122 A powerful, precise targeting system controlled by tumor deletions transforms CEA and MSLN CAR-T cells into tumor-selective agents

BackgroundMesothelin (MSLN) and carcinoembryonic antigen (CEA) are classic tumor-associated antigens that are expressed in many solid tumors including the majority of lung, colorectal and pancreatic cancers. However, both MSLN and CEA are also expressed in vital normal organs. This normal expression creates risk of serious inflammation for CEA- or MSLN-directed therapeutics. To date all active CEA- or MSLN-targeted investigational therapeutics have been toxic when administered systemically.MethodsWe have developed a safety mechanism to protect normal tissues without abrogating sensitivity of cytotoxic T cells directed at MLSN(+) or CEA(+) tumors in a subset of patients with defined loss of heterozygosity (LOH) in their tumors (figure 1). This dual-receptor (Tmod< sup >TM</sup >) system exploits common LOH at the HLA locus in cancer cells, allowing T cells to recognize the difference between tumor and normal tissue.1 2 T cells engineered with specific Tmod constructs contain: (i) a MSLN- or CEA-activated CAR; and, (ii) an inhibitory receptor gated by HLA-A*02. HLA-A*02 binding blocks T cell cytotoxicity, even in the presence of MSLN or CEA. The Tmod system is designed to treat heterozygous HLA class I patients, selected for HLA LOH. When HLA-A*02 is absent from tumors selected for LOH, the CARs are predicted to mediate potent killing of the A*02(-) malignant cells.ResultsThe Tmod system robustly protects surrogate normal cells even in mixed-cell populations in vitro while mediating robust cytotoxicity of tumor cells in xenograft models (see example in figure 2). The MSLN CAR can also be paired with other blockers, supporting scalability of the approach to patients beyond HLA-A*02 heterozygotes.Abstract 122 Figure 1Illustration of the Tmod T cell engaging with tumor cells with somatic loss of HLA-A*02 and with normal cells.Abstract 122 Figure 2Bioluminescence measurements show the average difference between the size of the MSLN(+)A*02(+) ‘normal’ graft compared to the MSLN(+)A*02(-) tumor graft on the two flanks of mice after T cell infusion. Both tumor and normal grafts are destroyed by CAR-Ts (CAR-3 and M5 benchmark) while the MSLN Tmod cells kill the tumor but not the normal graft.ConclusionsThe Tmod mechanism may provide an alternative route to leverage solid-tumor antigens such as MSLN and CEA in safer, more effective ways than previously possible.ReferencesHamburger AE, DiAndreth B, Cui J, et al. Engineered T cells directed at tumors with defined allelic loss. Mol Immunol 2020;128:298–310.Hwang MS, Mog BJ, Douglass J, et al. Targeting loss of heterozygosity for cancer-specific immunotherapy. Proc Natl Acad Sci U S A 2021;118(12):e2022410118.

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682 Antibody-mediated blockade of interleukin-10 receptor-alpha promotes the activation of immune cells from in vitro dissociated tumor samples

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0682 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A721-A721

Author(s):

Alexey Berezhnoy ◽

Kalpana Shah ◽

Daorong Liu ◽

Peter Lung ◽

Vatana Long ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Interferon Gamma ◽

