Expansion Of CTLs Research Articles

Comparison of Transplant Donor- and Third-Party Donor-Derived CMV-Specific T Cells for CMV Infection After Allogenic Stem Cell Transplantation

Background: Adoptive transfer of cytomegalovirus (CMV)-specific T cells (CTLs) from original transplant donors or third-party donors was effective for CMV infection after allogenic stem cell transplantation (allo-SCT), but the safety and antiviral activity of CTL types have not been compared. Additionally, mechanisms driving sustained antiviral immunity induced by these CTLs need to be established and compared. Methods: i) We compared antiviral abilities of transplant donor and third-party CTLs for CMV infection in two mouse models, compared the in vivo recovery of CMV-specific immunity and analyzed underlying mechanisms driving sustained antiviral immunity induced by these CTLs therapies. ii) We collected data from 31 patients receiving third-party CTLs and selected matched pairs of 62 individuals who received donor CTLs for refractory CMV infection after allo-SCT, compared the safety and efficacy of the CTLs types for CMV infection, and evaluated the recovery of virus-specific immunity in patients. Findings: i) In mouse models, we observed that both donor and third-party CTLs effectively combated systemic CMV infection by reducing CMV pathology and tumor burden 28 days post-infusion. The in vivo recovery of CMV-specific immunity after CTLs infusion was comparable in both groups. A detailed analysis of the source of recovered CTLs showed the proliferation and expansion of graft-derived endogenous CTLs in both groups. ii) In patients, adoptive therapy with donor or third-party CTLs had comparable clinical responses without significant therapy-related toxicity. We observed strong expansion of CD8 + tetramer + T cells and the proliferation of recipient’s endogenous CTLs after CTLs infusion, which were associated with a reduced or cleared of viral load. Interpretation: Adoptive therapy with transplant donor or third-party CTLs triggered comparable antiviral responses to CMV infection by helping to restore CMV-specific immunity. Funding: National Key Research and Development Program of China, National Natural Science Foundation of China. Declaration of Interest: The authors have no competing financial interests to declare. Ethical Approval: This study was approved by the ethics committee of Peking University People's Hospital

Abstract PR14: Identification of specificity TCR groups of tumor antigen specific T-cells

Abstract A major breakthrough in the treatment of advanced melanoma has been the development and FDA approval of immune checkpoint inhibitors. Approximately 20% of the patients who received anti-CTLA-4 or anti-PD-1 therapy have long-term remissions. At the core of the clinical success lies the fact that cancer patients bear T lymphocytes that recognize tumor-specific antigens. It has been proposed that efficacy of immune checkpoint inhibitors is attributable, at least in part, to diversification of T-cell receptor (TCR) repertoire in peripheral circulation. However, current TCR repertoire analyses mostly rely on counting unique TCR sequences, which does not consider the degenerate nature of TCR recognition, that is, many different TCRs can recognize the same antigen peptide and the same TCR can recognize different antigen peptides. This will create roadblocks to identify recurrent TCR clones that likely recognize the same clinically relevant tumor antigen. To understand how TCR sequences encode its specificity, our lab sequenced several thousands of TCRs from T-cells enriched by peptide-major histocompatibility complex multimer (pMHC)-guided cell sorting. We established a TCR repertoire database that contains our own sequences and published TCRs with known specificity. We used this database as the training dataset for a machine learning algorithm that classified TCRs (mainly TCR beta) based on their similarity and specificity. Although the orginal algorithm—termed Grouping of lymphocyte interactions by paratope hotspots(GLIPH)—was proficient in identifying TCR clusters in a small dataset such as those generated by single-cell TCR sequences, it failed to perform reliably when applied to larger data set generated by high-throughput bulk TCR beta sequences. Most importantly, the reference sequences used by GLIPH 1.0 are insufficient when the targeted TCR dataset is much larger. The new version of GLIPH (GLIPH 2.0) was significantly improved in performance and reliability. As a proof of concept, we applied GLIPH 2.0 to TCR-sequences generated from peripheral T-cells of a cohort of melanoma patients. Each of these patients received one infusion of poly-clonal CTLs specific for HLA-A2/MART1. These autologous MART-specific CTLs were generated by priming with MART1 peptide-pulsed dendritic cells and enriched by pMHC-tetramer-guided cell sorting. MART1 peptide stimulation promotes expansion of CTLs based on TCR clonality. However, the overlap of sequences among these polyclonal CTLs is marginal. These TCR sequences were then analyzed by GLIPH and we found that MART1-specific CTLs showed significant enrichment of CDR3 motifs. As a control, we analyzed peripheral TCR repertoire from a group HLA-A2+ healthy donors and found no significant convergence in TCR sequences. This proves that GLIPH 2.0 can reliably identify TCR convergence and antigen specificity CD3 motifs from large-scale TCR beta repertoires. This algorithm can further facilitate the identification of recurrent tumor antigens in melanoma patients receiving T-cell-based immunotherapy. Citation Format: Liang Chen, Chunlin Wang, Mark Davis. Identification of specificity TCR groups of tumor antigen specific T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR14.

Cytotoxic Immune Response Can be Obtained Earlier in Chronic Phase Chronic Myeloid Leukemia Patients Treated with Dasatinib Than with Other Tyrosine Kinase Inhibitors

