Cytotoxic T Lymphocytes Lines Research Articles

Immunogenicity profile in African green monkeys of a vaccine candidate based on a mutated form of human Interleukin-15

BackgroundInterleukin (IL)-15 is a proinflammatory T-cell growth factor overexpressed in several autoimmune diseases such as rheumatoid arthritis. Our initial strategy to neutralize the increased levels of IL-15 consisted in a vaccine candidate based on the recombinant modified human IL-15 (mhIL-15) mixed with the alum adjuvant. A previous study in non-human primates Macaca fascicularis has shown that vaccination induces neutralizing antibodies against native IL-15, without affecting animal behavior, clinical status, or the percentage of IL-15-dependent cell populations. However, the mhIL-15 used as an antigen was active in the IL-2-dependent cytotoxic T-cell line CTLL-2, which could hinder its therapeutic application. The current article evaluated the immunogenicity in African green monkeys of a vaccine candidate based on IL-15 mutant D8SQ108S, an inactive form of human IL-15.ResultsIL-15 D8SQ108S was inactive in the CTLL-2 bioassay but was able to competitively inhibit the biological activity of human IL-15. Immunization with 200 µg of IL-15 mutant combined with alum elicited anti-IL-15 IgG antibodies after the second and third immunizations. The median values of anti-IL-15 antibody titers were slightly higher than those generated in animals immunized with 200 µg of mhIL-15. The highest antibody titers were induced after the third immunization in monkeys vaccinated with 350 µg of IL-15 D8SQ108S. In addition, sera from immunized animals inhibited the biological activity of human IL-15 in CTLL-2 cells. The maximum neutralizing effect was observed after the third immunization in sera of monkeys vaccinated with the highest dose of the IL-15 mutant. These sera also inhibited the proliferative activity of simian IL-15 in the CTLL-2 bioassay and did not affect the IL-2-induced proliferation of the aforementioned T-cell line. Finally, it was observed that vaccination neither affects the animal behavior nor the general clinical parameters of immunized monkeys.ConclusionImmunization with inactive IL-15 D8SQ108S mixed with alum generated neutralizing antibodies specific for human IL-15 in African green monkeys. Based on this fact, the current vaccine candidate could be more effective than the one based on biologically active mhIL-15 for treating autoimmune disorders involving an uncontrolled overproduction of IL-15.

Hyperprogression of a mismatch repair-deficient colon cancer in a humanized mouse model following administration of immune checkpoint inhibitor pembrolizumab.

Immunotherapy is an established treatment modality in oncology. However, in addition to primary or acquired therapy resistance with immune checkpoint blockade (ICB), hyperprogressive disease (HPD) or hyperprogression (HP) with acceleration of tumor growth occurs in a subset of patients receiving ICB therapy. A validated and predictive animal model would help investigate HPD/HP to develop new approaches for this challenging clinical entity. Using human cytotoxic T-cell line TALL-104 injected intraperitoneally into immunodeficient NCRU-nude athymic mice bearing mismatch repair-deficient (MMR-d) human colon carcinoma HCT116 p53-null (but not wild-type p53) tumor xenograft, we observed accelerated tumor growth after PD-1 blockade with pembrolizumab administration. There was increased colon tumor cell proliferation as determined by immunohistochemical Ki67 staining of tumor sections. There was no increase in MDM2 or MDM4/MDMX in the p53-null HCT116 cells versus the wild-type p53-expressing isogenic tumor cells, suggesting the effects in this model may be MDM2 or MDM4/MDMX-independent. Human cytokine profiling revealed changes in IFN-γ, TRAIL-R2/TNFRSF10B, TRANCE/TNFSF11/RANK L, CCL2/JE/MCP-1, Chitinase 3-like 1, IL-4 and TNF-α. This represents a novel humanized HPD mouse model with a link to deficiency of the p53 pathway of tumor suppression in the setting of MMR-d. Our novel humanized preclinical TALL-104/p53-null HCT116 mouse model implicates p53-deficiency in an MMR-d tumor as a possible contributor to HPD/HP and may help with evaluating therapeutic strategies in cancer immunotherapy to extend clinical benefits of ICB’s in a broader patient population.

Defective Interferon Gamma Production by Tumor-Specific CD8+ T Cells Is Associated With 5'Methylcytosine-Guanine Hypermethylation of Interferon Gamma Promoter.

Interferon gamma (IFNγ) supports effector responses of CD8+ cytotoxic T lymphocytes (CTLs) and is a surrogate marker for detection of antigen-specific T cells. Here, we show that tumor-specific CTL clones have impaired IFNγ expression and production upon activation. Assessment of the relationship between IFNγ production and the 5′methylcytosine-guanine (CpG) dinucleotide methylation of the IFNγ promoter using bisulfite treatment has shown that IFNγ− CTL clones accumulates CpG hypermethylation within the promoter at key transcription factor binding sites (−186 and −54), known to be vital for transcription. We confirmed these findings using ex vivo isolated and short-term expanded bulk tumor-specific CTL lines from four cancer patients and demonstrated that IFNγ methylation inversely correlates with transcription, protein level, and cytotoxicity. Altogether, we propose that a sizeable portion of human tumor-specific CTLs are deficient in IFNγ response, contributed by CpG hypermethylation of the IFNγ promoter. Our findings have important implications for immunotherapy strategies and for methods to detect human antigen-specific T cells.

