immunomodulatory effects of selective HDAC6 inhibition by ACY241 and supports its potential role for improving tumor-specific CTL function and tumor cell recognition when used in combination with antigen-specific cancer vaccine. DisclosuresBae:OncoPep Inc.: Consultancy, Equity Ownership. Chauhan:Stemline Therapeutics: Consultancy. Hideshima:Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Munshi:OncoPep Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Anderson:OncoPep Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Identification and Validation of HLA-A24 XBP1us, XBP1sp, CD138, and CS1 Peptides to Generate Antigens Specific-Cytotoxic T Lymphocytes: Preclinical Basis for Vaccine Therapy in HLA-A24 Patients with Multiple Myeloma and Other Cancers

XBP1 (X-box binding protein 1), CD138 (Syndecan-1), and CS1 (CD2 subset 1, CRACC, SLAMF7, CD319) are highly expressed antigens associated with tumor pathogenesis in multiple myeloma cells and various solid tumor cells. We have previously reported on the use of HLA-A2 peptides specific to these antigens to generate antigen-specific and HLA-2-restricted cytotoxic T cells (CTL) in preclinical studies and phase I/II clinical trials in patients with smoldering MM. These studies extend this concept to HLA-A24 patients. Immunogenic HLA-A24 peptides specific to both the spliced and unspliced isoforms of XBP1, CD138, and CS1 were identified, synthesized, and evaluated in vitro for their ability to generate the antigen-specific cytotoxic T lymphocytes (CTL) targeting tumor cells. The specific epitopes were selected based on their binding affinity to HLA-A24, extended half-time disassociation rates, proteasomal C terminal cleavage, and TAP transport. We report here on novel highly immunogenic HLA-A24-specific peptides XBP1 US185 -193 (I S P W I L A V L), XBP1 SP223-231 (V Y P E G P S S L), CD138265-273 (I F A V C L V G F), and CS1240-248 (L F V L G L F L W), which induce antigen-specific CD3+CD8+ CTL against MM and various solid tumors. Each of the single peptide-specific CTL displayed immune functional activities including IFN-g/IL-2 cytokine production and cytotoxic activity against HLA-A24+ MM cells. Furthermore, our studies demonstrated that a cocktail of the four HLA-A24-specific peptides induced multipeptide-specific CD3+CD8+ CTL with functional anti-tumor properties against myeloma cells. Phenotypically, the multipeptide-specific CTL were central memory (CM; CCR7+CD45RO+/CD3+CD8+), effector memory (EM; CCR7-CD45RO+/CD3+CD8+) and terminal effector (TE; CCR7-CD45RO-/CD3+CD8+) cells expressing high levels of cell activation markers CD69, CD38, and CD40L. The multipeptide-specific effector and memory CTL subsets demonstrated HLA-A24-restricted and peptide-specific anti-tumor activities, and the highest activities were detected within the CM CTL subset. Treatment of the multipeptide-specific CTL or tumor target cells with different types of checkpoint inhibitors (a-PD1, a-TIM3, a-PDL-1, a-Galectin-9) or the immune modulator drug, lenalidomide, enhanced their anti-myeloma activities and antigen-specific CD3+CD8+ T cell proliferation to HLA-A24+ tumor cells. Therefore, a cocktail of the four immunogenic HLA-A24 epitopes provides the framework for development of a cancer vaccine based targeted immunotherapy. In conclusion, we report here on novel immunogenic HLA-A24-specific XBP1 unspliced, XBP1 spliced, CD138, and CS1 peptides that elicit CTL with anti-myeloma activities. These peptides will allow us to extend our immunotherapeutic vaccine approach beyond the current HLA-A2-specific peptides to HLA-A24 patients with smoldering myeloma, multiple myeloma, other plasma cell disorders and potentially with solid tumors. These preclinical studies also demonstrate that incorporation of immunomodulatory agents and/or checkpoint inhibitors can amplify and maintain antigen-specific memory CTL responses against tumor. DisclosuresBae:OncoPep Inc.: Consultancy, Equity Ownership. Raje:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Eli Lilly: Research Funding. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Hideshima:C4 Therapeutics: Equity Ownership; Acetylon: Consultancy. Chauhan:Stemline Therapeutics: Consultancy. Munshi:OncoPep Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Anderson:Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Regeneration of Tumor Antigen Specific CTLs Utilizing iPSC Technology for Off-the-Shelf Immunotherapy