Introduction: Generation of BCR-ABL fusion gene by reciprocal translocation of chromosomes 9 and 22 immortalizes hematopoietic stem cells by mechanisms such as activation of the JAK-STAT pathway, translational activation of BCL-XL, and inhibition of DNA repair, thereby leading to chronic myeloid leukemia (CML). Amazing improvement in the prognosis of CML has been achieved since the introduction of tyrosine kinase inhibitors (TKIs). Imatinib, a 1st-generation TKI, and dasatinib and nilotinib, 2nd-generation TKIs, are generally used for chronic phase (CP) CML as induction therapy. However, to date, no consensus about the cessation of TKIs in CP-CML patients has been obtained. We recently reported the case of a CP-CML patient with long-term complete molecular response (MR) after cessation of dasatinib, who has been maintaining memory CTLs with T cell receptor (TCR) clonality (Jo et al. Oncology Letters 15: 2935-2938, 2018). Here, we show that up-regulation of memory CTLs occurs early in dasatinib-treated patients compared with those treated with other TKIs.Methods: We examined the TCR V beta gene repertoire to analyze TCR clonality of CD8-positive T cells in TKI-treated CP-CML patients using flow cytometry.Results: Table 1 summarizes the data comparing patients treated with TKIs including dasatinib (Dasa group) and those treated with TKIs without dasatinib (non-Dasa group). Seven patients were treated with dasatinib only; 7, with imatinib only; 8, with multiple TKIs, including dasatinib; and 1, with multiple TKIs without dasatinib. The median age at first TKI administration was 57 years in the Dasa group and 69 years in the non-Dasa group. No significant statistical difference was observed in age at first TKI administration. The time of TCR clonality assay was significantly earlier in the Dasa group than in the non-Dasa group (P = 0.0013). There was no significant difference in the MR at the time of TCR clonality assay between the 2 groups. Figure 1 shows representative data of the TCR clonality assay of the patients in the non-Dasa group. We defined a TCR V beta gene percentage of 10% and above as being positive for monoclonality in this study. The time of analysis was at 116th month (Mo) after the 1st imatinib administration, and NK cell percentage was 30.2% at that time. TCR monoclonality was observed in neither effector CTLs (upper panel) nor memory CTLs (lower panel), although the patient had gained MR5. Figure 2 shows representative time-course data of the patients in the Dasa group. MR levels were MR4.5 (13th Mo), MR5 (16th Mo), and MR5 (19th Mo). Interestingly, memory CTL clonality with the TCR V beta 20 gene had already been observed in the 13th Mo, and it had been continuously observed in the 16th and 19th Mo. NK cell percentages were less than 24% throughout the observation period. Table 2 summarizes the CTL clonality assay results and NK cell percentages. There was no significant change in the NK cell percentages between the 2 groups. Although no statistical significance was observed in both effector and memory CTL clonality between the 2 groups, it is notable that approximately 73% and 87% positivity of effector and memory CTL clonality was observed in the Dasa group. Approximately 38% and 50% positivity of effector and memory CTL clonality was observed in the non-Dasa group, although the TKI exposure time for this group was significantly longer. Notably, the positive percentages of effector and memory CTL clonality in the non-Dasa group are quite similar to the overall probabilities of maintenance of deep MR reported in various imatinib-stop studies such as the STIM study (Mahon et al. Lancet Oncol 11: 1029-1035, 2010) and the TWISTER study (Ross et al. Blood 122: 515-522, 2013). These results suggest that acquisition of CTL clonality may correlate with treatment-free remission (TFR) in CP-CML patients treated with TKIs.Conclusions: Effector and memory CTL clonality was attained more rapidly and frequently in dasatinib-treated CP-CML patients than in patients treated with TKIs without dasatinib. There was no significant difference in the NK cell percentages. The positive percentages of CTL clonality resembled the percentages of TFR in various TKI-stop studies. These results suggest that the acquisition of CTL clonality may provide long-term remission and TFR to CP-CML patients and that cessation of TKIs should be considered in patients with clonal expansion of memory CTLs, irrespective of NK cells. [Display omitted] DisclosuresJo:Bristol-Myers Squibb: Honoraria.

Abstract 3758: TLR9 agonist SNA-induced innate and adaptive immune responses in tumor microenvironment enhance checkpoint inhibitor antitumor activity in mouse tumor models

Abstract Spherical Nucleic Acids (SNAs) are a novel class of therapeutic agents with oligonucleotides densely packed and radially oriented in 3D format around liposomal nanoparticles. As a result of the 3D-architecture, SNAs exhibit increased cellular uptake and nuclease stability compared with linear oligonucleotides. Checkpoint inhibitors (CPI) have shown antitumor efficacy in a variety of solid and liquid tumors. However, tumors with low immunogenicity and lacking pre-existing T lymphocyte infiltrates (TILs) in the tumor microenvironment (TME) are less responsive to CPI. TLR9 agonists induce potent innate and adaptive immune responses and have been shown to increase TIL and activate APC to secrete IFNs leading to activation and expansion of tumor-specific CTLs in TME increasing effectiveness of CPIs. In the present study, we have evaluated antitumor effects of SNA following intratumoral delivery in A20 lymphoma and CT26 colon cancer models. SNA was also studied in combination with anti-PD-1 or anti-PD-L1 in A20 and with IDO-1 inhibitor in CT26 models. We have also evaluated biomarkers in TME to elucidate the mechanism of SNA-mediated effects in enhancing anti-PD-1 efficacy. Monotherapy of SNA or oral dosing of IDO-1 inhibitor in CT26 tumor model resulted in a tumor growth inhibition (TGI) of up to 80% or 20%, respectively, compared with vehicle. The combination of SNA and IDO-1 inhibitor enhanced TGI up to 90% compared with either monotherapy. Mice bearing A20 tumors were treated with SNA, intraperitoneal dosing of anti-PD-1 or anti-PD-L1 or the combination of SNA/anti-PD-1 or SNA/anti-PD-L1. SNA alone showed potent dose-dependent TGI. Anti-PD-1 or anti-PD-L1 monotherapy resulted in modest TGI compared with vehicle. The combination of SNA with anti-PD-1 or anti-PD-L1 enhanced TGI compared with either monotherapy. FACS analysis of A20 tumor tissue following treatments revealed reduced T-regulatory cells and increased T-effector cells. Moreover, SNA treatment led to increased expression of IFN-inducible genes, ISG15, IRF7, MX1, and IP10 and a CD8 T cell marker CD8A in TME and maintained these gene expression levels when combined with anti-PD-1. These results establish the mode of action of TLR9 agonist SNA in TME in potentiating antitumor effects of anti-PD-1. These data demonstrate that the TLR9 agonist SNA showed potent antitumor activity as a result of increased IFN gene expression coupled with increased CD8+ T cells and decreased T-regulatory cells locally in the tumor and thereby enhancing efficacy of checkpoint inhibitors. A TLR9 agonist SNA, AST-008, is currently in clinical development. Citation Format: SubbaRao Nallagatla, Bart R. Anderson, Richard Kang, Ekambar R. Kandimalla. TLR9 agonist SNA-induced innate and adaptive immune responses in tumor microenvironment enhance checkpoint inhibitor antitumor activity in mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3758.