Identification of a novel HLA-A24-restricted cytotoxic T lymphocyte epitope peptide derived from mesothelin in pancreatic cancer.

Pancreatic cancer involves highly malignant tumors, and the development of new therapeutic strategies is critical. Mesothelin is overexpressed in infiltrating pancreatic cancer cells and plays an important role in the invasion and migration processes. In this study, we focused on mesothelin as a tumor-specific antigen target for a pancreatic cancer vaccine. We first investigated the mesothelin-derived epitope peptide restricted to HLA-A*2402. A total of 19 candidate peptides were synthesized, and we then determined their potential to induce peptide-specific cytotoxic T lymphocytes (CTLs). Peptide-specific CTLs were induced by five peptides derived from mesothelin, and these CTLs successfully exhibited peptide-specific IFN-γ production. After the expansion of each CTL, two CTL lines were established, which were induced by mesothelin-10-5 peptide (AFYPGYLCSL). These CTL lines exhibited peptide-specific cytotoxicity and IFN-γ production. Moreover, we were able to generate mesothelin-10-5 peptide-specific CTL clones. These CTL clones also had specific cytotoxic activity against HLA-A*2402-positive pancreatic cancer cells that endogenously expressed mesothelin. These results indicate that the mesothelin-10-5 peptide is a novel HLA-A*2402 restricted CTL epitope and that it is a promising candidate target for antigen-specific immunotherapy against pancreatic cancers.

Human CTL-based functional analysis shows the reliability of a munc13-4 protein expression assay for FHL3 diagnosis

AbstractFamilial hemophagocytic lymphohistiocytosis (FHL) is the major form of hereditary hemophagocytic lymphohistiocytosis (HLH); as such, it requires prompt and accurate diagnosis. We previously reported that FHL type 3 (FHL3) can be rapidly screened by detecting munc13-4 expression in platelets using flow cytometry; however, the reliability of the munc13-4 expression assay for FHL3 diagnosis is unclear. Regardless of the type of UNC13D mutation, all reported FHL3 cases examined for the munc13-4 protein showed significantly reduced expression. However, the translated munc13-4 protein of some reportedly disease-causing UNC13D missense variants has not been assessed in terms of expression or function; therefore, their clinical significance remains unclear. The aim of this study was to determine the reliability of a munc13-4 expression assay for screening FHL3. Between 2011 and 2016, 108 HLH patients were screened by this method in our laboratory, and all 15 FHL3 patients were diagnosed accurately. To further elucidate whether munc13-4 expression analysis can reliably identify FHL3 patients harboring missense mutations in UNC13D, we developed an alloantigen-specific cytotoxic T lymphocyte (CTL) line and a CTL line immortalized by Herpesvirus saimiri derived from FHL3 patients. We then performed a comprehensive functional analysis of UNC13D variants. Transient expression of UNC13D complementary DNA constructs in these cell lines enabled us to determine the pathogenicity of the reported UNC13D missense variants according to expression levels of their translated munc13-4 proteins. Taken together with previous findings, the results presented herein show that the munc13-4 protein expression assay is a reliable tool for FHL3 screening.

Genetic abrogation of immune checkpoints in antigen-specific cytotoxic T-lymphocyte as a potential alternative to blockade immunotherapy

T cell function can be compromised during chronic infections or through continuous exposure to tumor antigens by the action of immune checkpoint receptors, such as programmed cell death protein 1 (PD-1). Systemic administration of blocking antibodies against the PD-1 pathway can restore T cell function, and has been approved for the treatment of several malignancies, although there is a risk of adverse immune-related side-effects. We have developed a method for generating gene knockouts in human antigen (Ag)-specific cytotoxic T-Lymphocyte (CTLs) using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing. Using this method, we generated several transduced CD4+ or CD8+ antigen-specific polyclonal CTL lines and clones, and validated gene modifications of the PD-1 gene. We compared these T-cell lines and clones with control groups in the presence of programmed death-ligand 1 (PD-L1) and observed improved effector functions in the PD1-disrupted cell group. Overall, we have developed a versatile tool for functional genomics in human antigen-specific CTL studies. Furthermore, we provide an alternative strategy for current cell-based immunotherapy that will minimize the side effects caused by antibody blockade therapy.

Ex Vivo PD-L1/PD-1 Pathway Blockade Reverses Dysfunction of Circulating CEA-Specific T Cells in Pancreatic Cancer Patients.