In the tumor immunotherapy field tumor antigen specific T cell therapy is currently attracting attention. However, a critical issue still awaits resolution; it is difficult to efficiently expand tumor antigen specific T cells without exhaustion. To solve this problem, we are now utilizing induced pluripotent stem cell (iPSC) technology. When iPSCs are established from tumor antigen specific cytotoxic T cells (CTLs), T cells regenerated from these iPSCs (T-iPSCs) are expected to express the same T cell receptors (TCRs) as the original CTLs. Therefore, it will become possible to obtain de novo generated tumor antigen specific CTLs almost unlimitedly. In line with this concept, we have succeeded in establishing iPSCs from CTLs specific for the melanoma antigen MART-1 and regenerating MART-1 specific CTLs (Vizcardo et al, Cell Stem Cell, 2013).The strategy mentioned above was supposed to be applied as autologous transfusion. Autologous transfusion setting, however, has some problems; i) high cost to establish T-iPSCs from each patient, ii) heterogeneity of the quality as iPSCs, iii) difficulty in getting T-iPSCs with high affinity TCRs. To solve these problems, we are now planning to apply this method to the allogeneic transfusion setting. In allogeneic setting, we can select high quality T-iPSC clones with high affinity TCRs and such clones can be utilized to many patients with the same HLA type. In this context, it is preferable to establish T-iPSCs from HLA haplotype homozygous donors, since CTLs generated from these T-iPSCs can be administered to the patients carrying the same HLA haplotype at least on one allele. Thus by utilizing allogenic T-iPSCs the cost would be reduced and the quality of T-iPSCs can be ensured.Now we are trying to establish T-iPSCs from CTLs specific for various tumor antigen from healthy donors. We succeeded in establishing T-iPSCs from CTLs specific for EB virus related antigen LMP2 and cancer testis antigen WT1, both of which are restricted to HLA A2402, the commonest HLA-A in the Japanese. We improved in vitro culture condition and have succeeded in regenerating CD8 single positive cells expressing CD8 alpha-beta heterodimers. These regenerated CTLs exhibited very high antigen specific cytotoxicity comparable to the original CTLs. The CTLs derived from WT1 specific T-iPSCs were able to lyse some leukemia cell lines and primary leukemia cells which express endogenous WT1 protein. Administration of regenerated CTLs prolonged the survival of mice inoculated with leukemia cell line in in vivo xenograft model. In addition, we succeeded in transducing iPSCs derived from non-T cells with WT1-specific TCR genes (TCR-iPSCs) and generating CTLs from them. Generated CTLs from TCR-iPSCs exhibited comparable TCR specific cytotoxicity as regenerated CTLs from T-iPSCs. Using this method, it would be much easier to produce CTLs with defined high affinity TCRs specific for various tumor antigen.We are currently endeavoring to apply this method clinically. In the future, we intend to establish an allogeneic transfusion system, in which various tumor antigen specific T-iPSCs/TCR-iPSCs from a wide range of HLA haplotype homozygous donors will be lined up as a "T-iPS/TCR-iPSCs cell bank", with the aim of making off- the-shelf tumor immunotherapy a reality. DisclosuresKataoka:Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria. Ogawa:Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding. Kawamoto:Regcel: Research Funding.

Improvement of Antigen-Specific CTL Persistence By Transduction of Novel Artificial Adapter Molecule