Using anti-CD38 immunotherapy to enhance anti-tumor T-cell immunity in chronic lymphocytic leukemia (CLL)

Abstract Increased CD38 expression on chronic lymphocytic leukemia (CLL) cells correlates with a negative prognosis, however, its utility as a therapeutic target in this malignancy remains unknown. Our investigations show that the anti-CD38 therapeutic mAb, daratumumab (Dara) induces direct apoptosis of CLL cells and promotes their immune-mediated clearance (through ADCC). Notably, CD38 expression is quite high on a subset of CLL-cells (CD19+/CD38hi/CD24+/IL-10+; B-regulatory-like phenotype), which promote Treg expansion while prohibiting CD8+ cytotoxic T-cells (cTLs). Indeed, immunosuppression is a hallmark of CLL and patients often present with increased number of Tregs and Th2 cells but decreased effector T cells and cTLs. It has recently been reported that CD38hi Tregs are significantly immunosuppressive and when examining this population in peripheral blood mononuclear cells (PBMCs) from CLL patients (n=13), we noted that >50% of CD4+/CD25+/FoxP3+ cells were CD38hi (median MFI ~600). We hypothesized that Dara would kill CD38hi CLL cells à decreased CD38hi Treg formation/direct killing à expansion of cTLs. Indeed, ex-vivo treatment of CLL-patient derived PBMCs showed that Dara was highly lethal to both CD38hi “Breg-like” CLL cells and Tregs; associated with decrease in IL-10, increase in IFN-γ, effector T cells, Th17 subsets and proliferation of cTLs. Altogether this pointed towards recovery of anti-tumor immunity which was further evidenced by enhanced tumor-specific cytolytic activity of cTLs. Our pre-clinical data demonstrates that targeting CD38 in CLL has an anti-tumor effect via 1.) lethal effect on CLL-Bregs and Tregs and 2.) tumor-specific expansion of cTLs, which we will test clinically in an upcoming trial.

Abstract 625: Eradication of cancer cells by T-cell receptor-engineered T cells targeting neoantigens/oncoantigens

Abstract Cytotoxic T lymphocytes (CTLs) play critical roles in cancer-immune responses, and functional characterization of CTLs and their cancer-specific antigens will facilitate cancer immunotherapies. Immunogenic peptides, which can be derived from oncogenic proteins specifically expressed in cancer cells but not expressed in normal organs except testis (oncoantigens), or from peptides with somatic nonsynonymous mutations (neoantigens), are known as good targets for CTLs to eradicate cancer cells. In this study, we aimed to establish a method to efficiently identify oncoantigen/neoantigen-specific CTLs. Firstly, we screened candidate HLA-A2402-restricted oncoantigen/neoantigen peptides by in silico prediction of their binding affinity to MHC class I molecules. We conducted an in-vitro stimulation of CD8 lymphocytes carrying HLA-A24:02 allele by each peptide, and then confirmed clonal expansion of the peptide-specific CTLs by TCR repertoire sequencing analysis, interferon-γ enzyme-linked immunospot (ELISPOT) and/or peptide-HLA multimer assays. After identification of TCR alpha-beta pairs, we conducted retroviral transduction and prepared the TCR-engineered T cells to evaluate their cytotoxic activities against cancer cells. As oncoantigens, we isolated the CTLs for FOXM1 and UBE2T from healthy donors, and found these CTLs showed strong cytotoxicity against HLA-A2402-positive cancer cells expressing target proteins, but not against HLA-unmatched cancer cells. Similarly, the TCR-engineered T cells for FOXM1 and UBE2T showed killing effects for only HLA-A2402-positive cancer cells. Neoantigen-specific TCR-engineered CTLs also exhibited the mutated peptide-specific response, but did not cross-react to the nonmutated peptide. In addition, neoantigen-specific cytotoxicity was observed against HLA-A2402-positive cancer cells expressing the proteins with target somatic mutations. In conclusion, we developed the pipeline to screen possible onconatigens/neoantigens and establish antigen-specific TCR-engineered CTLs from peripheral blood lymphocytes. Our approach provides a promising strategy to develop personalized immunotherapies using onconatigen/neoantigen-reactive TCR-engineered T cells to treat cancer. Citation Format: Tatsuo Matsuda, Taigo Kato, Yuji Ikeda, Matthias Leisegang, Sachiko Yoshimura, Tetsuro Hikichi, Makiko Harada, Makda Zewde, Jae-Hyun Park, Hans Schreiber, Kazuma Kiyotani, Yusuke Nakamura. Eradication of cancer cells by T-cell receptor-engineered T cells targeting neoantigens/oncoantigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 625. doi:10.1158/1538-7445.AM2017-625

Control of cytotoxic T lymphocyte memory development by beta2-adrenergic receptor signaling

Abstract Cytotoxic T lymphocytes (CTLs) play a critical role in immune responses to intracellular pathogens. CTL responses are initiated by T cell receptor signaling, and they can be modulated by secreted factors, such as cytokines and other extracellular signals. Previous research from our lab demonstrated that the acute effector functions of CTLs are regulated by the neurotransmitter norepinephrine (NE) via beta2-adrenergic receptor (ADRB2) signaling in vitro. We now demonstrate that ADRB2 signaling intrinsically modulates in vivo memory development in response to viral infection using an adoptive transfer model. Expansion of ADRB2-deficient CTLs is unaffected during the initial priming phase in response to the model pathogen, vesicular stomatitis virus. However, ADRB2-deficient CTLs contract rapidly and do not yield any detectable pools of memory cells when compared to wild-type counterparts. Transcriptome analysis revealed a variety of differentially expressed genes, both expressed in resting naïve cells as well as in cells obtained during the primary expansion phase of infection. Among them, we found that the expression of the high affinity IL-2Ra (CD25) was reduced during the activation phase in ADRB2-deficient cells, which was confirmed at the protein level by flow cytometry. While IL-2 responsiveness is a key determinant in programming memory cell development, treatment with IL-2/anti-IL-2 complexes during early phases of infection failed to rescue memory formation of ADRB2-deficient cells, despite a restoration of CD25 expression on day 5 post-infection. Future experiments will dissect the role of ADRB2 signaling during CTL development on the capacity to form memory upon antigen exposure.

