Abstract 1134: PET-imaging of89Zr-labeled bispecific T-cell engagers in syngeneic tumor bearing mice

AbstractDetermination of treatment response to immunotherapy in glioblastoma multiforme (GBM) is a process which can take months. Detection of CD8+ T cell recruitment to the tumor with a noninvasive imaging modality such as positron emission tomography (PET) may allow for tumor characterization and early evaluation of therapeutic response to immunotherapy. In this study, we utilized 89Zr-labeled anti-CD8 cys-diabody-PET to provide proof-of-concept to detect CD8+ T cell immune response to oncolytic herpes simplex virus (oHSV) M002 immunotherapy in a syngeneic GBM model. Immunocompetent mice (n = 16) were implanted intracranially with GSC005 GBM tumors, and treated with intratumoral injection of oHSV M002 or saline control. An additional non-tumor bearing cohort (n = 4) receiving oHSV M002 treatment was also evaluated. Mice were injected with 89Zr-labeled anti-CD8 cys-diabody seven days post oHSV administration and imaged with a preclinical PET scanner. Standardized uptake value (SUV) was quantified. Ex vivo tissue analyses included autoradiography and immunohistochemistry. PET imaging showed significantly higher SUV in tumors which had been treated with M002 compared to those without M002 treatment (p = 0.0207) and the non-tumor bearing M002 treated group (p = 0.0021). Accumulation in target areas, especially the spleen, was significantly reduced by blocking with the non-labeled diabody (p < 0.001). Radioactive probe accumulation in brains was consistent with CD8+ cell trafficking patterns after oHSV treatment. This PET imaging strategy could aid in distinguishing responders from non-responders during immunotherapy of GBM.

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Specific cytostatic effect of lymph node cells from normal and T cell-deficient mice on syngeneic tumor target cellsin vitro and its specific abrogation by body fluids from syngeneic tumor-bearing mice

European Journal of Immunology ◽

10.1002/eji.1830030806 ◽

1973 ◽

Vol 3 (8) ◽

pp. 483-487 ◽

Cited By ~ 31

Author(s):

Eunice Chia ◽

H. Festenstein

Keyword(s):

Lymph Node ◽

T Cell ◽

Body Fluids ◽

Cytostatic Effect ◽

Tumor Target ◽

Tumor Bearing ◽

Syngeneic Tumor ◽

Lymph Node Cells ◽

Deficient Mice

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398 AGEN1181, an Fc engineered anti-CTLA-4 antibody, demonstrates clinical activity, alone or in combination with balstilimab (anti-PD-1), and broadens the therapeutic potential of CTLA-4 therapy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0398 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A423-A423

Author(s):

Steven O’Day ◽

Anthony El khoueiry ◽

Chethan Ramamurthy ◽

Andrea Bullock ◽

Irina Shapiro ◽

...

Keyword(s):