Expression Profiles ◽

Interleukin 10 ◽

Biologically Active ◽

List Type ◽

Receptor Alpha

BackgroundInterleukin-10 (IL-10) is a multifunctional cytokine that can mediate immune suppression or activation depending on the immunological context. Mouse studies have demonstrated that blockade of IL-10 enhances immune response against tumors and chronic viral infections;1 2 intriguingly, high concentrations of long-acting, pegylated IL-10 have also shown anti-tumor activity.3 Here we investigated IL-10 and IL-10 receptor-alpha (IL-10RA) expression profiles in normal and tumor tissues as well as the immunological effects of modulating the IL-10 pathway via antibody-mediated blockade of IL-10RA.MethodsIL-10 and IL-10RA mRNA are expressed by several tumors, including renal, lung, breast, and colon cancers. Fluorescent in-situ hybridization revealed that the majority of IL-10RA was expressed by CD3-negative tumor-infiltrating cells, localized in close proximity to T cells in the tumor microenvironment (TME). Immunohistochemistry studies confirmed expression of IL-10RA in the TME, while no expression was detected in healthy tissues. Furthermore, dissociated tumor cells produced biologically active levels of IL-10 in culture.ResultsMonoclonal antibodies (mAbs) against IL-10RA prevented IL-10 signaling and enhanced release of IL-12 by monocyte-derived dendritic cells activated with suboptimal LPS concentrations. The effect of IL-10RA blockade was greater than that observed with IL-10 neutralizing mAbs. In mixed lymphocyte reactions and superantigen-driven T-cell activation, IL-10RA blockade enhanced IL-2 secretion by T lymphocytes. Consistent with earlier observations in mouse models,4 the effect of IL-10RA blockade was nonredundant with blockade of the PD-1/PD-L1 axis, resulting in enhanced IL-2 and interferon-gamma secretion by T cells when both pathways were inhibited. Blockade of IL-10RA during CD3-redirected in vitro killing of tumor cells by PBMC induced IL-12 release as well as upregulation of CD86 and HLA-DR by CD3-negative cells. In in vitro dissociated tumor cells, IL-10RA blockade induced release of IL-2, interferon-gamma and other proinflammatory cytokines; additional PD-1/PD-L1 axis blockade further enhanced cytokine release.ConclusionsIn summary, antibody-mediated IL-10RA blockade can potentiate immune activation in the dissociated tumor cells and may be a valuable addition to cancer immunotherapies, including redirected T-cell killing and checkpoint blockade.ReferencesVicari A, et al. J. Exp. Med 2002;196:541–549.Ejrnaes M, et al. J Exp Med 2006;203:2461–72.Emmerich J, et al. Cancer Res 2012. 72:3570–3581.Brooks D, et al. Proc Natl Acad Sci U S A 2008;105:20428–33.

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154 Context-dependent reversible modulation of cJUN expression by CAR T cells for cancer treatment

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.154 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A164-A164

Author(s):

Zhifen Yang ◽

Francesco Marincola

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

List Type ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Context Dependent ◽

Fadu Cells

BackgroundOverexpression of canonical AP-1 factor cJUN was shown to prevent CAR T cell exhaustion and improve anti-tumor potency in vivo (1). However, its clinical utilization is limited by potential for transformation and oncogenic risk. Here, we present a conditional, antigen-dependent, non-editing CRISPR-activation (CRISPRa) circuit (RB-339) that delivers context-dependent upregulation of endogenous cJUN increasing CAR-T cell resistance to exhaustion.MethodsRB-339 is a CAR T cell engineered to conditionally turn on the transcription of the cJUN endogenous gene. The circuit includes a lentiviral construct encoding an anti-HER2 (4D5) single chain variable fragment, with CD28 and CD3ζ co-stimulatory domains linked to a tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the promoter region of cJUN. A second construct encodes linker for activation of T cells, complexed to nuclease-deactivated/dead Cas9 (dCas9)-VP64-p65-Rta transcriptional activator (VPR) via a TEV-cleavable linker (LdCV). Activation of CAR allows the release of dCas9 for nuclear localization and conditionally and reversibly induces the expression of cJUN. RB-339 was compared in vitro to control (cRB-339, lacking the cJUN sgRNA) and CAR-T cells engineered to constitutively express cJun.ResultsRB-339 induced cJUN upregulation upon stimulation with HER2-coated beads and this resulted in significantly elevated expression over a 6-day time course compared to the control cRB-339 (figure 1A-B). When HER2-coated beads were removed at day 3, cJUN expression returned to baseline parallel to cRB-339. The conditional upregulation of cJUN in RB-339 contrasted with the constitutive overexpression in the transgene carrying cells that was irrespective of antigen stimulation (figure 1C). Upon exposure to HER2+ FaDu cancer cells, RB-339 peaked at day 2 and declined afterwards when FaDu cells were killed at day 3; cJUN increased again at day 4 upon restimulation with FaDu cells at day 3 (figure 2). Such a dynamic induction of cJUN resulted in significantly enhanced CAR-T cells proliferation in RB-339 compared to the respective conventional CAR-T cells or cRB-339 (figure 3).Abstract 154 Figure 1Conditional upregulation of cJUN by RB-339 in vitro. RB-339 and its control cRB-339 were stimulated by HER2-coated or BSA-coated beads for either six days or three days followed by removal of beads at day 3 (figure 1A-B). Intracellular expression of cJUN was detected at indicated time points. Intracellular cJUN expression in overexpressed cJUN-CAR (figure 1C)Abstract 154 Figure 2Kinetics of cJUN upregulation in RB-339 upon exposure to HER2+ FaDu tumor cells. RB-339 and its control cRB-339 were stimulated by FaDu tumor cells for six days with or without restimulation at day 3Abstract 154 Figure 3Conditional upregulation of cJUN resulted in enhanced CAR-T proliferation in RB-339 in vitro after 6-day co-culture with FaDu tumor cells, compared to conventional HER2 CAR or cRB-339ConclusionsWe conclude that CAR-T engineered to conditionally express the canonical AP-1 factor cJUN increases expansion potential similarly to CAR-T cells engineered to constitutively express the cJun transgene. However, the context-dependent upregulation of cJUN limits the risk of oncogenic transformation. We are currently combining inducible and reversible cJUN and IL-12 upregulation for the generation of the next RB-339 product.ReferenceLynn RC, Weber EW, et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature 2019; 576(7786):293–300.