Purpose: Carcinoembryonic antigen (CEA) is a candidate target for cellular immunotherapy of pancreatic cancer. In this study, we have characterized the antigen-specific function of autologous cytotoxic T lymphocytes (CTL) specific for the HLA-A2-restricted peptide, pCEA691-699, isolated from the peripheral T-cell repertoire of pancreatic cancer patients and sought to determine if ex vivo PD-L1 and TIM-3 blockade could enhance CTL function.Experimental Design: CD8+ T-cell lines were generated from peripheral blood mononuclear cells of 18 HLA-A2+ patients with pancreatic cancer and from 15 healthy controls. In vitro peptide-specific responses were evaluated by flow cytometry after staining for intracellular cytokine production and carboxy fluorescein succinimydyl ester cytotoxicity assays using pancreatic cancer cell lines as targets.Results: Cytokine-secreting functional CEA691-specific CTL lines were successfully generated from 10 of 18 pancreatic cancer patients, with two CTL lines able to recognize and kill both CEA691 peptide-loaded T2 cells and CEA+ HLA-A2+ pancreatic cancer cell lines. In the presence of ex vivo PD-L1 blockade, functional CEA691-specific CD8+ T-cell responses, including IFNγ secretion and proliferation, were enhanced, and this effect was more pronounced on Ag-specific T cells isolated from tumor draining lymph nodes.Conclusions: These data demonstrate that CEA691-specific CTL can be readily expanded from the self-restricted T-cell repertoire of pancreatic cancer patients and that their function can be enhanced by PD-L1 blockade. Clin Cancer Res; 23(20); 6178-89. ©2017 AACR.

Obtention and characterization of the recombinant simian Interleukin-15 in Escherichia coli for the preclinical assessment of an IL-15-based therapeutic vaccine

ABSTRACTRecombinant simian IL-15 (siIL-15) was obtained for the preclinical assessment of an anti-human IL-15 vaccine. For this purpose, the cDNA from peripheral blood mononuclear cells of a Macaca fascicularis monkey was cloned into a pIL-2 vector. The siIL-15 was expressed in Escherichia coli strain W3110 as an insoluble protein which accounted for 13% of the total cellular proteins. Inclusion bodies were solubilized in an 8 M urea solution, which was purified by ion exchange and reverse phase chromatography up to 92% purity. The protein identity was validated by electrospray ionization-mass spectrometry, confirming the presence of the amino acids which distinguish the siIL-15 from human IL-15. The purified siIL-15 stimulates the proliferation of cytotoxic T-lymphocytes line (CTLL)-2 and Kit 225 cells with EC50 values of 3.1 and 32.5 ng/mL, respectively. Antisera from modified human IL-15-immunized macaques were reactive to human and simian IL-15 in enzyme-linked immunosorbent assays. Moreover, the anti-human IL-15 antibodies from immune sera inhibited siIL-15 activity in CTLL-2 and Kit 225 cells, supporting the activity and purity of recombinant siIL-15. These results indicate that the recombinant siIL-15 is biologically active in two IL-15-dependent cell lines, and it is also suitable for the preclinical evaluation of an IL-15-based therapeutic vaccine.

Induction and Analysis of Cytotoxic T-Lymphocytes that Recognize Autologous Oral Squamous Cell Carcinoma.

It is essential that cytotoxic T-lymphocytes (CTLs) recognize tumor-associated antigens (TAAs) and exhibit antitumor effects in immunological responses for tumor rejection. However, only a few cases with oral squamous cell carcinoma (OSCC) have been studied for these antitumor mechanisms. We established a cancer cell line and autologous CTL pair for identifying TAAs in OSCC, despite difficulties in establishing such a pair. A cell line (OTM) from a primary lesion and a CTL line (TcOTM) were derived from OSCC in a 64-year-old female patient. CTL clones were generated by repetitive limiting dilutions. Accurate characterization was performed by in vitro analysis. The TcOTM clone showed specific cytotoxic activity against OTM cells in an human leukocyte antigen (HLA)-A24-restricted manner. Furthermore, it exerted cytotoxicity against the allogenic HLA-A24 cell lines. These data indicated that the TcOTM clone recognized a TAA presented by HLA-A24 cells. This unique method will allow for identification of unknown TAAs for OSCC in the future.

Abstract 3767: Identification of a novel and a shared H3.3K27M mutation derived neoantigen epitope and H3.3K27M specific TCR engineered T cell therapy for glioma

