Antigen-specific Killing Research Articles

A novel antibody-TCR (AbTCR) platform combines Fab-based antigen recognition with gamma/delta-TCR signaling to facilitate T-cell cytotoxicity with low cytokine release

The clinical use of genetically modified T-cell therapies has led to unprecedented response rates in leukemia and lymphoma patients treated with anti-CD19 chimeric antigen receptor (CAR)-T. Despite this clinical success, FDA-approved T-cell therapies are currently limited to B-cell malignancies, and challenges remain with managing cytokine-related toxicities. We have designed a novel antibody-T-cell receptor (AbTCR) platform where we combined the Fab domain of an antibody with the γ and δ chains of the TCR as the effector domain. We demonstrate the ability of anti-CD19-AbTCR-T cells to trigger antigen-specific cytokine production, degranulation, and killing of CD19-positive cancer cells in vitro and in xenograft mouse models. By using the same anti-CD19 binding moiety on an AbTCR compared to a CAR platform, we demonstrate that AbTCR activates cytotoxic T-cell responses with a similar dose-response as CD28/CD3ζ CAR, yet does so with less cytokine release and results in T cells with a less exhausted phenotype. Moreover, in comparative studies with the clinically validated CD137 (4-1BB)-based CAR, CTL019, our anti-CD19-AbTCR shows less cytokine release and comparable tumor inhibition in a patient-derived xenograft leukemia model.

Abstract IA24: Translating fundamental immunobiology into adoptive T-cell therapy for ovarian cancer

Abstract In nearly half of all high-grade serous cancers, tumor-infiltrating T lymphocytes (TILs) invade and accumulate in tumor epithelial islets. These immunologically “hot” cancers are prognostic for patient survival and provide a strong rationale for immune-based treatment strategies that mobilize and enhance endogenous immune responses. In other cancers, such as melanoma and HPV-associated cervical cancer, TILs with autologous tumor reactivity can be isolated from resected tumors and expanded ex vivo for reinfusion to the patient in a process called adoptive immunotherapy. Adoptive immunotherapy using TILs in patients with these cancers frequently results in durable complete responses. In ovarian cancer patients, responses to TIL therapy have been less dramatic due in part to technical difficulties in identifying and expanding the tumor-reactive subset of TILs. In an effort to improve TIL therapy, we identified a surface biomarker of naturally occurring tumor reactive TILs in ovarian cancer and showed in preclinical models systems that these cells mediate enhanced TIL killing of autologous tumor cells ex vivo and in vivo, paving the way for more effective TIL therapy. Mass cytometry analysis revealed a complex network of immune inhibitory molecules expressed by tumor-reactive TILs that act in unison to dampen antitumor immunity, and for which clinical-grade blocking antibodies exist, rationalizing the use of TIL therapy in combination with immune checkpoint inhibitors for ovarian cancer. Facilitating adoptive T-cell therapy in the subset of patients with immunologically “cold” ovarian cancers relies upon the de novo induction of T cell antitumor reactivity. One approach relies on the stimulation of nonreactive endogenous immune cells with tumor antigens. Use of lysates generated from the patient’s own tumor provides the opportunity to vaccinate against shared antigens as well as patient-specific mutational antigens. In this prime-and-boost approach, a patient can first be vaccinated against patient-specific antigens to induce antitumor immunity, and the vaccine-primed T cells then harvested and expanded to high numbers outside of the body before reinfusion into the patient. The feasibility and safety of this approach has now been established in ovarian cancer with evidence of biologic activity. In the era of synthetic biology, “off-the-shelf” tumor-reactive T cells can now be generated de novo through the use of gene engineering. Using advanced gene-transfer systems and sophisticated cell-cultivation technologies, patients’ T cells are endowed with antigen-specificity and a capacity for enhanced antigen-dependent proliferation, activity, and survival, based on principles of fundamental immunology. In this way, engineered T cells can overcome issues of HLA loss by tumor cells as well as T-cell exhaustion. In preclinical xenograft models, gene-modified T cells exert antigen-specific killing of human ovarian cancer. Gene-engineered T cells are the subject of our ongoing clinical trials and pave a new route toward adoptive immunotherapy, which renders ovarian cancers immunologically “hot.” Citation Format: Daniel J. Powell, Jr. Translating fundamental immunobiology into adoptive T-cell therapy for ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr IA24.

Abstract 4935: High-throughput immune-oncology screen identifies EGFR inhibitors as potent enhancers of CTL antigen-specific tumor cell killing