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687 Enhancement of anti-tumor immunity by ICT01: a novel g9d2 T cell-activating antibody targeting butyrophilin-3A (BTN3A)

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0687 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A726-A726

Author(s):

Aude de Gassart ◽

Patrick Brune ◽

LE Suong ◽

Sophie Agaugue ◽

Emmanuel Valentin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

T Cell Activation ◽

Cell Subset ◽

List Type ◽

Human Cancers ◽

Wide Range

BackgroundgdT-cells are innate-like lymphocytes described as potent killer of cancer cells whose infiltration into tumors is associated with a positive prognosis.1 2 This supports gd T-cells use in cancer immunotherapy. BTN3A, which belongs to the B7-subfamily of Ig proteins, is required for the recognition of malignant or infected cells by human g9d2 T-cells by sensing intracellular accumulation of phosphoantigens.3 ImCheck Therapeutics is developing ICT01, a humanized anti-BTN3A (IgG1, Fc-silenced), g9d2 T-cell-activating antibody for the treatment of patients with solid or hematologic tumors.MethodsA complete IND-enabling program was conducted to characterize the preclinical activity and safety of ICT01. ICT01 effects on human and cynomolgus PBMCs were characterized in vitro using flow cytometry. ICT01-mediated killing activity of g9d2 T-cells was assessed using in vitro co-cultures with tumor and non-tumor cells. Immunocompromised mice bearing human tumors and adoptively transferred with human g9d2 T cells were used to assess ICT01 anti-tumor activity in vivo. The PK, PD and safety of intravenous ICT01 (0.1 to 100 mg/kg single- and repeated-dose) were evaluated in Cynomolgus monkeys.ResultsICT01 selectively binds to all three BTN3A isoforms with high affinity (<10nM). When assayed in human and cynomolgus PBMCs in vitro, ICT01 promoted a robust and specific activation of g9d2 T-cells as shown by concentration dependent increase in cell surface CD69 and CD25 and cytokines secretion (IFNγ, TNFα). In co-culture experiments, ~20% of target occupancy on tumor cells is sufficient for maximal g9d2 T-cell degranulation (e.g. CD107a/b expression). ICT01-activated g9d2 T-cells continuously and serially kill a wide range of tumor cells in multi-day co-culture conditions. In contrast, non-tumoral BTN3A-expressing B cells, HUVEC and fibroblasts were unaffected. In mouse AML and ovarian cancer models, repeated injections of ICT01 delayed tumor growth and significantly prolonged animal survival. In primates, ICT01 exposure and target engagement was dose-dependent, with all tested doses producing a specific g9d2 T cell activation and trafficking out of the circulation within 1 hour. ICT01 administration was well tolerated with no safety signals observed at doses up to 25 mg/kg/week based on clinical, laboratory, and anatomic pathology parameters.ConclusionsThe combined in vitro and in vivo pharmacology data provide evidence that ICT01 is an attractive and novel therapeutic approach for enhancing the innate anti-tumor potential of g9d2 T-cells by activating BTN3A. Importantly, ICT01 did not affect healthy BTN3A-expressing cells, and NHP studies confirmed ICT01 safety with a wide therapeutic index. Therefore, ICT01 is being tested in the ongoing EVICTION trial (NCT04243499).Ethics ApprovalPseudonymized samples isolated from healthy volunteers’ whole blood by ImCheck Therapeutics under the agreement n° 7173 between ImCheck Therapeutic SAS and EFS PACA (Etablissement Français du Sang Provence-Alpes-cote d’Azur)ReferencesGentles AJ, Newman AM, Liu CL, et al. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nature Medicine 2015;21(8):938–945.Tosolini M, Pont F, Poupot M, et al. Assessment of tumor-infiltrating TCRVγ9Vδ2 γδ lymphocyte abundance by deconvolution of human cancers microarrays. OncoImmunology. 2017;6(3):e1284723.Harly C, Guillaume Y, Nedellec S, et al. Key implication of CD277/butyrophilin-3 (BTN3A) in cellular stress sensing by a major human γδ T-cell subset. Blood 2012;120(11):2269–2279.