Local Activation of p53 in the Tumor Microenvironment Overcomes Immune Suppression and Enhances Antitumor Immunity.

Mutations in tumor suppressor p53 remain a vital mechanism of tumor escape from apoptosis and senescence. Emerging evidence suggests that p53 dysfunction also fuels inflammation and supports tumor immune evasion, thereby serving as an immunological driver of tumorigenesis. Therefore, targeting p53 in the tumor microenvironment (TME) also represents an immunologically desirable strategy for reversing immunosuppression and enhancing antitumor immunity. Using a pharmacological p53 activator nutlin-3a, we show that local p53 activation in TME comprising overt tumor-infiltrating leukocytes (TILeus) induces systemic antitumor immunity and tumor regression, but not in TME with scarce TILeus, such as B16 melanoma. Maneuvers that recruit leukocytes to TME, such as TLR3 ligand in B16 tumors, greatly enhanced nutlin-induced antitumor immunity and tumor control. Mechanistically, nutlin-3a-induced antitumor immunity was contingent on two nonredundant but immunologically synergistic p53-dependent processes: reversal of immunosuppression in the TME and induction of tumor immunogenic cell death, leading to activation and expansion of polyfunctional CD8 CTLs and tumor regression. Our study demonstrates that unlike conventional tumoricidal therapies, which rely on effective p53 targeting in each tumor cell and often associate with systemic toxicity, this immune-based strategy requires only limited local p53 activation to alter the immune landscape of TME and subsequently amplify immune response to systemic antitumor immunity. Hence, targeting the p53 pathway in TME can be exploited to reverse immunosuppression and augment therapeutic benefits beyond tumoricidal effects to harness tumor-specific, durable, and systemic antitumor immunity with minimal toxicity. Cancer Res; 77(9); 2292-305. ©2017 AACR.

CD4 T Helper Cells Instruct Lymphopenia-Induced Memory-Like CD8 T Cells for Control of Acute LCMV Infection.

Lymphopenic conditions lead to expansion of memory-like T cells (TML), which develop from naïve T cells by spontaneous proliferation. TML cells are often increased in the elderly population, AIDS patients, and patients recovering from radio- or chemotherapy. At present, it is unclear whether TML cells can efficiently respond to foreign antigen and participate in antiviral immunity. To address this question, we analyzed the immune response during acute low-dose infection with lymphocytic choriomeningitis virus-WE in T cell lymphopenic CD4Cre/R-diphtheria toxin alpha (DTA) mice in which most peripheral T cells show a TML phenotype. On day 8 after infection, the total number of effector T cells and polyfunctional IFN-γ and TNF-α producing CD8 T cells were three- to fivefold reduced in CD4Cre/R-DTA mice as compared to controls. Viral clearance and the humoral immune response were severely impaired in CD4Cre/R-DTA mice although CTLs efficiently killed transferred target cells in vivo. Transfer of naïve CD4 T cells but not anti-PD-L1 blockade restored the expansion of antigen-specific polyfunctional CD8 T cells and resulted in lower viral titers. This finding indicates that under lymphopenic conditions endogenous CD4 TML cell lack the capacity to promote expansion of CTLs. However, CD8 TML cells retain sufficient functional plasticity to participate in antiviral immunity in the presence of appropriate help by fully functional CD4 T cells. This capacity might be exploited to develop treatments for improvement of CD8 T cell functions under various clinical settings of lymphopenia.

Clinical Phase II Study of WT1 Peptide Vaccination in Pediatric Leukemia Patients after Stem Cell Transplantation and Analysis of Immunological Monitoring

IntroductionAllogeneic stem cell transplantation (allo-SCT) is a radical treatment for pediatric patients with relapsed and/or chemotherapy-resistant leukemia. Because these leukemia cells seem refractory to chemotherapy and irradiation, immunotherapy are needed. WT1 is highly expressed in pediatric leukemia and essential for leukemia cell growth, leading to the hypothesis that the WT1 protein-based immunization after allo-SCT may improve the clinical outcome of the SCT therapy. We investigated the clinical efficacy and immunological effect of immunotherapy targeting WT1 protein for pediatric patients after allo-SCT. MethodMajor inclusion criteria were as follows: Patients with HLA-A*2402 and/or HLA-*A0201 aged 20 years or younger and WT1 mRNA expression in leukemic cells; donors with HLA-A*2402 and/or HLA-*A0201. The HLA A*2402 restricted 9mer modified WT1 peptide or HLA A*0201 restricted natural WT1 peptide emulsified in Montanide ISA 51 adjuvant was injected intradermally. The dose of WT1 peptide depended on patient weight. The vaccinations were scheduled to be given weekly for 12 consecutive weeks, and if no recurrence and severe adverse effect were observed, vaccination was continued. Absolute number and frequency of WT1 specific cytotoxic T lymphocytes were analyzed by WT1 tetramer assay.ResultsA total of 18 patients with 9 AML/MDS, 8 ALL and 1 NHL were enrolled. Seven patients were those with high risk (HR) of relapse who had received SCT at 3rd CR or on disease. The other 11 patients were at standard risk (SR) but had either relapsed after SCT or shown induction failure. 16 had HLA-A*2402 and the other 2 had HLA-A*0201. 12 of the 18 patients received WT1 peptide vaccination monthly after the first 12-time consecutive vaccinations with one-week interval and are still in complete remission for 16-72 months (median 31 mo.). One patient discontinued the vaccination because of pancreatitis due to treatment with steroid for GVHD. One year probabilities of PFS is 66.7% in all, and it is notable that 4 of 7 HR patients are maintained in long-term CR. Although, of these 12 patients with persistence of CR, seven patients had high WT1 mRNA level before vaccination in peripheral blood (PB) or bone marrow (BM), WT1 mRNA levels decreased to within normal level after vaccination. WT1-specific CTLs in PB were detected and increased after vaccination in all cases and absolute numbers of these cells were significantly increased after vaccination in all cases (p< 0.05). The fold increase of WT1-specific CTL numbers after the vaccination was significantly higher in cases which maintained CR (p< 0.05). The WT1-specific CTLs with high tetramer staining were detected during the first 12-time vaccinations in 14 patients, and of these patients, 12 patients are still in CR. In the other 4 patients, only very few WT1 specific CTLs were detected, and three of the four patients showed recurrence. Five patients (1AMKL, 4 ALL) had recurrence. There was no significant difference in the fold increase of WT1-specific CTL numbers after the vaccination between cases with immunosuppressive agents for the prevention of GVHD and those without the agents.DiscussionWe reported the interim result of the phase II clinical study of WT1 peptide immunotherapy after SCT for pediatric patients, in which promising clinical efficacy of the therapy was suggested. In addition, we found that an increase in WT1-specific CTLs was associated with a decrease in WT1 mRNA, suggesting occurrence of WT1-driven graft versus leukemia (GVL) effect, which is consistent with strategies to enhance leukemia antigen-specific GVL response without deterioration of GVHD. The following three mechanisms are suggested. 1) After SCT, many kinds of cytokines are produced and proliferation of T lymphocytes occur; 2) lymphodepletion after SCT allows development of T cells specific to WT1 peptide; and resultantly, 3) efficient homeostatic expansion of WT1-specific CTLs is induced. Post-SCT vaccination with WT1 peptide can be a safe and effective strategy to prevent the recurrence of the disease without deterioration of GVHD in pediatric hematological malignancy. For avoiding late side effect due to preconditioning regimen and GVHD, effective post SCT immunotherapy is a promising strategy for pediatric patient. Larger studies are warranted on application of WT1 targeted immunotherapy to prevent recurrence after pediatric SCT DisclosuresNo relevant conflicts of interest to declare.