Introduction: Endogenous or gene-modified T cells with T cell receptors (TCRs) are major designs of cytotoxic T lymphocytes (CTL) therapies. However, gene-modified TCR-CTL therapies targeting on malignant tumors have been generally unsuccessful in previous clinical trials. The major reasons were poor expansion and short persistence of CTL. To overcome these problems, TCR-affinity have been genetically enhanced, but often resulted in life-threatening toxicities. We aimed to modify TCR-CTL in order to improve the function by enhancing intracellular signaling after TCR ligation without affinity modulation.Method: We generated three artificial T cell activating adapter molecules (ATAM), which are CD3z, CD3z/CD28, and CD3z/4-1BB. CD28 or 4-1BB intracellular domain was inserted in the middle of CD3z intracellular domain, so that they can assemble with internal TCR complex when stimulated with corresponding antigen. ATAM-T2A-tEGFR was packaged into retrovirus vector to transduce the T cells. In order to evaluate the functional alteration, we established ATAM-CMV CTL model. We stimulated T cells with CMV pp65 peptide to induce endogenous CMV CTL. On day 4 after stimulation, ATAMs were retrovirally transduced into CMV CTL. After a selection with anti-EGFR mAb, we achieved more than 90% purity of ATAM positive CMV CTL and used in 51Cr release assay, cytokine release assay, and the growth assay. In the growth assay, we stimulated the ATAM CMV-CTL weekly and assayed the viable cell number, tetramer positivity, phenotype, and apoptosis. Regarding these assay, we used HLA transduced K562 (HLA-K562) as APC.Results: First, we confirmed the expression of each ATAM on CD8+ T cells, which showed similar expression among each ATAM. To determine the cytotoxicity of CMV CTL according to each ATAM or control tEGFR transduction, we performed 51Cr release assay against HLA-K562 pulsed with or without CMV peptide. In the presence of CMV peptide, each ATAM-CMV CTL showed similar cytolytic activities to control CMV CTL. [specific lysis (%): tEGFR 32.0+-6.0, CD3z 32.4+-2.6, CD3z/CD28 31.1+-8.8, and CD3z/4-1BB 37.4+-1.5: at an E:T ratio = 3:1, respectively}. In the intracellular cytokine assay against HLA-K562 pulsed with CMV peptide, similar proportions of responder were positive for IFN-g and IL-2 in control and ATAM-CMV CTL. [IFN-g (%): tEGFR, 67.3+-9.4, CD3z, 69.0+-7.4, CD3z/CD28, 66.5+-8.0, and CD3z/4-1BB, 69.6+-7.9; IL-2 (%): tEGFR, 17.5+-5.0, CD3z, 17.7+-4.3, CD3z/CD28, 15.9+-4.3, and CD3z/4-1BB, 18.9+-4.8, respectively]. The cytotoxicity and cytokine secretion upon irrelevant stimulation were completely negative among each ATAM-CMV CTL. Although the surface phenotype was not changed in control CMV-CTL, that of ATAMs-CMV CTL was significantly expanded after antigen stimulation [central memory (CD45RA-CD62L+) (%): tEGFR, 6.0+-2.9, CD3z, 26.8+-11.1, CD3z/CD28, 19.4+-9.5, and CD3z/4-1BB, 35.8+-26.8: at 7 days after stimulation]. Regarding the cell growth, CD3z/CD28 and CD3z/4-1BB showed greater CTL expansion upon weekly antigen stimulation. The fold expansion of tEGFR, CD3z, CD3z/CD28, and CD3z/4-1BB-CMV CTL were 4.9+-0.7, 8.2+-2.4, 12.3+-1.1, and 13.0+-1.6 at day7, 1.6+-0.4, 8.0+-2.5, 18.6+-3.9, and 21.3+-7.7 at day14, and 0.4+-0.2, 1.4+-0.7, 5.1+-3.0, and 30.4+-22.9 at day21, respectively (Fig). Comparing with control, CD3z/4-1BB-CMV CTL showed continuous greater expansion after several stimulations (p<0.01: during day0 to day7, p<0.05: during day7 to day14). To examine the reason for the growth advantage, we investigated the apoptosis after the stimulation with DAPI/Annexin V staining. It revealed that the live cells after specific stimulation elevated in ATAMs-CMV CTL compared with control CMV CTL.Conclusion: We generated three ATAMs and successfully transduced into CMV CTL. Particularly, the whole cell growth and continuance of growth after weekly stimulation were significantly improved in CD3z/4-1BB CTL. This greater cell expansion may improve in vivo persistence and further effectiveness of TCR-CTL therapy. ATAM-CMV CTL did not recognize any APC without corresponding peptide, indicating that ATAM transduction could improve the CTL effectiveness without altering CTL specificity. Thus we believe that we could potentially apply ATAM transduction to other TCR-CTLs targeting malignant tumors safely. [Display omitted] DisclosuresKiyoi:Alexion Pharmaceuticals: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; AlexionpharmaLLC.: Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; MSD K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co.LTD.: Research Funding; Kyowa-Hakko Kirin Co.LTD.: Research Funding; Chugai Pharmaceutical Co. LTD.: Research Funding; Celgene Corporation: Consultancy.

Regeneration of CD8αβ T Cells from T-cell-Derived iPSC Imparts Potent Tumor Antigen-Specific Cytotoxicity.