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12 Development of an in vitro assay to assess bispecific T cell engager using T cells from CD3e humanized mice

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0012 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A12-A12

Author(s):

Jun Zhou ◽

Shuang Zhu ◽

Hongjuan Zhang ◽

Lei Zheng ◽

Mingfa Zang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Lymphoblastic Leukemia ◽

Humanized Mice ◽

Activation Markers ◽

In Vivo Models ◽

Vitro Assay ◽

Bite Antibody

BackgroundBispecific T cell engagers (BiTE) is a fast-growing class of immunotherapies. They are bispecific antibody that bind to T cell-surface protein (for example, CD3e) and a specific tumor associate antigen (TAA) on tumor cells, by which to redirect T cells against tumor cells in a MHC-independent manner. A successful example in the clinical is Blinatumomab, a BiTE antibody against CD3/CD19 approved in 2014 to treat acute lymphoblastic leukemia. Currently, many CD3-based BiTE are in clinical trials, including BCMAxCD3, Her2xCD3, CEAxCD3, and PSMAxCD3. To evaluate the efficacy of BiTE in vitro, human peripheral blood monocyte cells (hPBMC) are commonly being used as a source of T cells to co-culture with tumor cells. The disadvantage of using hPBMC is donor-to-donor variability and the availability of the original donor if a study needs to be repeated.MethodsTo overcome this, we proposed to replace hPBMC with T cells from human CD3e (hCD3) genetically engineered mouse models mice (GEMM) for in in vitro coculture assay. T cells were isolated from hCD3 GEMM mice using negative selection mouse T cell isolation kit. Conventional tumor cell lines or luciferase-engineered patient-derived-xenograft (PDX)-derived organoids (PDXO) expressing specific antigens are co-cultured with hCD3 T cells in 96-well plates in the presence of BiTE antibody.ResultsWe measured the killing of tumor cells using either flow cytometry or luciferase activity as readouts. To analyze tumor-reactivity of T cells to cancer cell line or organoids, IFN-gamma in the culture medium was measured and activation markers on T cells was assessed.ConclusionsOur data showed the feasibility of using humanized mice T cells as a replacement for hPBMCs to assess BiTE antibody in vitro. We are further validating the application of murine hCD3 T cells for in vivo models to test bispecific T cell engagers.

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P01.02 TLR-mediated suppression of the CCL22-CCR4 axis as a new target for tumor immunotherapy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-itoc8.6 ◽

2021 ◽

Vol 9 (Suppl 1) ◽

pp. A3.2-A4

Author(s):