Abstract Brain cancers are the leading cause of cancer related mortality in children and young adults with median overall survival of 9-10 months and hence represents a significant unmet medical need. Genome-wide sequencing efforts of pediatric gliomas have identified a recurrent and shared missense mutation in the gene encoding the replication-independent variant of histone 3, H3.3. Approximately 70% of diffuse intrinsic pontine gliomas (DIPG) and 50% of thalamic and other midline gliomas harbor the amino acid substitution from lysine (K) to methionine (M) at the position 27 of H3.3 gene. Tumor specific missense mutations are not subjected to self-tolerance and can be suitable targets (neoantigens) for cancer immunotherapy. Herein, we evaluated whether the H3.3K27M mutation can induce specific cytotoxic T lymphocyte (CTL) response in HLA-A2+ T cells. In vitro stimulation of HLA-A2+ donor derived CD8+ T cells with a synthetic peptide encompassing the H3.3K27M mutation (H3.3K27M epitope) induced CTL lines which recognized not only T2 cells loaded with the synthetic H3.3K27M epitope peptide but also lysed the HLA-A2+ DIPG cells which endogenously harbor the H3.3K27M mutation. On the other hand, the CTL lines did not react to either HLA-A2+ but H3.3K27M- negative DIPG cell lines or H3.3K27M positive but HLA-A2 negative DIPG cells. The H3.3K27M epitope peptide but not the non-mutant counterpart indicated an excellent binding affinity (Kd 151nM) to HLA-A2 based on competitive binding inhibition assay. From CTL clones with high and specific affinities to HLA-A2-H3.3K27M-tetramer, cDNAs for T cell receptor (TCR) alpha and beta chains were cloned into a retroviral vector. Human HLA-A2+ T cells transduced with the TCR demonstrated antigen specific reactivity as well as anti-glioma responses in vitro. Peptide titration assays suggested that the H3.3K27M specific TCR had the half-maximal reactivity for peptide recognition of around 100nM. Furthermore, critically important for safety of clinical application, alanine scanning demonstrated that the key amino acid sequence motif in the epitope of the TCR reactivity is not shared by any known human protein. Finally, intravenous administration of T cells transduced with H3.3K27M specific TCR significantly inhibited the growth of intracranial HLA-A2+ H3.3K27M positive glioma xenografts in immune deficient NSG mice. These data provide us with a strong basis for developing peptide based vaccines as well as adoptive transfer therapy using autologous T cells transduced with the H3.3K27M specific TCR. Acknowledgements: This study is supported by the NIH/NINDS (1RO1NS096954), V Foundation and Parker Institution for Cancer Immunotherapy. Citation Format: Zinal Chheda, Gary Kohanbash, John Sidney, Kaori Okada, Naznin Jahan, Diego Carrera, Payal Watchmaker, Kira Downey, Shuming Liu, Shruti Shrivastav, Sabine Mueller, Ian F. Pollack, Angel M. Carcaboso, Alessandro Sette, Yafei Hou, Hideho Okada. Identification of a novel and a shared H3.3K27M mutation derived neoantigen epitope and H3.3K27M specific TCR engineered T cell therapy for glioma [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 3767. doi:10.1158/1538-7445.AM2017-3767

Abstract 1704: UPR signaling promotes T-cell dysfunction to prevent immune-mediated cancer cell killing and immune checkpoint therapy resistance

Abstract A critical point in cancer progression is evading recognition by the immune system. Cancer cells accomplish this by stimulating immune checkpoint signals on effector T-cells. In patients with advanced melanoma treated with immune checkpoint inhibitors, 3-year survival increased by 20%. While immune checkpoint therapies are the new first treatment option for advanced melanoma in over a decade, their efficacy is limited because resistance often develops. Understanding the molecular mechanisms of immune checkpoint inhibitor resistance is critical to develop combinatorial drug therapy to potentiate therapeutic responsiveness. The unfolded protein response (UPR) is an endoplasmic reticulum (ER) stress pathway activated when unfolded/misfolded proteins accumulate within the ER. Highly secretory cell types, such as T-cells, have larger ER cell compartments and elevated UPR components to deal with the increased protein synthesis/folding required by these cell types. Therefore, these cell types may be highly sensitive to ER stress. Our data demonstrates elevated UPR signaling components as a driver of T-cell exhaustion/dysfunction. Using a previously established T-cell exhaustion protocol, we stimulated naïve T-cells with antibodies to CD3/CD28 for 5 days and co-cultured them with MDA-MB-231 breast cancer cells, Mun2b melanoma cell line, or CMI patient-derived melanoma cell line. Exhausted T-cells displayed an increased PD-1 and PERK expression, suggesting that UPR signaling is activated during T-cell exhaustion. Treatment of naïve T-cells with DTT, a chemical agent that stimulates ER stress, also induced PD-1 and PERK compared with vehicle-treated T-cells. Gene expression analysis of T-cells indicate that co-culture with cancer cells, not CD3/CD28 activation, elevates T-cell UPR gene expression. Furthermore, induction of ER stress through low-dose DTT treatment decreased cytotoxic T-cell mediated cancer cell death, further supporting our hypothesis of ER stress inducing T-cell exhaustion. Inhibition of PERK by RNAi in TALL-104, a human cytotoxic T-cell line, enhanced T-cell mediated cancer cell clearance when exposed to ER stress-inducing agents, suggesting that PERK may represent a novel target to prevent T-cell exhaustion or restore T-cell effector capabilities. PERK inhibition in the patient-derived melanoma cells did not negatively affect T-cell-mediated killing, suggesting that systemic PERK inhibition may be an effective therapeutic strategy to enhance anti-tumor immune responses. Matched PBMC from melanoma patients before treatment or after ipilimumab therapy resistance indicated increased UPR signaling components in PBMC samples from patients after ipilimumab resistance when compared with PBMC samples before therapy, supporting a novel role UPR signaling in anti-CTLA4 therapy resistance. Citation Format: Yismeilin R. Feliz Mosquea, David R. Soto Pantoja, Adam Wilson, Pierre L. Triozzi, Katherine L. Cook. UPR signaling promotes T-cell dysfunction to prevent immune-mediated cancer cell killing and immune checkpoint therapy resistance [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 1704. doi:10.1158/1538-7445.AM2017-1704

OS09.4 Identification of a novel H3.3.K27M mutation-derived neoantigen epitope and cloning of H3.3.K27M-specific T-cell receptor for T-cell therapy in gliomas