Although adoptive transfer of cytotoxic T lymphocytes (CTL) offer a promising cancer therapeutic direction, the generation of antigen-specific CTL from patients has faced difficulty in efficient expansion in ex vivo culture. To resolve this issue, several groups have proposed that induced pluripotent stem cell technology be applied for the expansion of antigen-specific CTL, which retain expression of the same T-cell receptor as original CTL. However, in these previous studies, the regenerated CTL are mostly of the CD8αα+ innate type and have less antigen-specific cytotoxic activity than primary CTL. Here we report that, by stimulating purified iPSC-derived CD4/CD8 double-positive cells with anti-CD3 antibody, T cells expressing CD8αβ were generated and exhibited improved antigen-specific cytotoxicity compared with CD8αα+ CTL. Failure of CD8αβ T-cell production using the previous method was found to be due to killing of double-positive cells by the double-negative cells in the mixed cultures. We found that WT1 tumor antigen-specific CTL regenerated by this method prolonged the survival of mice bearing WT1-expressing leukemic cells. Implementation of our methods may offer a useful clinical tool. Cancer Res; 76(23); 6839-50. ©2016 AACR.

Protective efficacy and hepatitis B virus clearance in mice enhanced by cell-mediated immunity with novel prime-boost regimens.

In this study, anti-hepatitis B virus (HBV) immunity was evaluated in mice using several regimens of the HBV recombinant protein vaccine HBSS1 that expressed in CHO cells containing S (1-223 aa) and preS1 (21-47 aa) and recombinant adenovirus rAdSS1 vaccine. Further, the protective efficacy of these vaccine regimens was studied in a mouse model. High titres of antigen-specific antibodies and neutralizing activity were elicited in mice after vaccination. However, robust multi-antigen (preS1 and S)-specific cell-mediated immunity (CMI) was only detected in mice primed with HBSS1 and boosted with rAdSS1. Moreover, functional T-cell responses with high levels of cytokines and antigen-specific cytotoxic T-cell responses (CD107a+ CD8+ ) were also detected in the mice. Rapid clearance of hepatitis B surface antigen and HBV DNA in blood and significantly decreased hepatitis B envelope antigen levels were observed in mice immunized with the heterogeneous prime-boost vaccine after hepatitis B virus challenge by hydrodynamic injection (HI) of pCS-HBV1.3. The clearance of HBV correlated well with antigen-specific CMI (Th1 and CTL responses) and cytokine profiles (IFN-γ, TNF-α, IL-2) elicited by vaccination. Taken together, our results might contribute to the development of new human HBV vaccines and a better understanding of the mechanisms underlying immune protection and clearance of hepatitis B virus infection.

T-Cell Therapy Using Interleukin-21-Primed Cytotoxic T-Cell Lymphocytes Combined With Cytotoxic T-Cell Lymphocyte Antigen-4 Blockade Results in Long-Term Cell Persistence and Durable Tumor Regression.

Purpose Peripheral blood-derived antigen-specific cytotoxic T cells (CTLs) provide a readily available source of effector cells that can be administered with minimal toxicity in an outpatient setting. In metastatic melanoma, this approach results in measurable albeit modest clinical responses in patients resistant to conventional therapy. We reasoned that concurrent cytotoxic T-cell lymphocyte antigen-4 (CTLA-4) checkpoint blockade might enhance the antitumor activity of adoptively transferred CTLs. Patients and Methods Autologous MART1-specific CTLs were generated by priming with peptide-pulsed dendritic cells in the presence of interleukin-21 and enriched by peptide-major histocompatibility complex multimer-guided cell sorting. This expeditiously yielded polyclonal CTL lines uniformly expressing markers associated with an enhanced survival potential. In this first-in-human strategy, 10 patients with stage IV melanoma received the MART1-specific CTLs followed by a standard course of anti-CTLA-4 (ipilimumab). Results The toxicity profile of the combined treatment was comparable to that of ipilimumab monotherapy. Evaluation of best responses at 12 weeks yielded two continuous complete remissions, one partial response (PR) using RECIST criteria (two PRs using immune-related response criteria), and three instances of stable disease. Infused CTLs persisted with frequencies up to 2.9% of CD8+ T cells for as long as the patients were monitored (up to 40 weeks). In patients who experienced complete remissions, PRs, or stable disease, the persisting CTLs acquired phenotypic and functional characteristics of long-lived memory cells. Moreover, these patients also developed responses to nontargeted tumor antigens (epitope spreading). Conclusion We demonstrate that combining antigen-specific CTLs with CTLA-4 blockade is safe and produces durable clinical responses, likely reflecting both enhanced activity of transferred cells and improved recruitment of new responses, highlighting the promise of this strategy.