J Grün ◽

I Piseddu ◽

C Perleberg ◽

N Röhrle ◽

S Endres ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

B Cells ◽

Regulatory T Cells ◽

Type I ◽

List Type ◽

Gm Csf

BackgroundUnmethylated CpG-DNA is a potent ligand for the endosomal Toll-like-receptor-9, important for the immune activation to pathogen-associated molecules.1 CpG and other TLR-ligands show effective immunotherapeutic capacities in cancer treatment by inducing an antitumorigenic immunity.2 They are able to reduce tumor progression by reduction of intratumoral secretion of the immunoregulating chemokine CCL223 and subsequent recruitment of immunosuppressive regulatory T cells (Treg), which express CCR4 the only so far known receptor for CCL22.4 Our recent work has shown that CCL22 secretion by dendritic cells (DC) in the lymph node, mediates tolerance by inducing DC-Treg contacts.5 Indeed, in the absence of CCL22, immune responses to vaccination were stronger and resulted in tumor rejection.6 Therefore, we are aiming to investigate the effects of TLR-ligands on systemic CCL22 levels, elucidating all involved mechanisms to identify new targets for cancer immunotherapy.Materials and MethodsT, B and CD11c+ DCs of wildtype (wt) and RAG1-/- mice were isolated from splenocytes by magnetic-activated cell sorting for in vitro assays. Different co-cultures were incubated with CpG and GM-CSF, known as an CCL22 inducer.5 For in vivo experiments, wt mice were treated with CpG, R484 or poly(I:C) alone and in combination with GM-CSF. CCL22-levels in a number of organs were analyzed.ResultsAnalyzing the different immune cell compartments in vitro, we found that DCs in whole splenocytes secrete CCL22 during culture while DC cultured alone showed no CCL22 secretion. When treated with CpG, CCL22-levels were reduced in splenocytes, while it was induced in DC culture alone. The same results were seen when RAG splenocytes, that lack functional B and T cells, were cultured with CpG. CpG treated B cells were able to suppress CCL22 secretion by DC unlike T cells alone. Co-cultures of T and B cells treated with CpG, however, induced the strongest CCL22 suppression in DC. In vivo, we could show that all TLR ligands tested reduced CCL22 in a number of organs significantly. Furthermore, CpG showed the strongest suppression of CCL22 even in the presence of the CCL22 inducer GM-CSF.5ConclusionsWe could show that B cells with T cells mediate CCL22 suppression by TLR ligands. The fact that CpG was able to reduce CCL22 levels even in the presence of the inducer GM-CSF demonstrates the potent CCL22 suppressive capacity of TLR ligands.ReferencesO’Neill LA, et al. The history of toll-like receptors – redefining innate immunity. Nat Rev Immunol 2013;13(6):453–60.Rothenfusser S, et al. Recent advances in immunostimulatory CpG oligonucleotides. Curr Opin Mol Ther 2003;5(2):98–106.Wang S, et al. Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells. Proc Natl Acad Sci U S A 2016;113(46): E7240–E7249.Rapp M, et al. CCL22 controls immunity by promoting regulatory T cell communication with dendritic cells in lymph nodes. J Exp Med 2019;216(5):1170–1181.Piseddu I, et al. Constitutive expression of CCL22 is mediated by T cell-derived GM-CSF. J Immunol 2020;205(8):2056–2065.Anz D, et al. Suppression of intratumoral CCL22 by type i interferon inhibits migration of regulatory T cells and blocks cancer progression. Cancer Res 2015;75(21):4483–93.Disclosure InformationJ. Grün: None. I. Piseddu: None. C. Perleberg: None. N. Röhrle: None. S. Endres: None. D. Anz: None.

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A Nanobody Against Cytotoxic T-Lymphocyte Associated Antigen-4 Increases the Anti-Tumor Effects of Specific CD8+ T Cells

Journal of Biomedical Nanotechnology ◽

10.1166/jbn.2019.2859 ◽

2019 ◽

Vol 15 (11) ◽

pp. 2229-2239 ◽

Cited By ~ 3

Author(s):

Zhuoran Tang ◽

Fengzhen Mo ◽

Aiqun Liu ◽

Siliang Duan ◽

Xiaomei Yang ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Tumor Cells ◽

Cytotoxic T Lymphocyte ◽

Ex Vivo ◽

T Cell Proliferation ◽

Tumor Cell Apoptosis ◽

Xenograft Models

Adoptive cell-based immunotherapy typically utilizes cytotoxic T lymphocytes (CTLs), expanding these cells ex vivo. Such expansion is traditionally accomplished through the use of autologous APCs that are capable of interactions with T cells. However, incidental inhibitory program such as CTLA-4 pathway can impair T cell proliferation. We therefore designed a nanobody which is specific for CTLA-4 (CTLA-4 Nb 16), and we then used this molecule to assess its ability to disrupt CTLA-4 signaling and thereby overcome negative costimulation of T cells. With CTLA-4 Nb16 stimulation, dendritic cell/hepatocellular carcinoma fusion cells (DC/HepG2-FCs) enhanced autologous CD8+ T cell proliferation and production of IFN-γ in vitro, thereby leading to enhanced killing of tumor cells. Using this approach in the context of adoptive CD8+ immunotherapy led to a marked suppression of tumor growth in murine NOD/SCID hepatocarcinoma or breast cancer xenograft models. We also observed significantly increased tumor cell apoptosis, and corresponding increases in murine survival. These findings thus demonstrate that in response to nanobody stimulation, DC/tumor cells-FC-induced specific CTLs exhibit superior anti-tumor efficacy, making this a potentially valuable means of achieving better adoptive immunotherapy outcomes in cancer patients.

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In Vitro Expansion of Autologous Follicular Lymphoma-Specific T Cells for Adoptive Transfer.