Initial IL7R signaling regulate the induction of effector CD8+ T cells and subsequent anti-tumor immune response in lymphopenic host (CCR3P.208)

Abstract Induction of lymphopenia and adoptive transfer of T cells are followed by lymphopenia-induced proliferation (LIP), and generated a anti-tumor immune response. However, the distinct role of redundant IL7 in lymphopenic host and subsequent LIP of T cells for anti-tumor immune response has been unclear, since multiple mechanisms are involved in the anti-tumor immune response under lymphopenic conditions. To clarify the correlation between LIP and the anti-tumor effect, LIP was inhibited by IL7R blockade with anti-IL7Ra mAb at various stages, and the anti-tumor effect was assessed. When LIP was occurred, the growth of transplanted LLC expressing model antigen gp33, was markedly regressed. Using IL7R blockade with mAb at various stages, we found that IL7R signaling at the start of LIP is crucial for the anti-tumor immune response. Although some reports focused on the benefit of IL7 for the improvement of effector T cell function, later IL7R blockade did not arrest the expansion of CTLs, and anti-tumor effect was occurred. IFN-g production also depend on IL7R signaling during induction phase, but not later phase, and, the expression of co-inhibitory molecules, PD-1, on CTLs was unchanged in IL7R blockade hosts. In conclusion, our results demonstrated that initial IL7R signaling and subsequent LIP regulated the expansion of IL7Ra+ effector precursors mainly, but that the effector function of CTL precursors and migration into tumor did not require IL7R signaling.

Highlights of This Issue

In clinical studies, the MET, VEGFR2, and RET inhibitor cabozantinib induced marked responses based on bone scans of men with metastatic castration-resistant prostate cancer. Dai and colleagues explored the mechanisms underlying cabozantinib's effects on prostate cancer bone metastases utilizing a combination of in vitro studies and in vivo murine models. Cabozantinib exhibited both direct antitumor effects as well as a unique impact on important components of the bone microenvironment: osteoblasts and osteoclasts. These results indicate that simultaneously targeting the tumor and both key cell types in the bone microenvironment can have an important therapeutic impact on prostate cancer bone metastases.The PI3K pathway is de-regulated in multiple ways in NSCLC. However, the role of PI3Kβ and its relation to PTEN, a negative regulator of the PI3K pathway, in NSCLC remains unclear. Using an immunohistochemistry approach utilizing two independent patient cohorts, Cumberbatch and colleagues have demonstrated that PI3Kβ protein expression level is significantly higher in NSCLC with squamous histology, and this higher expression is significantly inversely correlated with the expression of PTEN. These data identify for the first time a subset of NSCLC patients for whom inhibitors of PI3Kβ may be predicted to achieve the greatest clinical benefit.The signalling pathway of SDF-1α and CXCR7 in tumor development and metastasis is only marginally understood in contrast to the interplay of SDF-1α with its second receptor CXCR4. Heckmann and colleagues show that stimulation by SDF-1α although inducing invasion of colon cancer cells with lentiviral CXCR7- or CXCR4-overexpression evoked a completely different and partly antidromic gene expression pattern in both cell entities. Furthermore, SDF-1α stimulation resulted in increased sensitivity against 5-FU of CXCR4 cells; CXCR7 cells were more chemoresistant. These opposing results for CXCR4- or CXCR7-overexpressing cells demand an unexpected attention in the clinical application of chemokine receptor antagonists.Radiation therapy (RT) is widely used to treat various solid cancers, but in addition to its antitumor effects, it recruits immunosuppressive cells. To counteract this effect, we used a combination treatment of RT and intratumoral antigenic peptide delivery. We found that the adjuvant effect generated by RT was sufficient for intratumoral vaccination to elicit priming and expansion of antigen-specific CTLs. This led to CTL-mediated killing of the immunosuppressive stromal cells in tumors, resulting in synergistic therapeutic antitumor effects. These data serve as an important foundation for future clinical translation of RT combined with a therapeutic HPV vaccine to control cancer.