Abstract As immune checkpoint blocking antibodies increasing become foundational therapies for the treatment of cancer, there is a pressing need to identify compounds that synergize with checkpoint blockade as the basis of combinatorial treatment regimens. We have developed a screening assay in which a luciferized tumor cell line expressing a model antigen is co-cultured with a transgenic CD8+ T cell specifically recognizing the model antigen in a H-2b-restricted manner. The target tumor cell/T cell assay was screened with a small molecule library to identify compounds that inhibit or enhance T cell-mediated killing of tumor cells in an antigen-dependent manner. The EGFR inhibitor Erlotinib was the top hit that enhanced T cell killing of tumor cells. Subsequent experiments with Erlotinib and additional EGFR inhibitors validated the screen result. EGFR inhibitors increase both basal and IFN-γ-induced antigen processing and presentation of MHC class-I, which enhanced recognition and lysis by CD8+ cytotoxic T lymphocytes. The tumor cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen utilizing the same target tumor cell/T cell assay identified sgRNAs targeting EGFR as sensitizing tumor cells to T cell-mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in the MC38 syngeneic colon cancer model that was superior to PD-1 blockade or EGFR inhibition alone, further validating EGFR inhibitors as immunomodulatory agents that enhance PD-1 checkpoint blockade. This novel target tumor cell/T cell assay can be screened in high-throughput with small molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds AND target genes, respectively, that enhance or inhibit T cell recognition and killing of tumor cells. Citation Format: Patrick H. Lizotte, Troy Luster, Megan E. Cavanaugh, Luke J. Taus, Abha Dhaneshwar, Naomi Mayman, Aaron Yang, Mark Bittinger, Paul Kirschmeier, Nathanael S. Gray, David A. Barbie, Pasi A. Janne. High-throughput immune-oncology screen identifies EGFR inhibitors as potent enhancers of CTL antigen-specific tumor cell killing [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 4935.

Abstract 2783: Empowering therapeutic monoclonal antibodies with IFN-alpha for cancer immunotherapy

Abstract Type 1 IFNs stimulates secretion of IP-10 (CXCL10) which is a critical chemokine to recruit effector T cells to the tumor microenvironment and IP-10 knockout mice exhibit a phenotype with compromised effector T cell generation and trafficking. Type 1 IFNs also induces MHC class 1 upregulation on tumor cells which can enhance anti-tumor CD8 T cell effector response in the tumor microenvironment. Although type 1 IFNs show great promise in potentiating anti-tumor immune response, systemic delivery of type 1 IFNs is associated with toxicity thereby limiting clinical application. In this study, we fused tumor targeting antibodies with IFN-alpha and showed that the fusion proteins can be produced with high yields and purity. IFN fusions selectively induced IP-10 secretion from antigen positive tumor cells, which was critical in recruiting the effector T cells to the tumor microenvironment. Further, we establish a real time in vitro antigen specific CTL killing assay using IncuCyte Zoom and tested the anti-tumor efficacy of anti-PDL1-IFN-alpha fusion using this system. We found anti-PDL1-IFN-alpha as low as 10 pM exhibits potent activity in potentiating OT1 CD8 T cells killing against OVA expressing tumor cells, while control IFN fusion did not exhibit any activity in the same experiment. Furthermore, IFN-alpha fusion antibody was well tolerated in vivo and demonstrated anti-tumor efficacy in an anti-PD-L1 resistant syngeneic mouse tumor model. Our data supports the hypothesis of targeting type 1 IFN to the tumor microenvironment may enhance effector T cell responses in non-inflamed tumors. Citation Format: Jun Guo, Yu Xiao, Ramesh B. Lyer, Marc R. Lake, Uri S. Ladror, Xin Lu, Bill Pappano, John E. Harlan, Medha J. Tomlinson, Gail T. Bukofzer, Cherrie K. Donawho, Alexander R. Shoemaker, Tzu-Hsuan Huang. Empowering therapeutic monoclonal antibodies with IFN-alpha for cancer immunotherapy [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 2783.

Abstract 3754: The novel hexavalent human CD137/4-1BB agonist HERA-CD137L promotes anti-cancer immunity by activating CD8 T cells while regulatory T cells are not affected

Abstract CD137/4-1BB is an inducible costimulatory receptor mainly expressed on immune cells following activation. Binding of the cognate ligand CD137L/4-1BBL leads to receptor trimerization and activation of signaling cascades involved in expansion and survival of T cells and myeloid cells as well as memory formation and protection against autoimmunity. Apogenix´ HERA technology is based on trivalent single-chain molecular mimics of the TNF-SF receptor binding domains fused to a silenced human IgG1-Fc-domain which serves exclusively as a dimerization scaffold. This results in hexavalent agonists replicating the natural receptor binding mode. Due to their inherent high receptor clustering capacity, HERA compounds are true agonists and their biological activity is, in contrast to bivalent anti-TNFR-SF antibodies, independent of FcR-mediated crosslinking events. HERA-CD137L was produced in CHO-S cells and purified resulting in highly pure non-aggregating protein lots. PK studies in mice demonstrated a terminal half-life of 75 hours indicating excellent in vivo stability. To study the effects on immune cells in vitro, T cells were isolated from healthy-donor buffy coats and stimulated with anti-CD3 antibody alone or in combination with HERA-CD137L. Using multicolor flow cytometry, we confirmed that expression of CD137 increased on CD8+ T cells following stimulation with anti-CD3 antibody. In accord with upregulation of the activation markers CD25 and CD69 and the memory marker CD45RO, treatment with HERA-CD137L enhanced proliferation of both CD4+ and CD8+ T cells, as determined by CFSE analysis. Intracellular accumulation of IFN-γ, TNF-α, Granzyme B and Perforin upon CD137 ligation was observed in CD8+ but not CD4+ T cells. HERA-CD137L treatment of THP-1 monocytes co-cultured with primary T cells also increased their cytotoxic activity against multiple tumor cell lines, including colorectal and mammary, as shown in a real-time live cell analysis (RTCA) assay. Primary human monocytes express low levels of CD137 and differentiation to macrophages in vitro did not increase expression. However, pro-inflammatory cytokines such as TNF-α and the chemokine CCL4 were secreted after stimulation with HERA-CD137L. RTCA assays further demonstrated enhanced antigen-specific killing of MDA-MB231 tumor cells by HERA-CD137L treated T cells. HERA-CD137L conveys its activity through effector T cell proliferation while regulatory T (Treg) cell proliferation or production of anti-inflammatory cytokines are not altered in Treg cell cultures. In contrast, HERA-CD137L prevented Treg-mediated suppression of effector T cells. Based on the in vitro data presented, HERA-CD137L is a promising candidate to promote anti-tumor immune responses either as single agent or in combination with other IO-compounds. Citation Format: Meinolf Thiemann, Jaromir Sykora, David M. Richards, Christian Merz, Viola Marschall, Mauricio Redondo Mueller, Julian P. Sefrin, Karl Heinonen, Harald Fricke, Christian Gieffers, Oliver Hill. The novel hexavalent human CD137/4-1BB agonist HERA-CD137L promotes anti-cancer immunity by activating CD8 T cells while regulatory T cells are not affected [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 3754.