Blood ◽

10.1182/blood.v106.11.1482.1482 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1482-1482

Author(s):

Seung-Tae Lee ◽

Yun Fang Jiang ◽

Soung-Chul Cha ◽

Hong Qin ◽

Larry W. Kwak ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Follicular Lymphoma ◽

Tumor Cells ◽

Cd4 T Cells ◽

Cd40 Ligand ◽

Autologous Tumor ◽

T Cell Lines

Abstract Advanced stage follicular lymphoma remains an incurable disease with a median survival of 8 to 10 years that has not significantly changed over the last four decades. Therefore, novel treatment options are necessary to improve the clinical outcome in these patients. The observation of spontaneous regressions in a small percentage of patients suggested that augmenting the host immune response could potentially control this malignancy. Strategies using active specific immunotherapy with idiotype vaccines led to induction of clinical and molecular responses in a few patients but have met with only limited success possibly due to the low frequency of antigen-specific T cells induced in the patients. In contrast to active immunization, T cells of a given specificity and function may be selected and expanded in vitro to the desired number for adoptive cell transfer. Towards this goal, we stimulated tumor infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs) from five follicular lymphoma patients with CD40 ligand-activated autologous tumor cells at approximately ten-day intervals in the presence of IL-2 and IL-15. After four rounds of stimulations, T cell lines generated from 3/5 patients recognized autologous unmodified tumor cells by producing significant amounts of TNF-α, GM-CSF and/or IFN-γ. By phenotypic analysis, the T cell lines were predominantly CD4+ T cells (> 70%), and intracellular cytokine assay showed that up to 40% of the CD4+ T cells were tumor-reactive. The inhibition of cytokine production by anti-HLA class II but not class I blocking antibodies confirmed that the CD4+ T cells were tumor-reactive. Further characterization revealed that the T cells from one patient recognized autologous tumor but not autologous normal B cells suggesting that they were tumor-specific. While in a second patient CD4+ T cell clones generated from the T cell line by limiting dilution recognized autologous tumor and autologous normal B cells but not autologous monocytes suggesting that they were B cell lineage-specific. We conclude that follicular lymphoma-specific T cells exist and can be efficiently expanded in vitro from both TILs and PBMCs using CD40 ligand-activated autologous tumor cells for adoptive T cell therapy. Additionally, identification of antigens recognized by these T cells could lead to development of novel immunotherapeutic strategies for lymphomas.

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Antigen-dependent costimulation to improve T-cell therapy for cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.7_suppl.151 ◽

2017 ◽

Vol 35 (7_suppl) ◽

pp. 151-151

Author(s):

Christopher C. DeRenzo ◽

Phuong Nguyen ◽

Stephen Gottschalk

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Tumor Cells ◽

T Cell Activation ◽

Tumor Antigens ◽

Single Chain ◽

Antitumor Effects ◽

T Cell Therapy ◽

Potential Strategy

151 Background: T-cell therapy for cancer faces several challenges, including limited T-cell expansion at tumor sites, and lack of unique tumor antigens that are not expressed in normal tissues. To overcome the first obstacle, we developed Engager (ENG) T cells, which secrete bispecific molecules consisting of single chain variable fragments specific for CD3 and a tumor antigen. ENG T cells have the unique ability to redirect bystander T cells to tumors, amplifying antitumor effects. Costimulatory chimeric antigen receptors (CoCARs) are one potential strategy to restrict full T-cell activation to tumor sites that express a unique "antigen address." The goal of this project was now to generate T cells that express engager molecules and CoCARs (ENG/CoCAR T cells), which recognize distinct tumor antigens, and evaluate their effector function. Methods: We focused on two tumor antigens, EphA2 and HER2, which are expressed in a broad range of solid tumors. RD114-pseudotyped retroviral particles encoding an EphA2-ENG or a HER2-CoCAR were used to transduce CD3/CD28-activated human T cells. Transduced T cells were cocultured with EphA2+/HER2- or EphA2+/HER2+ tumor cells. Results: Both EphA2-ENG and EphA2-ENG/HER2-CoCAR T cells were activated by EphA2+ targets, as judged by IFNγ secretion. EphA2-ENG T cells secreted little IL-2 and died after one stimulation with EphA2+/HER2- or EphA2+/HER2+ tumor cells. In contrast, EphA2-ENG/HER2-CoCAR T cells secreted high levels of IL-2 and proliferated when stimulated with EphA2+/HER2+ cells. Little IL-2 secretion and no proliferation was observed after stimulation of the same T cells with EphA2+/HER2- cells, indicating these T cells are only fully activated in the presence of both target antigens. Upon repeated stimulation with EphA2+/HER2+ tumor cells, EphA2-ENG/HER2-CoCAR T cells continued to secrete IL-2 and proliferate without the addition of external cytokines for at least 10 weeks. Conclusions: EphA2-ENG/HER2-CoCAR T cells demonstrated robust dual antigen dependent IL-2 secretion, and continued proliferation upon repeat stimulation with EphA2+/HER2+ cells. Thus, providing antigen-specific costimulation is a potential strategy to improve the safety and efficacy of T-cell therapy for cancer.