Enhanced Cancer Radiotherapy through Immunosuppressive Stromal Cell Destruction in Tumors

Radiotherapy kills cancer cells by causing DNA damage, and stimulates a systemic antitumor immune response by releasing tumor antigen and endogenous adjuvant within the tumor microenvironment. However, radiotherapy also induces the recruitment of immunosuppressive myeloid cells, which can interfere with the antitumor immune responses elicited by apoptotic tumor cells. We hypothesized that local delivery of vaccine following radiotherapy will lead to the priming of antigen-specific CTL immune responses and render immunosuppressive myeloid cells susceptible to killing by the activated CTLs. Using several antigenic systems, we tested whether intratumoral injection of antigenic peptide/protein in irradiated tumors would be able to prime CTLs as well as load myeloid cells with antigen, rendering them susceptible to antigen-specific CTL killing. We show that by combining radiotherapy and targeted antigenic peptide delivery to the tumor, the adjuvant effect generated by radiotherapy itself was sufficient to elicit the priming and expansion of antigen-specific CTLs, through the type I IFN-dependent pathway, leading to synergistic therapeutic antitumor effects compared with either treatment alone. In addition, using two different types of transgenic mice, we demonstrated that CTL-mediated killing of stromal cells in tumors by our approach is important for tumor control. Finally, we confirmed the efficacy of this approach in our preclinical model using two clinically tested therapeutic human papilloma virus (HPV) vaccines. These data serve as an important foundation for the future clinical translation of radiotherapy combined with a clinically tested therapeutic HPV vaccine for the control of HPV-associated cancers.

IL-12 is required for mTOR regulation of memory CTLs during virus infection (P6232)

Abstract Inflammatory cytokines are critical in the induction of memory CTLs, and inhibition of mTOR by rapamycin can enhance memory CTL generation. However, it is unknown if important inflammatory cytokines such as IL-12 are involved in the regulation of memory CTLs by mTOR during infection. We show here that inhibition of mTOR by rapamycin represses CTLs expansion and enhances memory CTL generation during vaccinia virus infection in mice. In addition, rapamycin promotes central memory phenotype in CTLs. However, the absence of IL-12 signal in CTLs diminishes rapamycin regulation on both CTL expansion and subsequent memory CTL formation, despite that rapamycin substantially increases memory CTLs when IL-12 signal is missing. Moreover, rapamycin-regulated memory CTLs in the absence of IL-12 are impaired in secondary expansion upon pathogen challenge, and subsequently form abolished secondary memory. In conclusion, IL-12 contributes to the strength of rapamycin regulation on primary memory CTLs, and lack of IL-12 signal leads to impaired secondary expansion of memory CTLs. Thus, the IL-12 signal is required for mTOR regulation on functional memory CTLs.

Abstract 4576: Immunization with CPG in combination with MHC class I and class II peptides stimulates rapid and strong tumor antigen-specific CTL responses in melanoma patients.

Abstract There is now ample evidence that the toll-like receptor 9 ligand CPG/PF-3512676 represents a potent adjuvant for peptide/protein-based cancer vaccines capable of stimulating ex vivo detectable tumor antigen (TA)-specific CD8+ T cell responses in patients with advanced melanoma. Although CD4+ T cells appeared to promote the expansion and functions of CD8+ T cells in experimental models, melanoma-associated helper peptides have been shown to paradoxically decrease CD8+ T cell responses to a multipeptide melanoma vaccine emulsified in Montanide, possibly by inducing antigen-specific Tregs. To further investigate this question, we have performed one Phase I study of immunization where patients with advanced melanoma were immunized with CPG, Montanide and the HLA-A2-restricted analog peptide NY-ESO-1 157-165V either alone (Arm 1, n=5) or in combination with the pan-DR CD4 helper peptide NY-ESO-1 119-143 (Arm 2, n=7). Patients in both arms exhibited a rapid increase of NY-ESO-1-specific CD8+ T cells that were detectable ex vivo by flow cytometry. Strikingly, the mean frequency of vaccine-induced NY-ESO-1-specific CD8+ T cells after 6 biweekly immunizations was five times higher in patients who also received the helper peptide. Melanoma patients immunized with the helper peptide (Arm 2) developed a rapid increase of Foxp3− NY-ESO-1-specific CD4+ T cells that produced Th-1 type cytokines and IL-21 ex vivo. As compared to Arm 1, melanoma patients included in Arm 2 developed vaccine-induced NY-ESO-1-specific CD8+ T cells that were more differentiated, produced more IFN-γ and exhibited higher cytolytic functions in ex vivo assays. Collectively, our data support the inclusion of well-defined MHC class II peptides in combination with CPG/PF-3512676 and MHC class I peptides in multipeptide melanoma vaccines to better augment the expansion, differentiation, IFN-γ production and lytic function of TA-specific CTLs in patients with advanced melanoma. This work was supported by the National Institutes of Health/National Cancer Institute grants R01CA90360 and R01CA157467 (to H.M. Zarour). Citation Format: Julien Fourcade, Zhaojun Sun, Ornella Pagliano, Joe-Marc Chauvin, Cindy Sander, Stergios Moschos, Ahmad Tarhini, Hussein Tawbi, John M. Kirkwood, Hassane M. Zarour. Immunization with CPG in combination with MHC class I and class II peptides stimulates rapid and strong tumor antigen-specific CTL responses in melanoma patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4576. doi:10.1158/1538-7445.AM2013-4576

Transduction of Malignant Plasma Cells with Three Costimulatory Molecules (TRICOM) Elicits Myeloma-Specific Immune Response in Vitro – a Promising Strategy for Immunotherapy