Brain cancers are the leading cause of cancer related mortality and morbidity in children and young adults. Children with diffuse intrinsic brainstem malignant gliomas have one-year progression-free survival rates below 25% and median overall survival of 9 to 10 months with current treatment options and hence represent a significant unmet medical need. Genome-wide sequencing efforts of pediatric gliomas have identified a recurrent and shared missense mutation in the gene encoding the replication-independent variant of histone 3, H3.3. Approximately 70% of diffuse intrinsic pontine gliomas (DIPG) and 50% of thalamic and other midline gliomas harbor the amino-acid substitution from lysine (K) to methionine (M) at the position 27 of H3.3 gene (H3.3.K27M mutation). Tumor-specific missense mutations are not subjected to self-tolerance and can be suitable targets (i.e. neoantigen) for cancer immunotherapy. We evaluated whether the H3.3.K27M mutation can induce specific cytotoxic T lymphocyte (CTL) response in HLA-A2+ human T cells. In vitro stimulation of HLA-A2+ donor-derived CD8+ T-cells with a synthetic peptide encompassing the H3.3.K27M mutation (H3.3.K27M epitope) induced CTL lines which recognized not only T2 cells loaded with the synthetic H3.3.K27M epitope peptide but also lysed HLA-A2+ DIPG cell lines which endogenously harbor the H3.3.K27M mutation. On the other hand, the CTL lines did not react to either HLA-A2+ H3.3.K27M -negative DIPG cell lines or H3.3K27M-positive but HLA-A2-negative cells. The H3.3.K27M epitope peptide, but not the non-mutant counterpart, indicated an excellent affinity (Kd 151nM) to HLA-A2 based on competitive binding inhibition assay. From CTL clones with high and specific affinities to HLA-A2-H3.3.K27M-tetramer, cDNAs for T cell receptor (TCR) α- and β-chains were cloned into a retroviral vector. Human HLA-A2+ T-cells transduced with the TCR demonstrated antigen-specific reactivity as well as anti-glioma responses in vitro. Peptide titration assay suggested that the H3.3.K27M-specific TCR had the half-maximal reactivity for peptide recognition of around 100nM. Furthermore, critically important for safety of clinical application, alanine scanning demonstrated that the key amino-acid sequence motif in the epitope for the TCR reactivity is not shared by any known human protein. Finally, intravenous administration of T-cells transduced with the H3.3.K27M-specific TCR significantly inhibited the growth of intracranial HLA-A2+ H3.3.K27M-positive glioma xenografts in immune-deficient mice. These data provide us with a strong basis for developing peptide-based vaccines as well as adoptive transfer therapy using autologous T-cells transduced with the H3.3.K27M-specific TCR.

Targeting T-Cell Receptor β-Constant Domain for Immunotherapy of T-Cell Malignancies

T-cell lymphomas and leukemias are aggressive, treatment-resistant cancers with poor prognosis. Immunotherapeutic approaches have been limited by a lack of target antigens discriminating malignant from healthy T-cells. While treatment of B-cell cancers has been enhanced by targeting pan B-cell antigens, an equivalent approach is not possible for T-cell malignancies since profound T-cell depletion, unlike B-cell depletion, would be prohibitively toxic. We propose an immunotherapeutic strategy for targeting a pan T-cell antigen without causing severe depletion of normal T-cells.The α/β T-cell receptor (TCR) is a pan T-cell antigen, expressed on >90% of T-cell lymphomas and all normal T-cells. An overlooked feature of the TCR is that the β-constant region comprises 2 functionally identical genes: TRBC1 and TRBC2. Each T-cell expresses only one of these. Hence, normal T-cells will be a mixture of individual cells expressing either TRBC1 or 2, while a clonal T-cell cancer will express TRBC1 or 2 in its entirety.Despite almost identical amino acid sequences, we identified an antibody with unique TRBC1 specificity. Flow cytometry (FACS) of T-cells in normal donors (n = 27) and patients with T-cell cancers (n = 18) revealed all subjects had TRBC1 and 2 cells in both CD4 and CD8 compartments, with median TRBC1 expression of 35% (range 25-47%). In addition, we examined viral-specific T-cells in healthy volunteers, by generation of Epstein Barr virus-specific primary cytotoxic T-cell lines (3 donors) or by identification of cytomegalovirus-specific (3 donors) or adenovirus-specific (5 donors) T-cells by peptide stimulation. We demonstrated similar TRBC1: 2 ratios in viral-specific cells, suggesting that depletion of either subset would not remove viral immunity. Next, using FACS and immunohistochemistry, we showed that TCR+ cell lines (n = 8) and primary T-cell lymphomas and leukemias (n = 55) across a wide range of histological subtypes were entirely restricted to one compartment (34% TRBC1).As proof of concept for TRBC-selective therapy, we developed anti-TRBC1 chimeric antigen receptor (CAR) T-cells. After retroviral transduction of healthy donor T-cells, comprising mixed TRBC1/2 populations, 90% of T-cells expressed CAR on the cell surface. No detectable TRBC1 T-cells remained in the culture, suggesting selective depletion of this population. Anti-TRBC1 CAR T-cells secreted interferon-γ in response to TRBC1-expressing target cell lines (p<0.001) or autologous normal TRBC1+ cells (p<0.001), and not TRBC2 cell lines or autologous normal TRBC2 cells. Anti-TRBC1 CAR killed multiple TRBC1 cell lines (p<0.001) and autologous normal TRBC1 cells (p<0.001), and not TRBC2 cell lines or autologous normal TRBC2 cells. These cell-line based findings were confirmed using primary cells from two patients with TRBC1+ adult T-cell leukaemia/lymphoma. We demonstrated specific tumour kill by allogeneic or autologous T-cells in vitro, despite partial downregulation of surface TCR by tumour cells. We developed a xenograft murine model of disseminated T-cell leukemia by engrafting engineered firefly luciferase+ TRBC1+ Jurkat cells in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Bioluminescent imaging and FACS of marrow at 5 days following IV T-cell injection showed that while mice treated with untransduced T-cells progressed, mice receving anti-TRBC1 CAR T-cells had disease clearance (p<0.0001). In a further model, mice were engrafted with equal proportions of TRBC1-Jurkat and TRBC2-Jurkat cells. FACS analysis of bone marrow at 5 days following T-cell injection demonstrated specific eradication of TRBC1 and not TRBC2 tumour by anti-TRBC1 CAR (p<0.001).In summary, we have demonstrated a novel approach to investigation and targeting of T-cell malignancies by distinguishing between two possible TCR β-chain constant regions. Using CART-cells targeting TRBC1 we have demonstrated proof of concept for anti-TRBC immunotherapy. Unlike non-selective approaches targeting the entire T-cell population, TRBC targeting could eradicate a T-cell tumour while preserving sufficient normal T-cells to maintain cellular immunity. DisclosuresMaciocia:Autolus: Equity Ownership, Patents & Royalties: TRBC1 and 2 Targeting for the Diagnosis and Treatment of T-cell Malignancies. Philip:Autolus: Equity Ownership. Onuoha:Autolus: Employment, Equity Ownership. Pule:Amgen: Honoraria; Roche: Honoraria; UCL Business: Patents & Royalties; Autolus Ltd: Employment, Equity Ownership, Research Funding.