PO-407 Identification of unique antigens on prostate cancer stem cells for cytotoxic T cell recognition

IntroductionCancer stem cells (CSCs) are more resistant to chemo- and radiation therapy than the bulk of the tumour. CSCs, surviving traditional therapies, possess self-renewal capacity and contribute to tumour re-growth. We are investigating the susceptibility of CSCs to antigen-specific killing by T cells. We generated a dataset incorporating all HLA Class I epitopes of the DU145 prostate cancer cell line. We aim to identify novel CSC epitopes which may serve as T cell targets.Material and methodsWe isolated HLA Class I:peptide complexes from DU145 cells by immunoaffinity precipitation and analysed the epitope sequences by liquid chromatography tandem-mass spectrometry (LC-MS/MS). We selected epitopes in the dataset which showed low gene expression in healthy tissues (www.GTexportal.org) and high predicted HLA-binding affinity (http://tools.immuneepitope.org/mhci/). DU145 CSCs were isolated based on aldehyde dehydrogenase (ALDH) activity using FACS. ALDHhigh and ALDHlow DU145 cells were determined to have CSC or non-CSC characteristics, respectively, by in vivo tumour initiation. Gene expression of the epitopes was measured by qPCR in ALDHhigh and ALDHlow DU145 cells. Protein expression was determined by immunofluorescence. We performed homology modelling of the HLA:peptide interfaces and quantified the binding interactions using PISA interface analysis software. We measured T cell activation by selected peptides analysing cytokine release by flow cytometry.Results and discussionsWe selected 42 epitopes from the DU145 dataset with low global tissue expression and predicted HLA binding percentile rank <1%. We identified 11 CSC epitopes by qPCR; 6 upregulated in ALDHhigh DU145 cells (e.g. TACSTD2) and 5 abundant in both ALDHhigh and ALDHlow DU145 cells (e.g. XPO1). We identified 3 HLA:epitope complexes for which the PISA P-value indicated high affinity interface interactions. CD8+ T cells that release cytokines in response to one of these epitopes have been identified. The antigen in question is upregulated in the prostate and testis, thus it is a promising T cell target on prostate CSCs.ConclusionWe have identified prostate CSC epitopes from proteins involved in vascular regulation, calcium signalling and nuclear export. This highlights the potential for targeting CSC epitopes from proteins other than those used for CSC isolation and maybe shared with non cancer stem cells. Our ongoing work involves tetramer based isolation of CSC epitope-specific cytotoxic T cells.