Abstract 1908 Introduction:Tumor vaccines hold promise as a means of eliciting anti-myeloma immunity and controlling disease that may be resistant to chemotherapy and biologic therapy. We have developed a whole cell tumor vaccine, whereby patient derived plasma cells are transduced with an attenuated vaccinia vector that contains transgenes for the costimulatory molecules B7.1 (CD80), ICAM-1 (CD54), and LFA-3 (CD58), designated TRIad of COstimulatory Molecules (TRICOM). In this manner, a broad array of tumor antigens, including those which may be specific to a given patient, are presented in the context of costimulatory molecules that have been shown to be synergistic in the stimulation of the effector T-cells. In the present study, we evaluated the phenotype and functional characteristics of TRICOM transduced primary myeloma cells. Methods and results:Plasma cells were isolated from bone marrow aspirates obtained from patients with multiple myeloma following Ficoll density centrifugation. Bone marrow derived mononuclear cells were infected with a replication-defective poxviral vector, the modified vaccinia Ankara strain (MVA), encoding TRICOM, or a control empty MVA vector. The expression of costimulatory molecules was assessed using flow cytometric analysis 3 hrs following viral infection. Viral transduction using the TRICOM vector at the dose of 20 MOI (multiplicity of infection) increased the mean percentage of CD38+ cells expressing CD80, CD54 and CD58 from a minimal baseline level (below 5%) to 70%, 56% and 47%, respectively (n=4). Transduction with control MVA vector did not augment expression of costimulatory molecules on plasma cells (mean percent expression of CD80, CD54 and CD58 of 2.6%, 2.7% and 3.8%, respectively, n=4). Of note, compared to CD38+ plasma cells, the CD38 negative fraction of bone marrow derived mononuclear cells demonstrated a significantly lower TRICOM transduction efficiency (mean percent expression of CD80, CD54 and CD58 of 16%, 17% and 16%, respectively, n=4, p<0.05 compared to CD38+ plasma cells).The ability of MVA-TRICOM transduced plasma cells to stimulate autologous T cell populations in vitro was assessed. Patient derived T-cells were purified from the non-adherent portion of PBMC by magnetic bead separation. MVA-TRICOM or empty MVA vector infected plasma cells were irradiated with 20Gy and co-cultured with autologous T cells at a 10:1 ratio of effector cells to vaccine for 7 days. MVA-TRICOM transduced plasma cells potently stimulated activated T cell responses, as assessed by the percentage of CD4+/CD25+/CD69+ T-cells (mean 7.8% of activated T-cells with TRICOM vaccine vs. 2.7% with control vaccine, n=3, p<0.05). In contrast, vaccine stimulation did not result in regulatory T-cell expansion, assessed as the percentage of cells co-expressing CD4,CD25 and FoxP3 (2.4% vs. 2.3%, for TRICOM and control vaccine, respectively, n=3). In concert with these findings, vaccine stimulation resulted in a polarization towards Th1 cytokine secretion, with 7.9% of CD4+ T-cells expressing intracellular IFN-γ after stimulation with TRICOM vaccine as compared to 5.4% after stimulation with the control vaccine (n=3, p<0.05). To further assess the expansion of tumor specific T cell populations, the ability of vaccine stimulated T cells to kill autologous tumor was assessed in a cell-based fluorogenic cytotoxicity assay. MVA-TRICOM transduced plasma cells potently stimulate the expansion of myeloma specific CTLs with the capacity to lyse autologous tumor targets. Mean CTL lysis was 20% and 8% for vaccine stimulated and unstimulated T cells respectively (n=2). Conclusions:Malignant plasma cells transduced with MVA-TRICOM strongly express costimulatory molecules, and potently stimulate activated, tumor reactive T cell populations. This preclinical data serves as a platform for developing a phase 1 clinical trial evaluating the use of MVA-TRICOM transduced autologous plasma cells in patients with multiple myeloma. Disclosures:No relevant conflicts of interest to declare.

Dendritic Cell Editing by Activated Natural Killer Cells Results in a More Protective Cancer-Specific Immune Response

Over the last decade, several studies have extensively reported that activated natural killer (NK) cells can kill autologous immature dendritic cells (DCs) in vitro, whereas they spare fully activated DCs. This led to the proposal that activated NK cells might select a more immunogenic subset of DCs during a protective immune response. However, there is no demonstration that autologous DC killing by NK cells is an event occurring in vivo and, consequently, the functional relevance of this killing remains elusive. Here we report that a significant decrease of CD11c+ DCs was observed in draining lymph nodes of mice inoculated with MHC-devoid cells as NK cell targets able to induce NK cell activation. This in vivo DC editing by NK cells was perforin-dependent and it was functionally relevant, since residual lymph node DCs displayed an improved capability to induce T cell proliferation. In addition, in a model of anti-cancer vaccination, the administration of MHC-devoid cells together with tumor cells increased the number of tumor-specific CTLs and resulted in a significant increase in survival of mice upon challenge with a lethal dose of tumor cells. Depletion of NK cells or the use of perforin knockout mice strongly decreased the tumor-specific CTL expansion and its protective role against tumor cell challenge. As a whole, our data support the hypothesis that NK cell-mediated DC killing takes place in vivo and is able to promote expansion of cancer-specific CTLs. Our results also indicate that cancer vaccines could be improved by strategies aimed at activating NK cells.

Abstract 3508: Zoledronate induce expansion of glypican-3 (GPC3) peptide specific cytotoxic T lLymphocytes sufficient for adoptive cancer immunotherapy

Abstract BACKGROUND Glypican-3 (GPC3) is an onco-fetal antigen which is overexpressed in human hepatocellular carcinoma (HCC), and is only expressed in the placenta and embryonic liver among normal tissues. Previously, we completed a phase I clinical trial of HLA-A2-restricted GPC3144-152 (FVGEFFTDV) and A24-restricted GPC3298-306 (EYILSLEEL) peptide vaccine in 33 patients with advanced HCC. This clinical trial of GPC3-derived peptide vaccine showed the vaccination to be safe, and indicates many immunological responses. To develop more efficient immunotherapy, we investigated large scale expansion of GPC3 peptide specific CTLs for adoptive immunotherapy. PURPOSE In this study, we investigated the efficiency of new method to induce expansion of GPC3 peptide specific CTLs. METHODS Treatment of human peripheral blood mononuclear cells (PBMCs) with zoledronate is a method that enables large-scale γδ T cell expansion. To induce expansion of GPC3 peptide specific CTLs and γδ T cells, the PBMCs of patients vaccinated with GPC3 peptide were cultured with GPC3 peptide and zoledronate for 14 days. We used CD8+ and CD8− cells that isolated from cultured cells using CD8 microbeads at day 14 as a effector cells. GPC3 peptide specificity and cytotoxic activity of CTLs were analysed by Dextramer assay, cytotoxicity assay and IFN-γ ELISPOT assay. We used human liver cancer cell line SK-Hep-1 (GPC3−, HLA-A*02:01/24:02) and a human GPC3 gene transfectant, SK-Hep-1 [[Unsupported Character - &amp;#8260;]] hGPC3 (GPC3+, HLA-A*02:01/24:02) as target cells. T2 (HLA-A*02:01, TAP−) was pulsed with GPC3 peptide or HIV peptide at room temperature for 1h. To evaluate antitumor activity in vivo, we inoculated SK-Hep-1/hGPC3 and SK-Hep-1/vec cells at the right flank of NOD/SCID mice. We injected the CD8+, CD8− or all cells as effector cells intravenously. RESULTS The expansion of Lymphocytes from a few PBMCs of vaccinated patients with advanced HCC using zoledronate yields cell numbers sufficient for adoptive transfer. The rate of increase of GPC3 specific CTLs was approximate 490 to 170,000-fold, whereas the rate of increase of total cell number was approximate 13 to 1,000-fold. These CD8+ cells including CTLs showed GPC3 specific cytotoxicity against SK-Hep-1/hGPC3 and T2 pulsed with GPC3 peptide, but not against SK-Hep-1/vec and T2 pulsed with HIV peptide, whereas CD8− cells including γδ T cells showed cytotoxicity against SK-Hep-1/hGPC3 and SK-Hep-1/vec while did not show GPC3 specificity. Furthermore, adoptive cell transfer of CD8+ cells, CD8− cells and total cells after expansion significantly inhibited tumor growth in NOD/SCID mouse model. CONCLUSION This study indicates that zoledronate is useful to large-scale expand antigen-specific CTLs and γδ T cells for adoptive immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3508. doi:1538-7445.AM2012-3508