T Cell ◽

Mouse Models ◽

T Cell Activation ◽

Cell Activation ◽

Tumor Control ◽

Clinical Activity ◽

Small Subset ◽

Tumor Bearing Mouse ◽

High Affinity ◽

Tumor Bearing

BackgroundImmune checkpoint therapies targeting CTLA-4, alone, or in combination with anti-PD-1 have shown durable responses in cancer patients. However, responses are limited to a small subset of patients in the most common immunogenic cancers. Here we describe, a novel anti-CTLA-4 antibody, AGEN1181, with enhanced FcyR-dependent functionality that harnesses a novel mechanism of action to promote superior T cell activation and anti-cancer immunity. Concordant with preclinical findings, we report preliminary safety, pharmacodynamic and efficacy data from a phase 1 study of AGEN1181 (NCT03860272), alone or in combination with balstilimab (anti-PD-1 antibody) in a range of immunogenic and non-immunogenic tumors.MethodsThe functional activity of AGEN1181 or AGEN1181-like mouse surrogate were assessed in primary cell-based assays or in PD-1 refractory syngeneic tumor-bearing mouse models (B16F10 or KPC pancreatic tumor). Efficacy was evaluated as monotherapy, or in combination with anti-PD-1, focal radiation or chemotherapy. In an ongoing phase I study, AGEN1181 is administered intravenously once every 3- or 6-weeks as monotherapy (0.1–4 mg/kg), or every 6-weeks (1–4 mg/kg) in combination with balstilimab (3 mg/kg) dosed every 2 weeks. Dose-limiting toxicities were evaluated in the first 28 days of treatment. Neoantigen burden was assessed from pre-treatment tumor biopsy, as available, by next-generation sequencing. Fcγ receptor genotyping was assessed by real-time PCR. Immunophenotyping of peripheral blood mononuclear cells collected pre- and post-treatment were analyzed by flow cytometry.ResultsPreclinically, AGEN1181 demonstrated superior T cell activation than a standard IgG1 anti-CTLA-4 analogue in donors expressing either the low or high affinity FcγRIIIA. In poorly immunogenic tumor-bearing mouse models, AGEN1181-like surrogate demonstrated robust tumor control in combination with anti-PD-1 and focal radiation or chemotherapy. As of August 25th, 2020, we observed a clinical benefit rate of 63–53% at 6 and 12 weeks respectively among evaluable treated patients. We observed two durable responses in patients with endometrial cancer that were BRCA-, microsatellite stable and PD-L1 negative. These patients progressed on prior PD-1 therapy or chemoradiation respectively. Notably, responders expressed either the low or high affinity FcγRIIIA. AGEN1181 showed potent dose-dependent increases in peripheral CD4+Ki67+, CD4+ICOS+ and CD4+HLA-DR+ T-cells. Treatment was well tolerated through the highest dose tested. Grade 3 or greater immune-related adverse events occurred in 28.5% patients and were consistent with CTLA-4 therapies.ConclusionsAGEN1181 is designed to expand the benefit of anti-CTLA-4 therapy to a broader patient population. AGEN1181, alone or in combination with balstilimab, demonstrates clinical activity in heavily pretreated patients.Trial RegistrationNCT03860272

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49 In situ phenotypic analysis of T cells in the tumor microenvironment of a pre-clinical model of non-small cell lung cancer (NSCLC) by tissue sectioning and whole-mount immunofluorescence imaging

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0049 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A52-A52

Author(s):

Elen Torres ◽

Stefani Spranger

Keyword(s):

T Cells ◽

Spatial Distribution ◽

T Cell ◽

Spatial Location ◽

Cell Infiltration ◽

Cell Populations ◽

Tumor Bearing ◽

T Cell Infiltration ◽

Volume Imaging ◽

Whole Mount

BackgroundUnderstanding the interactions between tumor and immune cells is critical for improving current immunotherapies. Pre-clinical and clinical evidence has shown that failed T cell infiltration into lung cancer lesions might be associated with low responsiveness towards checkpoint blockade.1 For this reason, it is necessary to characterize not only the phenotype of T cells in tumor-bearing lungs but also their spatial location in the tumor microenvironment (TME). Multiplex immunofluorescence staining allows the simultaneous use of several cell markers to study the state and the spatial location of cell populations in the tissue of interest. Although this technique is usually applied to thin tissue sections (5 to 12 µm), the analysis of large tissue volumes may provide a better understanding of the spatial distribution of cells in relation to the TME. Here, we analyzed the number and spatial distribution of cytotoxic T cells and other immune cells in the TME of tumor-bearing lungs, using both 12 µm sections and whole-mount preparations imaged by confocal microscopy.MethodsLung tumors were induced in C57BL/6 mice by tail vein injection of a cancer cell line derived from KrasG12D/+ and Tp53-/- mice. Lung tissue with a diverse degree of T cell infiltration was collected after 21 days post tumor induction. Tissue was fixed in 4% PFA, followed by snap-frozen for sectioning. Whole-mount preparations were processed according to Weizhe Li et al. (2019) 2 for tissue clearing and multiplex volume imaging. T cells were labeled with CD8 and FOXP3 antibodies to identify cytotoxic or regulatory T cells, respectively. Tumor cells were labeled with a pan-Keratin antibody. Images were acquired using a Leica SP8 confocal microscope. FIJI3 and IMARIS were used for image processing.ResultsWe identified both cytotoxic and regulatory T cell populations in the TME using thin sections and whole-mount. However, using whole-mount after tissue clearing allowed us to better evaluate the spatial distribution of the T cell populations in relation to the tumor structure. Furthermore, tissue clearance facilitates the imaging of larger volumes using multiplex immunofluorescence.ConclusionsAnalysis of large lung tissue volumes provides a better understanding of the location of immune cell populations in relation to the TME and allows to study heterogeneous immune infiltration on a per-lesion base. This valuable information will improve the characterization of the TME and the definition of cancer-immune phenotypes in NSCLC.ReferencesTeng MW, et al., Classifying cancers based on T-cell infiltration and PD-L1. Cancer Res 2015;75(11): p. 2139–45.Li W, Germain RN, and Gerner MY. High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging. Nat Protoc 2019;14(6): p. 1708–1733.Schindelin J, et al, Fiji: an open-source platform for biological-image analysis. Nat Methods 2012;9(7): p. 676–82.