Literature Highlights: Impactful Papers Published Elsewhere

Engineering safer immunolocks and keys (by Wapcaplet via Wikimedia Commons)IL2 therapy is used with caution due to adverse events at IL2 concentrations that are clinically useful. A modified IL2 receptor, which only binds to a modified form of IL2, was inserted into T cells destined for adoptive antitumor therapy. The modified IL2 was designed to only bind the modified receptor, and, as administration of this modified IL2 activates only the transferred T cells, the IL2 provides not only therapeutic benefit but also is not associated with identifiable toxicity.Sockolosky JT, …, Garcia KC. Science 2018 Mar 2;359:1037–42.Fibroblast engulfing debris (From Lakins et al.)Tumor fibroblasts were found to engulf and process tumor cell debris, then cross-present it to T cells. However, instead of activating T cells, this presentation is suppressive and reduces T-cell viability. The T-cell death results from increased expression of FasL and PD-L2 on the fibroblasts that, in turn, activates Fas and PD-1 pathways in the T cells. Blockade of these interactions restored antigen-specific T-cell killing of tumor cells.Lakins MA, …, Shields JD. Nat Commun 2018 Mar 5. DOI: 10.1038/s41467-018-03347-0.Tumor-associated macrophage clusters (from Perry et al., Fig. 2)Immunotherapeutics are hampered by suppressive elements in the tumor microenvironment, including M2 (suppressive) macrophages. Perry et al., using an authochthonous mouse model (mutant Braf/Pten) of melanoma, and Hoves et al., primarily with the MC38 model, show that activating CD40 while inhibiting CSF-1R signaling changes transcriptional modules, reduces M2s, increases inflammatory macrophages, and improves tumor control. The effects are strong, but not permanent, and both CD8+ T cells and macrophages are necessary for tumor eradication.Perry CJ, …, Kaech SM. J Exp Med 2018 Feb 7;215:877–93.Hoves S, …, Ries CH. J Exp Med 2018 Feb 7;215:859–76.Fc affinity affects checkpoint blockade effectiveness (from Arce Vargas et al.)Optimization of the interaction between human FcγRs and anti– CTLA-4 in humanized mice expressing human FcγRs efficiently depletes Tregs and controls tumor growth. However, removal of Tregs is only important in tumors that are highly inflamed and infiltrated by CD8+ T cells. Patients carrying FcγR polymorphisms with higher binding affinities for IgGs, and that have higher neoantigen burdens, show greater responses to CTLA-4 blockade.Arce Vargas F, …, Quezada SA. Cancer Cell 2018 Mar 22. DOI: 10.1016/j.ccell.2018.02.010.4-1BB–activated mitochondria (from Teijeira et al., Fig. 6)Removing checks on T-cell activation is often insufficient for reactivating CD8+ T cells. Inducing 4-1BB signaling while blocking PD-1 (Menk et al.) or during adoptive T-cell therapy (Teijeira et al.) induces robust antitumor immunity in mouse models of melanoma or colon cancer. 4-1BB appears to regulate mitochondrial mass and function, reprogramming the metabolic state of antitumor T cells to counter exhaustion.Menk AV, …, Delgoffe GM. J Exp Med 2018 Mar 6. DOI: 10.1084/jem.20171068.Teijeira A, …, Melero I. Cancer Immunol Res 2018 Apr. DOI: 10.1158/2326-6066.CIR-17-0767.Core of hydrogel (by Edgar181 via Wikimedia Commons)Surgery removes primary tumors, but the wound healing process produces an immunosuppressive environment that may be conducive to regrowth of residual tumor cells and distal metastasis. Placing a hydrogel laced with agonists for innate immune responses into post-surgical wounds from which tumors had been extracted resulted in activation of innate and specific antitumor responses and a decrease in metastasis.Park CG, …, Goldberg MS. Sci Transl Med 2018 Mar 21;10:eaar1916.

The natural HLA ligandome of glioblastoma stem-like cells: antigen discovery for T cell-based immunotherapy.

Glioblastoma is the most frequent malignant primary brain tumor. In a hierarchical tumor model, glioblastoma stem-like cells (GSC) play a major role in tumor initiation and maintenance as well as in therapy resistance and recurrence. Thus, targeting this cellular subset may be key to effective immunotherapy. Here, we present a mass spectrometry-based analysis of HLA-presented peptidomes of GSC and glioblastoma patient specimens. Based on the analysis of patient samples (n = 9) and GSC (n = 3), we performed comparative HLA peptidome profiling against a dataset of normal human tissues. Using this immunopeptidome-centric approach we could clearly delineate a subset of naturally presented, GSC-associated HLA ligands, which might serve as highly specific targets for T cell-based immunotherapy. In total, we identified 17 antigens represented by 41 different HLA ligands showing natural and exclusive presentation both on GSC and patient samples. Importantly, in vitro immunogenicity and antigen-specific target cell killing assays suggest these peptides to be epitopes of functional CD8+ T cell responses, thus rendering them prime candidates for antigen-specific immunotherapy of glioblastoma.

Host immune responses to Toxoplasma gondii.

Toxoplasma gondii can infect homoeothermic animals including humans and cause lethal toxoplasmosis in immunocompromised individuals. When hosts are infected with T. gondii, the cells induce immune responses against T. gondii. The pathogen infection is recognized by immune sensors that directly detect T. gondii structural components, leading to production of pro-inflammatory cytokines and chemokines. Antigen-presenting cells such as macrophages and dendritic cells strongly activate T cells and induce development of Th1 cells and antigen-specific killer CD8 T cells. These T cells and Group 1 innate lymphoid cells are main producers of IFN-γ, which robustly stimulates cell-autonomous immunity in cells infected with T. gondii. IFN-γ-inducible effectors such as IFN-inducible GTPases, inducible nitric oxide synthase and indoleamine-2,3-dioxygenase differentially play important roles in suppression of T. gondii growth and its direct killing in anti-T. gondii cell-autonomous immune responses. In this review, we will describe our current knowledge of innate, adaptive and IFN-γ-mediated cell-autonomous immunity against T. gondii infection.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

ID:3002 Notch-mediated control of memory T cells against cancer cells

T cells recognize an antigen presented by self-MHC, and the part of initially activated T cells differentiate toward memory T cells. T cells also recognize cancer cells leading to generation of memory T cells against cancer-derived antigens although the activity of T cells are frequently suppressed by various factors. The release from T cell inhibitory factors could allow T cells to respond to cancer cells. However, it remains unclear which molecules are required for long-term survival of memory T cells and generation of memory T cells against cancer cells. Notch functions as a regulator for fate decision, activation and survival of immune cells. We have demonstrated the roles of Notch in mature T cell differentiation and found that Notch signaling is essential for the maintenance of memory CD4 T cells. The inhibition of Notch disturbs the survival of memory CD4 T cells. The effect of Notch on T cell survival depended on glucose uptake through cell surface Glut1 expression. We revealed that Notch is crucial for the long-term survival of memory T cells against cancer cells and suppression of Notch signaling reduced the tumor antigen-specific killing of cancer cells. Those data demonstrate that Notch is pivotal for the maintenance of memory T cells against cancer cells and suggest that activation of Notch signaling might be advantageous to cancer immunotherapy.