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Ectonucleosidase CD39 Is Highly Expressed on ATLL Cells and Suppresses the Immune Response through the Adenosine Pathway

Blood ◽

10.1182/blood-2018-99-118555 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3720-3720

Author(s):

Yasuhiro Nagate ◽

Sachiko Ezoe ◽

Jiro Fujita ◽

Takafumi Yokota ◽

Michiko Ichii ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Line ◽

Tumor Cells ◽

Research Funding ◽

Poly I:C ◽

Acute Type ◽

Atll Cell

Abstract Background: Adult T-cell leukemia/lymphoma (ATLL) is a mature T-cell neoplasm, linked to the human T-cell lymphotropic virus, HTLV-1. Patients with ATLL are often at the risk of opportunistic infections. Some studies suggested that ATLL cells originate from HTLV-1-infected regulatory T cells (Tregs). It could be possible that this immunocompromised state is caused by the function of ATLL cells having similar phenotypes with Tregs. In this study, we examined the expression of immunosuppressive molecules associated with Tregs in ATLL cells, and analyzed their roles in the function of ATLL cells. Methods: The protocol of this study was approved by the Investigational Review Board of Osaka University Hospital. Peripheral blood mononuclear cells (PBMCs) were collected from 10 asymptomatic HTLV-1 carriers and 22 ATLL patients (1 with smoldering type, 5 with chronic type, 2 with lymphoma type, and 14 with acute type) after getting informed consent. PBMCs from 3 ATLL patients were separated into CD4+ CD7- CADM1+ATLL cells and adjacent CD4+CD7+ CADM1-normal T cells using Fluorescence-activated Cell Sorter (FACS), and cells in each fraction were subjected to total RNA sequencing experiments. Based on the results, we examined the expression patterns of CD39 and CD73 in HTLV-1 carriers or each type of ATLL patients, and also analyzed the immune functions of these molecules in ATLL tumor cells. Results: We compared whole transcriptome of ATLL cells and normal CD4+cells. Bioinformatic analyses showed that many genes associated with immunosuppressive functions were elevated or downregulated in ATLL cells. Among these genes we focused on CD39, CD73 and CD26, because they have recently been reported to be strongly associated with the functions of Tregs. CD39, expressed on normal Tregs, and extrinsic CD73 have immunosuppressive potential by catalyzing adenosine from extracellular ATP, and CD26 has opposite potential by resolving adenosine, which have a strong anti-inflammatory function and plays major role in Treg-mediated immunosuppression. We found that all of 4 ATLL cell lines (MJ, MT1, MT2, MT4) expressed CD39, but not CD73 just as human effector Tregs. Tumor cells from 12 acute ATLL patients (86%) and 2 chronic ATLL patients (40%) expressed CD39, but the expressions of CD73 were various. Also in asymptomatic carriers, we could detect CD39 and/or CD73 positive in CD7- CADM1+ abnormal fraction of CD4+cells. On the other hand, CD26, normally expressed on human CD4+Th cells other than effector Tregs, was negative in ATLL cell lines and primary ATLL cells except for cells in abnormal fraction of one asymptomatic carrier. CD39 negative cases in chronic/smoldering type tended to show slower disease progression after the blood collection. Next, the role of CD39 and/or CD73 in ATLL cells was assessed in vitro and in vivo. As expected, CD39+ ATLL cells converted significantly more extracellular ATP than CD39- ATLL cells, and mass spectrometry analysis of AMP/adenosine concentration identified the AMPase activity of CD73+ ATLL cells. Furthermore, we established CD39 knockout (KO) cells from ATL cell-line MJ using CRISPR/Cas9 system, and performed in vitro suppression assays for assessment of immunosuppressive function. Although wild type MJ suppressed the growth of normal CD4+ and CD8+ T cells, KO MJ did little. Next, we analyzed the role of CD39 in the progression of tumor cells in vivo. We transplanted mouse T-cell lymphoma cell-line EG7-OVA artificially expressing CD39 or mock into mice subcutaneously. The coinjection of immunoadjuvant poly(I:C) significantly suppressed the tumor growth of mock cells, but the tumor sizes of CD39 expressing cells were almost the same as those of mock cells without poly(I:C) injection (Figure). Conclusion: In this study, we reported that most of ATLL cells in acute type patients express CD39+ CD26- just as Tregs, and that CD39- KO of ATLL cell line cancelled its immunosuppressive effects, and forcibly expressed CD39 on tumor cells rejected the anti-tumor immunity in vivo. From these data, we clarified the pathological mechanism of immunosuppressive function in ATLL cells, and also showed that CD39 expression could be used as a prognostic clue and be a new therapeutic target of ATLL. Disclosures Ezoe: TAIHO Phamaceutical Co., Ltd.: Research Funding. Yokota:Celgene: Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer Inc.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; MSD K.K.: Research Funding. Ichii:Novartis Pharma K.K.: Speakers Bureau; Kowa Pharmaceutical Co.,LTD.: Speakers Bureau; Celgene K.K.: Speakers Bureau. Shibayama:Novartis Pharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Fujimoto Pharmaceutical: Honoraria, Research Funding; Jansen Pharmaceutical K.K: Honoraria; Ono Pharmaceutical Co.,LTD: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K: Honoraria, Research Funding. Oritani:Novartis Pharma: Speakers Bureau. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