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.

Identification of a novel feline large granular lymphoma cell line (S87) as non-MHC-restricted cytotoxic T-cell line and assessment of its genetic instability

Feline large granular lymphocyte lymphomas are rare but very aggressive tumors with a poor prognosis. In this study, a cell line from an abdominal effusion of a cat with large granular lymphoma was characterized. Immunophenotype staining was positive for CD3 and CD45R, and negative for CD4, CD8, CD56, CD79α, BLA.36 and NK1. A TCR γ gene rearrangement was detectable by PARR. Neither FeLV antigen nor exogenous FeLV provirus could be detected. A chromosomal instability associated with a centrosome hyperamplification could also be determined. The cell line is able to lyse target cells without antigen presentation or interaction with antigen presenting cells. Therefore, these cells were classified as genetically instable non-MHC-restricted cytotoxic T cells with large granular lymphocyte morphology.

The IDO1 selective inhibitor epacadostat enhances dendritic cell immunogenicity and lytic ability of tumor antigen-specific T cells.

Epacadostat is a novel inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1) that suppresses systemic tryptophan catabolism and is currently being evaluated in ongoing clinical trials. We investigated the effects of epacadostat on (a) human dendritic cells (DCs) with respect to maturation and ability to activate human tumor antigen-specific cytotoxic T-cell (CTL) lines, and subsequent T-cell lysis of tumor cells, (b) human regulatory T cells (Tregs), and (c) human peripheral blood mononuclear cells (PBMCs) in vitro. Simultaneous treatment with epacadostat and IFN-γ plus lipopolysaccharide (LPS) did not change the phenotype of matured human DCs, and as expected decreased the tryptophan breakdown and kynurenine production. Peptide-specific T-cell lines stimulated with DCs pulsed with peptide produced significantly more IFN-γ, TNFα, GM-CSF and IL-8 if the DCs were treated with epacadostat. These T cells also displayed higher levels of tumor cell lysis on a per cell basis. Epacadostat also significantly decreased Treg proliferation induced by IDO production from IFN-γ plus LPS matured human DCs, although the Treg phenotype did not change. Multicolor flow cytometry was performed on human PBMCs treated with epacadostat; analysis of 123 discrete immune cell subsets revealed no changes in major immune cell types, an increase in activated CD83+ conventional DCs, and a decrease in immature activated Tim3+ NK cells. These studies show for the first time several effects of epacadostat on human DCs, and subsequent effects on CTL and Tregs, and provide a rationale as to how epacadostat could potentially increase the efficacy of immunotherapeutics, including cancer vaccines.

Abstract A117: Novel and shared neoantigen for glioma T cell therapy derived from histone 3 variant H3.3 K27M mutation