Hyperglycaemia does not affect antigen-specific activation and cytolytic killing by CD8+ T cells in vivo.

Metabolism is of central importance for T cell survival and differentiation. It is well known that T cells cannot function in the absence of glucose, but it is less clear how they respond to excessive levels of glucose. In the present study, we investigated how increasing levels of glucose affect T-cell-mediated immune responses. We examined the effects of increased levels of glucose on CD8+ T-cell behaviour in vitro by assessing activation and cytokine production, as well as oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and intracellular signalling. In addition, we assessed in vivo proliferation, cytokine production and cytolytic activity of cells in chemically induced diabetic C57BL/6 mice. Elevated levels of glucose in in vitro cultures had modest effects on proliferation and cytokine production, while in vivo hyperglycaemia had no effect on CD8+ T-cell proliferation, interferon γ (IFNγ) production or cytolytic killing.

RNA-transfection of \u03b3/\u03b4 T cells with a chimeric antigen receptor or an \u03b1/\u03b2 T-cell receptor: a safer alternative to genetically engineered \u03b1/\u03b2 T cells for the immunotherapy of melanoma

BackgroundAdoptive T-cell therapy relying on conventional T cells transduced with T-cell receptors (TCRs) or chimeric antigen receptors (CARs) has caused substantial tumor regression in several clinical trials. However, genetically engineered T cells have been associated with serious side-effects due to off-target toxicities and massive cytokine release. To obviate these concerns, we established a protocol adaptable to GMP to expand and transiently transfect γ/δ T cells with mRNA.MethodsPBMC from healthy donors were stimulated using zoledronic-acid or OKT3 to expand γ/δ T cells and bulk T cells, respectively. Additionally, CD8+ T cells and γ/δ T cells were MACS-isolated from PBMC and expanded with OKT3. Next, these four populations were electroporated with RNA encoding a gp100/HLA-A2-specific TCR or a CAR specific for MCSP. Thereafter, receptor expression, antigen-specific cytokine secretion, specific cytotoxicity, and killing of the endogenous γ/δ T cell-target Daudi were analyzed.ResultsUsing zoledronic-acid in average 6 million of γ/δ T cells with a purity of 85% were generated from one million PBMC. MACS-isolation and OKT3-mediated expansion of γ/δ T cells yielded approximately ten times less cells. OKT3-expanded and CD8+ MACS-isolated conventional T cells behaved correspondingly similar. All employed T cells were efficiently transfected with the TCR or the CAR. Upon respective stimulation, γ/δ T cells produced IFNγ and TNF, but little IL-2 and the zoledronic-acid expanded T cells exceeded MACS-γ/δ T cells in antigen-specific cytokine secretion. While the cytokine production of γ/δ T cells was in general lower than that of conventional T cells, specific cytotoxicity against melanoma cell lines was similar. In contrast to OKT3-expanded and MACS-CD8+ T cells, mock-electroporated γ/δ T cells also lysed tumor cells reflecting the γ/δ T cell-intrinsic anti-tumor activity. After transfection, γ/δ T cells were still able to kill MHC-deficient Daudi cells.ConclusionWe present a protocol adaptable to GMP for the expansion of γ/δ T cells and their subsequent RNA-transfection with tumor-specific TCRs or CARs. Given the transient receptor expression, the reduced cytokine release, and the equivalent cytotoxicity, these γ/δ T cells may represent a safer complementation to genetically engineered conventional T cells in the immunotherapy of melanoma (Exper Dermatol 26: 157, 2017, J Investig Dermatol 136: A173, 2016).

Abstract 3996: MDSC depletion combined with CTLA-4 blockade cause tumor regression in a syngeneic model of oral cavity cancer