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377 AGEN2373 is a CD137 agonist antibody designed to leverage optimal CD137 and FcγR co-targeting to promote antitumor immunologic effects

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0377 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A402-A402

Author(s):

Claire Galand ◽

Vignesh Venkatraman ◽

Marilyn Marques ◽

James Strauss ◽

Richard Carvajal ◽

...

Keyword(s):

T Cell ◽

Cell Expansion ◽

Phase 1 ◽

Cross Linking ◽

Agonist Activity ◽

Critical Factors ◽

Tumor Bearing ◽

Potent Agonist ◽

Agonist Antibody ◽

Crd Iv

BackgroundCD137 (4-1BB) represents a costimulatory pathway that promotes T, NK, and dendritic cell effector functions favorable for antitumor immunity. The extracellular domain of CD137, comprised of four cysteine-rich domains (CRD-I, CRD-II, CRD-III, CRD-IV), trimerizes upon binding to CD137 ligand (CD137L) to induce cell stimulatory transcriptional and epigenetic changes.1 2 The investigation of CD137-targeting agonist antibody, urelumab (CRD-I-binding, IgG4), in human subjects showed immunologic and pharmacodynamic effects, but poor efficacy due to dose-limiting liver toxicity.3 Preclinical studies using a murine surrogate antibody, clone 3H3 (CRD-I-binding, rIgG2a), also demonstrated hepatotoxicity that correlated with activation of CD137-expressing myeloid cells and memory CD8+ T cells.4 5 In contrast, utomilumab (CRD-II/III-binding, IgG2) showed acceptable tolerability, but limited clinical efficacy.6 7 These and more recent findings implicate epitope and Fc gamma receptor (FcγR)-dependent antibody cross-linking as critical factors for CD137 therapeutic antibody design.MethodsWe investigated the molecular and cellular effects of AGEN2373 (CRD-IV-binding, IgG1), a conditionally active CD137-targeting agonist antibody designed to bind and induce CD137 signaling upon FcγR cross-linking while permitting ligand binding to CD137. The role of epitope and FcγR binding as critical factors for anti-CD137 therapeutic activity were elucidated in primary cell-based assays and syngeneic tumor-bearing mouse models using anti-mouse antibody clones S3B1 (CRD-IV-binding) and 3H3, surrogates of AGEN2373 and urelumab, respectively. In an ongoing phase 1 trial (NCT04121676), we evaluated the safety and tolerability of AGEN2373.ResultsAGEN2373 bound with high-affinity to CD137 CRD-IV and promoted potent agonist activity of CD137 that was conditionally dependent on Fc-dependent antibody cross-linking. AGEN2373 surrogate, S3B1, showed comparable binding and cross-link dependent agonist activity. In CT26 tumor-bearing mice, S3B1 and 3H3 demonstrated complete tumor control that was not reproducible with a Fc-silent S3B1 antibody. The Fc-dependent activity of S3B1 correlated with induced immunologic changes in the TME including CD8 T cell expansion, NK cell activation, and Treg depletion. Patients with advanced solid cancers, treated with AGEN2373 up to 1 mg/kg every 4 weeks, demonstrate clinical activity with no evidence of hepatotoxicity.ConclusionsConditional and potent agonist activity of AGEN2373 is dependent on binding to CD137 CRD-IV and FcγR. Preclinically, our data demonstrate that AGEN2373-like murine surrogate antibodies promote potent immune activation and anti-tumor immunity. Phase 1 clinical trials investigating the safety and efficacy of AGEN2373, alone or combination with balstilimab (anti-PD-1), are underway.Trial RegistrationNCT04121676ReferencesWen TJ, Bukczynski and Watts TH. 4-1BB ligand-mediated costimulation of human T cells induces CD4 and CD8 T cell expansion, cytokine production, and the development of cytolytic effector function. J Immunol 2002;168(10): p. 4897–906.Bitra A, et al. Crystal structures of the human 4-1BB receptor bound to its ligand 4-1BBL reveal covalent receptor dimerization as a potential signaling amplifier. J Biol Chem 2018;293(26): p. 9958–9969.Segal NH, et al., Results from an integrated safety analysis of urelumab, an agonist anti-CD137 monoclonal antibody. Clin Cancer Res 2017;23(8): p. 1929–1936.Bartkowiak T, et al., Activation of 4-1BB on liver myeloid cells triggers hepatitis via an interleukin-27-dependent pathway. Clin Cancer Res 2018;24(5): p. 1138–1151.Lin GH, et al., GITR-dependent regulation of 4-1BB expression: implications for T cell memory and anti-4-1BB-induced pathology. J Immunol 2013;190(9): p. 4627–39.Segal, N.H., et al., Phase I study of single-agent utomilumab (PF-05082566), a 4-1BB/CD137 agonist, in patients with advanced cancer. Clin Cancer Res 2018;24(8): p. 1816–1823.Li Y, et al., Limited Cross-Linking of 4-1BB by 4-1BB ligand and the agonist monoclonal antibody utomilumab. Cell Rep 2018;25(4): p. 909–920 e4.