Abstract Malignant gliomas, such as glioblastoma (GBM) and diffuse intrinsic pontine gliomas (DIPG), are lethal brain tumors in both adults and children. Indeed, brain tumors are the leading cause of cancer-related mortality and morbidity in children. Children with DIPG have one-year progression-free survival rates below 25%, and median overall survival of 9 to 10 months with current treatment. Recent genetic studies have revealed that malignant gliomas in children and young adults often show shared missense mutations, which encodes the replication-independent histone 3 variant H3.3. Approximately 30 % of overall GBM and over 70% of DIPG cases harbor the amino-acid substitution from lysine (K) to methionine (M) at the position 27 of H3.3. The H3.3 K27M mutation in DIPG is universally associated with shorter survival compared with patients with non-mutated H3.3. T-cells of the adaptive immune system are normally tolerant to wild-type self-proteins, but can recognize mutated peptide epitopes as non-self. Hence, tumor-specific missense mutations can be suitable targets (i.e. neoantigens) for cancer immunotherapy, such as cancer vaccines and adoptive T cell transfer therapy. We evaluated whether H3.3-derived peptides that encompass the H3.3 K27M mutation can induce specific cytotoxic T lymphocyte (CTL) responses in human leukocyte antigen (HLA)-A2+ CD8+ T-cells. For prediction of HLA-A2-binding epitopes, an algorithm integrating peptide binding to HLA (NetMHC 3.4 server) and a proteosomal cleavage site prediction system (http://paproc.de/) was used. Four candidate peptides encompassing different amino-acid positions around the H3.3 K27M mutation were synthesized, and peptide-specific CTL lines and clones were generated from peripheral blood mononuclear cells of HLA-A2+ donors by in vitro stimulation with each of the synthetic peptides. One of the 4 peptides (the H3.3.K27M epitope, hereafter) induced CTL lines which recognized not only the synthetic peptide loaded on T2 cells but also lysed HLA-A2+ DIPG cell lines which endogenously harbor the H3.3.K27M mutation. On the other hand, CTL lines did not react to HLA-A2+, H3.3 K27M mutation-negative cells or HLA-A2-negative, H3.3 K27M mutation+ cells. Furthermore, CTL clones with high and specific affinities to HLA-A2-H3.3.K27M-tetramer were successfully obtained, and α- and β-chain cDNAs from high-affinity T cell receptors (TCR)s were cloned into a lentiviral vector. Additional studies are underway to determine antigen specificity, key epitope residues in the epitope and possible cross-reactivity to naturally existing variants using T-cells transduced with the lentiviral vector encoding the TCR. Assessments of in vivo immune responses to the epitope peptide and preclinical confirmation for absence of autoimmunity are also underway using HLA-A2-transgenic mice. These data provide us with a strong basis for developing peptide-based vaccines as well as adoptive transfer therapy using autologous T-cells transduced with the TCR. Our experience with conducting immunotherapy trials in these patients will facilitate the translation. Citation Format: Yafei Hou, Gary Kohanbash, Kaori Okada, Shruti Shrivastav, Matthew Smith-Cohn, Theodore Nicolaides, Sabine Mueller, Angel Montero Carcaboso, Ian F. Pollack, Hideho Okada. Novel and shared neoantigen for glioma T cell therapy derived from histone 3 variant H3.3 K27M mutation. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A117.

Abstract B099: Immortalized HMy2/solid tumor hybridomas induce two different tumor antigen-specific T cell clones in vitro

Abstract Introduction: Adoptive T-cell immunotherapy represents a promising approach, as a mean of cancer immunotherapy, for management of different types of malignant tumors. This immunotherapeutic approach has been used with significant clinical benefits in EBV-associated lymphoid disorders, myeloma, and leukemia, in addition to a group of solid tumors. Different studies have demonstrated the probability of generating cytotoxic T-lymphocytes (CTLs) lines to high manufacturing standards, as well as the tolerability and therapeutic benefit of the approach. In our previous studies we verified that the EBV-associated B lymphoblastoid cell line (LCL), HMy2, fusion to hematologic tumor cells and cell lines in vitro produced stable hybridoma cells that grew spontaneously and immortally in tissue culture, expressed relevant tumor partner antigens, and showed an enhanced ability to stimulate allogeneic and semi-autologous T cell responses. Moreover, these studies prospered in the induction of hematologic tumor antigen-specific CTLs in peripheral blood lymphocytes (PBL) from healthy donors, and semi-autologous cancer patients as detected by HLA-A2-peptide pentamer staining and cellular cytotoxicity. In the current study, we aimed to further investigate newly generated solid/HMy2 hybrids for their abilities to stimulate naive HLA-A2+ T cells of healthy donors to induce new relevant TAA-specific, HLA-A2-restricted T cell clones of therapeutic value in management of hepatocellular and pancreatic adenocarcinomas. Materials and methods: Two tumor cell lines (HepG2; HCC cell line, and Panc-1; pancreatic tumor cell line) were used separately in generation of two distinct hybrids by fusion to HMy2, using PEG/ DMSO as fusogen and fusion protocol was followed as mentioned before. Fusion cells' (FCs) growth was selected physically using deferential detachment capabilities and then chemically by 3 days incubation in HAT supplemented growth media (RPMI-1640). Moreover, relevant CD markers and surface TAAs expression of the FCs were investigated to evaluate the fusion efficiency. Hybrid cells' viability under different tissue culture conditions, phenotypic properties (including CD markers, HLAs, and co-stimulatory molecules expression) and relevant TAAs expression profile were assessed using flowcytometric analysis and RT-PCR respectively. Two immunogenic, HLA-A2-restricted and high expressed antigens were selected (GPC3 and CEA for HCC and Panc-1 hybrids respectively) and targeted to induce T cell response against. Subsequently, depending on their phenotype, two FC clones were selected for stimulation of naïve allogeneic T cells, isolated from healthy HLA-A2+ donors, in vitro for several rounds, and the outgrowing T cells were phenotyped and analysed regarding T cell subtype, differentiation pattern, &amp; γ-IFN release, cellular cytotoxic avidity and efficacy as well as HLA-A2-restricted peptide-specificity. Results: The generated hybrid cell lines grew continuously in tissue culture in loosely adherent pattern and expressed CD40, CD80, CD86, HLA class I and HLA class II and the level of expression were higher in the hybrid cell lines than in the parent tumor cells. T cell proliferation assays showed enhanced ability of both fusion clones to stimulate both CD4 and CD8 subtypes of both central and effector cells, however, with slight dominance of effector than central counterparts Two HLA-A2-restricted peptide-specific cytotoxic T cell clones (against HLA-A*02:01-GPC3 144-152 and HLA-A*02:01-CEA 694-702) were produced for the first time using this technique and demonstrated abundant &amp; γ-IFN production and cytotoxic activity upon recognition of their target cells. Conclusion: Hybrid cell lines, such as those described, could be used as cost effective and feasible in vitro stimulators of semi-autologous, antigen-specific CTLs for adoptive T cell immunotherapy for not only hematologic but also for certain solid tumors. Citation Format: Yehia Mohamed, Michael Browning. Immortalized HMy2/solid tumor hybridomas induce two different tumor antigen-specific T cell clones in vitro. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B099.