Abstract Carcinogen-associated head and neck cancers are a heterogeneous group of aggressive cancers with a high recurrence rate and poor 5-year survival. Many of these cancers are T-cell inflamed but only a subset of patients respond to checkpoint blocking immunotherapy. Local immunosuppression mediated by both the tumor cells and other infiltrating immune cells are likely a major mechanism of resistance to immunotherapy. The mouse oral cancer (MOC) model includes genetically defined cell lines derived from carcinogen-induced tumors that generate tumors with variable T-cell inflamed phenotypes when transplanted into wild-type C57BL/6 mice. Mice bearing MOC1 tumors display a T-cell inflamed phenotype but do not significantly respond to PD-L1 checkpoint inhibition alone, which models a significant subset of human cancers from the oral cavity. Conversely, MOC2 tumors are non-T-cell inflamed. Using these MOC models, we have demonstrated an inverse relationship between accumulation of granulocytic MDSCs (gMDSCs) and effector immune cells (CD8/4+ T-cells, and NK cells) in the tumor microenvironment. Functionally, these gMDSCs suppressed CD8+ T-cell proliferation, IFNγ production, and antigen-specific killing. Depletion of these gMDSCs with a systemic anti-Ly6G antibody (1A8) did not change CD8/4+ T-cell or NK cell accumulation but restored tumor T-cell and NK cell activation and draining lymph node antigen-specific T-lymphocyte activation that was lost with tumor progression. Depletion of gMDSCs also significantly sensitized established MOC1 tumors to CTLA4-based checkpoint inhibition. CTLA-4 blockade alone induced tumor regression in a subset of mice and a marked delay in others. However, gMDSC depletion plus CTLA-4 blockade resulted in complete tumor rejection and development of immunologic memory in all treated mice. Peripheral gMDSCs were found to express the chemokine receptor CXCR2 but no other myeloid chemokine receptors, and expression of CXCR2 ligands CXCL1 and CXCL2 increased within the tumor microenvironment during tumor progression. Macrophage chemotaxis components CSF1R and CSF1 were not significantly expressed in these models. TCGA data analysis demonstrated an MDSC-rich gene expression profile in &amp;gt;60% of head and neck cancers, along with significant CTLA4, CXCR2, CXCL1, and CXCL8 (IL-8) expression within both HPV-positive and negative head and neck cancers. Functional inhibition or elimination of MDSCs from the tumor microenvironment may significantly reverse local immunosuppression and enhance responses to checkpoint inhibition in patients with head and neck cancer. Citation Format: Paul Esteban Clavijo, Ellen Moore, Jianhong Chen, Ruth Davis, Carter Van Waes, Zhong Chen, Clint Allen. MDSC depletion combined with CTLA-4 blockade cause tumor regression in a syngeneic model of oral cavity cancer [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 3996. doi:10.1158/1538-7445.AM2017-3996

Expansion of cancer germline antigen-specific cytotoxic T lymphocytes for immunotherapy.

The cancer germline antigens MAGE-A1, MAGE-A3, and NY-ESO-1 can be used to target relapsed or therapy-resistant malignant solid tumors, and previous studies have demonstrated that these antigens can be epigenetically upregulated on the surface of tumor cells following exposure to low-dose demethylating chemotherapy agents, such as decitabine. The extent to which cancer germline antigen cytotoxic T lymphocytes can be reliably expanded from healthy donors has not been well characterized, specifically in terms of whether these T cells consistently kill antigen-bearing targets or simply produce interferon-γ in the presence of the antigen. Cancer germline antigen cytotoxic T lymphocytes were generated using conventional method and high-density lymphocyte culture method. We demonstrate that there is no difference in the extent of antigen-specific killing with or without CD25 depletion when interleukin-21 is added to the cultures. Cancer germline antigen-specific killer cells could be expanded from 8/12 healthy donors using overlapping peptide mixes derived from MAGE-A1, MAGE-A3, and NY-ESO-1 and from 7/9 healthy donors using HLA-restricted epitopes. Furthermore, cytotoxic T lymphocyte derived from 4/5 patients displayed specific cytotoxicity of target cells expressing respective cancer germline antigen and HLA partially matched tumor lines. High-density lymphocyte culture prior to stimulation with cancer germline antigen peptides resulted in antigen-specific cytotoxic T lymphocyte from healthy donors and patients from whom cancer germline antigen cytotoxic T lymphocyte culture with conventional methods was not feasible. These data demonstrate that MAGE-A1-, MAGE-A3-, and NY-ESO-1-specific T cells with antigen-specific cytotoxicity can be cultured from healthy donors and patient-derived cells making adoptive immunotherapy with these cytotoxic T lymphocyte feasible.

Synergistic targeting of breast cancer stem-like cells by human γδ T cells and CD8+ T cells.

The inherent resistance of cancer stem cells (CSCs) to existing therapies has largely hampered the development of effective treatments for advanced malignancy. To help develop novel immunotherapy approaches that efficiently target CSCs, an experimental model allowing reliable distinction of CSCs and non-CSCs was set up to study their interaction with non-MHC-restricted γδ T cells and antigen-specific CD8+ T cells. Stable lines with characteristics of breast CSC-like cells were generated from ras-transformed human mammary epithelial (HMLER) cells as confirmed by their CD44hi CD24lo GD2+ phenotype, their mesenchymal morphology in culture and their capacity to form mammospheres under non-adherent conditions, as well as their potent tumorigenicity, self-renewal and differentiation in xenografted mice. The resistance of CSC-like cells to γδ T cells could be overcome by inhibition of farnesyl pyrophosphate synthase (FPPS) through pretreatment with zoledronate or with FPPS-targeting short hairpin RNA. γδ T cells induced upregulation of MHC class I and CD54/ICAM-1 on CSC-like cells and thereby increased the susceptibility to antigen-specific killing by CD8+ T cells. Alternatively, γδ T-cell responses could be specifically directed against CSC-like cells using the humanised anti-GD2 monoclonal antibody hu14.18K322A. Our findings identify a powerful synergism between MHC-restricted and non-MHC-restricted T cells in the eradication of cancer cells including breast CSCs. Our research suggests that novel immunotherapies may benefit from a two-pronged approach combining γδ T-cell and CD8+ T-cell targeting strategies that triggers effective innate-like and tumour-specific adaptive responses.