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ADG152, a Novel CD20xCD3 T Cell Engager Prodrug with Enhanced Therapeutic Index, Demonstrates Strong Anti-Tumor Activity with Improved Safety

Blood ◽

10.1182/blood-2021-147573 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1204-1204

Author(s):

Bin Cai ◽

Aaron N Nguyen ◽

Songmao Zheng ◽

Jianfeng Shi ◽

Guizhong Liu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Board Of Directors ◽

Tumor Cells ◽

Cytokine Release ◽

Publicly Traded ◽

Advisory Committees ◽

The Past

Abstract Recent clinical data illustrate the effectiveness of CD20xCD3 T cell engagers (TCEs) that redirect the patient's endogenous T cells to eliminate CD20-positive tumor cells. While several of these products have demonstrated promising clinical activities in B-cell malignancies, their potential therapeutic utility is limited by cytokine release syndrome (CRS), even after strategies such as step-up dosing are implemented. ADG152 is a novel CD20xCD3 TCE prodrug engineered using Adagene's SAFEbody technology to minimize or eliminate CRS and on-target/off-tumor toxicities. The anti-CD20 arm of ADG152 has been engineered for enhanced binding to CD20 compared to other clinical stage or approved antibodies, while its anti-CD3 arm has modulated affinity for CD3 and is also masked by a conditionally activable peptide. In normal tissues and in circulation, the masking moiety on the anti-CD3 arm can function to block the binding of ADG152 to T cells; however, in an activable condition such as the tumor microenvironment where protease activity has been reported to be elevated, the masked antibody can be activated, enabling the activated ADG152 to simultaneously engage T cells and neighboring CD20-expressing tumor cells. In vitro studies showed that ADG152 has enhanced binding to human B cells and CD20-positive Raji tumor cells compared with the benchmark CD20xCD3 TCE plamotamab. On the other hand, ADG152 has significantly reduced binding to the human CD3 δ/ε protein dimer and no binding to human CD3+, CD4+, and CD8+ T cells isolated from PBMCs of normal human donors. Consistent with these results, ADG152 shows significantly decreased ability (more than 100-fold) compared with the benchmark and the unmasked parental molecule to activate CD8+ T cells and to induce T cell-mediated killing in the presence of tumor cells in vitro. ADG152 demonstrated strong anti-tumor effects in vivo. In a human PBMC-engrafted Raji xenograft mouse tumor model, dosing with ADG152 resulted in almost complete tumor growth inhibition at 1.5 mg/kg. In exploratory toxicology studies in cynomolgus monkeys, ADG152 resulted in significantly less cytokine release in monkey blood compared with benchmark, giving ~100-fold safety margin for ADG152 for cytokine induction (Figure). In addition, ADG152 was as effective as the benchmark at inducing B cell depletion from peripheral blood of cynomolgus monkeys. In summary, the preclinical characterization of ADG152 demonstrates that our SAFEbody platform can be used to engineer safe and potent bispecific T cell engagers with increased therapeutic index by allowing for strong anti-tumor activities in mice at doses with minimal cytokine release in monkeys, thereby supporting its advancement to clinical development either as a single agent or in combination with other therapies for the treatment of CD20-expressing B cell malignancies. Figure 1 Figure 1. Disclosures Cai: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Nguyen: Sparcbio, LLC: Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Zheng: Janssen Pharmaceuticals: Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Shi: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Liu: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Li: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Du: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Frankel: Cytovia Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company; Adagene Inc.: Consultancy, Current equity holder in publicly-traded company; Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; IMV: Consultancy; Precision Biosciences: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Sutro: Membership on an entity's Board of Directors or advisory committees; Immunai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Minerva Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Myeloid Therapeutics: Consultancy; RAPT Therapeutics: Consultancy; Syros: Consultancy. Luo: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Xu: Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company.

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