IMPS-30NOVEL AND SHARED NEOANTIGEN FOR GLIOMA T CELL THERAPY DERIVED FROM HISTONE 3 VARIANT H3.3 K27M MUTATION

Recent genetic studies have revealed that malignant gliomas in children and young adults often show shared missense mutations, which encodes the replication-independent histone 3 variant H3.3. Approximately 30 % of overall glioblastoma (GBM) and over 70% of diffuse intrinsic pontine gliomas (DIPG) cases harbor the amino-acid substitution from lysine (K) to methionine (M) at the position 27 of H3.3. T-cells are normally tolerant to wild-type self-proteins, but can recognize mutated peptide epitopes as non-self. Hence, tumor-specific missense mutations can be suitable targets (i.e. neoantigens) for cancer immunotherapy. We evaluated whether H3.3-derived peptides that encompass the H3.3 K27M mutation can induce specific cytotoxic T lymphocyte (CTL) responses in human leukocyte antigen (HLA)-A2+ CD8+ T-cells. Four candidate peptides encompassing different amino-acid positions around the H3.3 K27M mutation were synthesized, and peptide-specific CTL lines and clones were generated from peripheral blood mononuclear cells of HLA-A2+ donors by in vitro stimulation with each of the synthetic peptides. One of the 4 peptides (the H3.3.K27M epitope, hereafter) induced CTL lines which recognized not only the synthetic peptide loaded on T2 cells but also lysed HLA-A2+ DIPG cell lines which endogenously harbor the H3.3.K27M mutation. On the other hand, CTL lines did not react to HLA-A2+, H3.3 K27M mutation-negative cells or HLA-A2-negative, H3.3 K27M mutation+ cells. Furthermore, CTL clones with high and specific affinities to HLA-A2-H3.3.K27M-tetramer were successfully obtained, and α- and β-chain cDNAs from high-affinity T cell receptor (TCR)s were cloned into a lentiviral vector. Additional studies are underway to determine antigen specificity, key epitope residues in the epitope and possible cross-reactivity to naturally existing variants using T-cells transduced with the lentiviral vector encoding the TCR. These data provide us with a strong basis for developing peptide-based vaccines as well as adoptive transfer therapy using autologous T-cells transduced with the TCR.

Patient-individualized CD8⁺ cytolytic T-cell therapy effectively combats minimal residual leukemia in immunodeficient mice.

Adoptive transfer of donor-derived cytolytic T-lymphocytes (CTL) has evolved as a promising strategy to improve graft-versus-leukemia (GvL) effects in allogeneic hematopoietic stem-cell transplantation. However, durable clinical responses are often hampered by limited capability of transferred T cells to establish effective and sustained antitumor immunity in vivo. We therefore analyzed GvL responses of acute myeloid leukemia (AML)-reactive CD8(+) CTL with central and effector memory phenotype in a new allogeneic donor-patient specific humanized mouse model. CTL lines and clones obtained upon stimulation of naive CD45RA(+) donor CD8(+) T cells with either single HLA antigen-mismatched or HLA-matched primary AML blasts, respectively, elicited strong leukemia reactivity during cytokine-optimized short to intermediate (i.e., 2-8 weeks) culture periods. Single doses of CTL were intravenously infused into NOD/scidIL2Rcg(null) mice when engraftment with patient AML reached bone marrow infiltration of 1-5%, clinically defining minimal residual disease status. This treatment resulted in complete regression of HLA-mismatched and strong reduction of HLA-matched AML infiltration, respectively. Most importantly, mice receiving AML-reactive CTL showed significantly prolonged survival. Transferred CTL were detectable in murine bone marrow and spleen and demonstrated sustained AML-reactivity ex vivo. Moreover, injections with human IL-15 clearly promoted CTL persistence. In summary, we show that naive donor-derived CD8(+) CTL effectively combat patient AML blasts in immunodeficient mice. The donor-patient specific humanized mouse model appears suitable to evaluate therapeutic efficacy of AML-reactive CTL before adoptive transfer into patients. It may further help to identify powerful leukemia rejection antigens and T-cell receptors for redirecting immunity to leukemias even in a patient-individualized manner.