Artificial Thymic Organoids Permit Allelic Exclusion and Efficient Generation of Naïve TCR-Engineered T-Cells from Human Hematopoietic Stem Cells In Vitro

Engineered adoptive immunotherapies have shown unprecedented activity in the treatment of cancer and chronic viral infections. Current approaches rely on individualized ex vivo genetic modification of autologous T cells due to the risk of graft-versus-host disease from allogeneic T cells. These processes furthermore require activation and prolonged expansion of T cells, which may reduce in vivo efficacy and persistence. Direct in vitro differentiation of engineered T cells from hematopoietic stem and progenitor cells (HSPCs) may overcome these problems by permitting the suppression of endogenous TCR expression through allelic exclusion, and the de novo generation of naïve antigen-specific T cells. Existing methods of in vitro human T cell differentiation are subject to wide experimental variability and do not adequately support the positive selection of immature T cell precursors to mature T cells, and thus have not been suitable for clinical-scale production of engineered T cells.We report here the preclinical development of an artificial thymic organoid (ATO) system using off-the-shelf, serum-free components and a standardized stromal cell line that supports highly efficient in vitro differentiation and positive selection of native and TCR-engineered human T cells from cord blood (CB), bone marrow, and mobilized peripheral blood CD34+ HSPCs, and purified CD34+CD38- hematopoietic stem cells. ATOs closely recapitulated thymic T cell commitment and differentiation, resulting in greater than 80% CD7+CD5+ T-lineage cells and 50% CD4+CD8+ double positive (DP) T cell precursors by 4 weeks. By 6 weeks, 30-40% of ATO cells were CD3+TCRαβ+ T cells, of which 20-30% were mature CD8 single positive (SP) T cells. CD4SP cells were generated at a lower frequency and later in culture (2-14% of CD3+TCRαβ+ cells). ATO-derived T cells exhibited a naïve CD45RA+CD27+CCR7+CD62L+ phenotype, a diverse, thymic-like TCR repertoire, and robust TCR-dependent cytokine release and proliferation.Transduction of CB CD34+ HSPCs with an HLA-A*02:01-restricted αβ TCR specific for NY-ESO-1 resulted in a markedly increased cell output per ATO (>400-fold, relative to input HSPCs) and enhanced generation of naïve CD3+TCRαβ+CD8αβ+ conventional T cells, the majority of which were antigen-specific by tetramer staining. Positive selection of TCR-engineered naïve T cells could be further enhanced by expression of cognate HLA-A*02:01 in ATO stromal cells. ATO-derived TCR-engineered T cells exhibited a near complete lack of endogenous TCR Vβ expression, consistent with induction of allelic exclusion by the exogenous TCR during T cell development. ATO-derived engineered T cells underwent antigen-specific cytotoxic priming, polyfunctional cytokine release, and proliferation in response to artificial APCs; and exhibited antigen-specific killing of NY-ESO-1+ tumor cells in vitro and in vivo.ATOs thus present a highly efficient off-the-shelf platform for the generation of clinically relevant numbers of naïve and potentially non-alloreactive engineered T cells for adoptive immunotherapy. Clinical translation of the ATO system will be aided by its simplicity, scalability, use of serum-free components, and compatibility with irradiated stromal cells. In addition, genetic manipulation of stem or stromal cell components can be easily incorporated into the system to further enhance downstream T cell engraftment or function. DisclosuresSeet:Kite Pharma: Patents & Royalties: Kite Pharma holds an exclusive license to certain intellectual property. Montel-Hagen:Kite Pharma: Patents & Royalties: Kite Pharma holds an exclusive license to certain intellectual property. Crooks:Kite Pharma: Patents & Royalties: Kite Pharma holds an exclusive license to certain intellectual property, Research Funding.

Systemic infection ruined NK cell-mediated help on influenza virus-specific CTLs

Abstract Host resistance against influenza A virus infection relies on the efficient collaboration of innate and adaptive immune response. Being a major component of innate immune system, NK cells have been confirmed to limit the replication and spread of influenza virus during early infection. However, their roles in the generation of virus-specific CTLs response still remain exclusive. Consistent with our in vitro study on human NK cells, here we firstly confirmed that murine NK cells could be directly infected with mouse-adaptive influenza A PR8 virus in vivo under both systemic and local infection. However, only systemic infection resulted in severe reduction and dysfunction of spleen NK cells, which was accompanied with the poor antigen-specific killing and IFN-γ secretion rather than the decrease in quantity of CTLs during the acute phase of infection. Importantly, the adoptive transfer of spleen NK cells from mice suffering from local infection rescued the impaired CTLs in systemic influenza virus-infected mice, whereas no similar effects could be observed by using NK cells from healthy mice. Taken together, our results demonstrated that NK cell-mediated help on the generation of functional influenza virus-specific CTLs was infection-dependent and determined by the dose of infection. This study provided some important information for developing novel NK-cell-targeted strategy against severe influenza infection.