Improved Efficacy of Dendritic Cell–Based Immunotherapy by Cutaneous Laser Illumination

The present study investigates a convenient laser-based approach to enhance dendritic cell (DC) migration and improve DC-based immunotherapy in murine models. Influence of laser illumination on dermal tissue microenvironment and migration of DCs following intradermal injection were determined by whole-mount immunohistochemistry, transmission electron microscope, and flow cytometry. We also investigated in vivo expansion of CTLs by flow cytometry, CTL activity by in vitro CTL assay, and antitumor efficacy of DC immunization following cutaneous laser illumination in both preventive and therapeutic tumor models. Laser illumination was found to significantly enlarge perforations in the perilymphatic basement membrane, disarray collagen fibers, and disrupt cell-matrix interactions in the dermis. The altered dermal tissue microenvironment permitted more efficient migration of intradermally injected DCs from the dermis to the draining lymph nodes (dLN). Laser illumination also slightly but significantly enhanced the expression of costimulatory molecule CD80 and MHC I on inoculated DCs. As a result, more vigorous expansion of tumor-specific IFN-γ(+)CD8(+) T lymphocytes and enhanced CTL activity against 4T1 but not irrelevant tumor cells were obtained in the laser-treated group over the control group. Laser-augmented DC immunization also completely abrogated early growth of 4T1 tumor and B16F10 melanoma in preventive tumor models and significantly extended the survival of 4T1-resected mice in a therapeutic tumor model. These data suggest a simple, safe, laser-based approach to significantly enhance DC-based immunotherapy.

Role of IL-15 in the Stimulation of Human CD8+ CMV-Reactive Human T Cells in a Hu-PBL Mouse Model Preconditioned with Self-Differentiated Human Dendritic Cells Programmed with Lentiviral Vectors

Abstract 2966IL-15 is known to be a key factor in the homeostatic stimulation of T cells and maintenance of central memory lymphocytes involved in long-lasting immune responses. In addition, IL-15 may enhance NK cell function to protect against infection and relapse after allo-HCT. Thus, IL-15 ectopic overexpression provided by dendritic cell (DC) immunotherapy after bone marrow transplantation or donor lymphocyte infusion may direct prompt and durable immune responses against leukemia relapse or viral reactivation.Here, we show that overnight transduction of monocytes with tricistronic lentiviral vectors (LV) co-expressing GM-CSF/IL-4/IL-15 induced their differentiation into highly viable DC that expressed the major DC markers CD209 (75%), CD86 (95%), HLA-DR (90%) for up to 21 days in culture. These so-called SmartDC/IL-15 maintained stable secretion of several inflammatory and Th1 cytokines (TNF α, MCP-1, IL-1 β, IL-8). A single in vitro priming of T cells obtained from CMV seropositive donors with autologous SmartDC/IL-15 loaded with CMV-pp65 peptides led to expansion of higher frequencies pp65 tetramer-positive CTLs as compared with SmartDC that were not engineered to express IL-15 (26.5% vs. 16.5% respectively). Importantly, IL-15 expression in Smart-DCs led to the enrichment of T cells with effector memory (80% vs. 65%) and central memory (8% vs. 6%) as compared with SmartDC controls. SmartDCs/IL-15 presented high viability in vivo (>6 weeks) in immune deficient NOD.Rag1—/—.IL2r γ—/— (NRG) mice. We further evaluated the effects of SmartDCs/IL-15 on lymphatic and anti-CMV immune reconstitution in a humanized peripheral blood lymphocyte (Hu-PBL) mouse model. NRG mice preconditioned with unloaded or pp65-loaded SmartDCs/IL-15 were infused 7 days later with autologous luciferase-labeled T cells for optical imaging analysis. At day 21 after fLUC-T cells infusion, mice injected with pp65-loaded SmartDCs/IL-15 showed higher bioluminescence signal from expanded T cells in spleens (3-fold) and injection site (2-fold) as compared with control SmartDCs. Human T cells recovered from spleens of SmartDC-IL15 showed enhanced frequencies of effector memory (58% vs. 30%) and central memory (5.6% vs. 4%) phenotype as compared with SmartDC controls.Remarkably, bioluminescent T cells co-localized with the site of SmartDC/IL-15 injection in a pp65-dependent manner and enhanced the bio-distribution of human T cells towards other rudimentary lymphatic structures found in NRG mice. Anti-pp65 reactivity in vivo was confirmed by flow cytometry analyses by tetramer staining in peripheral blood. Thus, SmartDCs/IL-15 induced accelerated anti-CMV immune responses and triggered lymphatic regeneration and repopulation in vivo, which may be explored clinically to enhance homeostatic and antigen-dependent immune reconstitution after transplantation. Disclosures:No relevant conflicts of interest to declare.