Abstract B126: Regeneration of tumor antigen specific CTLs utilizing iPSC technology

Abstract Current cancer immunotherapies mainly aim to activate and expand tumor antigen specific cytotoxic T lymphocytes (CTLs) in vitro or in vivo, but these methods are not so successuful because of the difficulty in preparing sufficient number of CTLs. We are trying to overcome this problem by utilizing the induced pluripotent stem cell (iPSC) technology. Our strategy is based on the concept that i) when iPSCs are produced from antigen specific T cells, the rearranged configurations of T cell receptor (TCR) genes are inherited to the iPSCs (which we call T-iPSCs), and that ii) when T cells are regenerated from such T-iPSCs, all of them should come to express the same TCR as the original one. Therefore it will become possible to obtain de novo generated tumor specific CTLs almost unlimitedly, because it is possible to make a mass-culture of cells at the iPSC level. In line with this concept, we have succeeded in establishing T-iPSCs from CTLs specific for the melanoma antigen MART-1 derived from tumor infiltarating lymphocytes, and in regenerating MART-1 specific T cells from such T-iPSCs (Vizcardo et al, Cell Stem Cell, 2013). The strategy mentioned above has been conducted in the category of autologous transplantation. Autologous transplantation setting, however, has some problems; i) high cost to establish T-iPSCs from each patients, ii) heterogeneneity of the quality as iPSCs, iii) difficulty in getting high affinity TCR. To solve these problems we are now planing to apply this method to the allogeneic transplantation setting. In allogeneic setting, we can select high quality T-iPSC clone and such a clone can be used to many other patients. It is preferable to produce T-iPSCs from a HLA haplotype homo donor, since CTLs generated from these T-iPSCs can be administerd to the patients carrying the same HLA haplotype on one allele. Thus by utilizing allogenic T-iPSCs the cost would be reduced and the quality of T-iPSCs can be ensured. Now we are trying to establish T-iPSCs from CTLs specific for various cancer antigen from healthy donors. We succeded in establishing T-iPSCs from CTLs specific for EB virus related antigen LMP2 and cancer testis antigen WT1, both of which are restricted to HLA A2402, the commonest HLA-A in the Japanese. We improved in vitro culture condition and have succeded in regenerating CD8 single positive cells expressing CD8 alpha-beta heterodimers. These regenerated CTLs exhibited very high antigen specific killing activity comparable to the original CTLs. The CTLs derived from WT1 specific T-iPSCs were able to kill some leukemia cell lines which express endogenous WT1 protein. They can be expanded more than ten thousand fold by repeated TCR stimulation without reducing their killing activity. We are now examining in vivo killing activity of these CTLs using zenograft model. This method could bring about a breakthrough in cancer immunotherapy. Citation Format: Takuya Maeda, Seiji Nagano, Hiroshi Ichise, Kyoko Masuda, Toshio Kitawaki, Akifumi Kondo-Takaori, Hiroshi Kawamoto. Regeneration of tumor antigen specific CTLs utilizing iPSC technology. [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 B126.

Abstract B128: Chimeric antigen receptor transduced gamma delta T lymphocytes provide enhanced tumor specificity

Abstract Human gamma delta T lymphocytes (δγT) have many properties of innate lymphocytes including antibody dependent cell mediated cytotoxicity, innate cytotoxicity and capacity for professional antigen presentation. Chimeric antigen receptors (CARs), which combine within a single molecule an scFv targeting a tumor associated antigen with T cell receptor signaling and co-stimulation, are capable of imparting potent effector function but are dependent on a high degree of target antigen specificity to avoid unwanted toxicity. We hypothesized that transduction of δγT with CARs would provide a mechanism to both enhance effector function and increase target antigen specificity. We have focused on the subset of human peripheral blood γδT harbor the Vγ9Vδ2 TCR, as these cells have innate effector function, mediated by the TCR recognition of stressed cells, against a wide range of human cancers. Moreover, the Vγ9Vδ2 subset can be readily activated and expanded from peripheral blood using zoledronic acid, a clinical agent that leads to engagement of the Vγ9Vδ2 TCR and that can be used within the GMP setting. Using GD2 as a model antigen, expressed on both cancers and peripheral nerves, we generated anti-GD2 CARs employing a novel scFv that is not prone to tonic signaling. Following stimulation with zoledronate, PBMC were transduced on day 5 with gamma-retroviral vectors expressing CARs, and left to expand for 2 weeks in the absence of antigenic target. Between 10-100 fold expansion was observed and the cell product was typically over 90% pure for Vγ9Vδ2 T cells, with transduction efficiency around 50%. Compared with αβT-CAR cells generated following stimulation of PBMC with CD3/CD28 beads, both CAR-expressing cell populations were a similar mixture of central and effector memory cells and had equivalent dim expression of exhaustion markers. Using a GD2-CAR with CD3-zeta and CD28 endodomains, γδT-CAR and αβT-CAR cells showed equivalent antigen-specific killing but γδT-CAR cells retained additional innate killing of a range of cancer cells. Both populations were capable of equivalent secondary antigen-specific expansion and interferon gamma secretion. To exploit the innate target recognition of the Vγ9Vδ2 cells through its TCR, we designed CARs for use in γδT cells that provided only antigen-dependent costimulation. Such CARs retain antigen specific target recognition through the TCR but avoid off target toxicity marking a potential new paradigm for enhancing cancer specificity. Citation Format: Jonathan Fisher, anna capsomidis, Barry Flutter, Gabriel Benthal, Rebcca Wallace, Kenth Gustafsson, Karin Straathof, Martin Pule, John Anderson. Chimeric antigen receptor transduced gamma delta T lymphocytes provide enhanced tumor specificity. [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 B128.