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253 Anti-TIGIT antibodies require enhanced FcγR co-engagement for optimal T and NK cell-dependent anti-tumor immunity

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0253 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A275-A275

Author(s):

Rebecca Ward ◽

Elena Paltrinieri ◽

Marilyn Marques ◽

Priyadarshini Iyer ◽

Sylvia Dietrich ◽

...

Keyword(s):

T Cell ◽

Tumor Microenvironment ◽

Tumor Immunity ◽

Cell Activation ◽

Nk Cell ◽

Tumor Control ◽

Tumor Bearing ◽

Anti Tumor Activity ◽

Ct26 Tumor ◽

Dependent Mechanism

BackgroundT-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an important negative regulator of the immune response to cancer that contributes to resistance/relapse to anti-PD-1 therapy.1 In clinical trials, anti-human (h) TIGIT antibodies have shown promising activity in combination with anti-PD-1/PD-L1 antibodies for the treatment of various solid tumors.2 However, the optimal format for anti-TIGIT antibodies remains controversial. Here we describe a novel Fcγ receptor (FcγR)-dependent mechanism of action that is critical for enhancing T and NK cell anti-tumor immunity, and, further informs on the optimal design of anti-TIGIT antibodies.MethodsWe investigated a panel of Fc-silent, Fc-competent, and Fc-engineered anti-mouse (m) TIGIT antibody variants in syngeneic murine CT26 tumor-bearing or B16F10 pseudo-metastases models. To further elucidate the relative contribution of T and NK cells in controlling tumor growth, we assessed the activity of Fc-engineered anti-TIGIT antibodies in NK cell-depleted or T cell-deficient (Nu-Foxn1nu) CT26 tumor-bearing mice. Immune-related pharmacodynamic changes in the tumor microenvironment were assessed by flow cytometry. We further validated these findings in primary human T and NK cell activation assays using Fc-engineered anti-human TIGIT antibodies.ResultsThe Fc-engineered anti-mTIGIT antibody, which demonstrates enhanced binding to mouse FcγRIV, was the only variant to deliver single agent anti-tumor activity. The Fc-enhanced variant outperformed the Fc-competent variant while the Fc-inert variant had no anti-tumor activity. Tumor control by anti-mTIGIT antibodies was not dependent on Treg depletion, but rather on increased frequency of CD8+ T cells and activated NK cells (Ki67, IFNγ, CD107a and TRAIL) in the tumor microenvironment. Concordant with observations in the mouse, Fc-engineered anti-hTIGIT antibodies with improved binding to FcγRIIIA demonstrate superior T and NK cell activation in PBMC-based assays compared to a standard hIgG1 variant. Notably, superior activity of the Fc-engineered anti-hTIGIT antibody was observed from PBMC donors that express either high or low affinity FcγRIIIA. Blockade of FcγRIIIA or depletion of CD14+ and CD56+ cells reduced the functional activity of the Fc-enhanced anti-TIGIT antibody, confirming the requirement for FcγR co-engagement to maximize T cell responses.ConclusionsOur data demonstrate the importance of FcγR co-engagement by anti-TIGIT antibodies to promote immune activation and tumor control. First generation anti-TIGIT antibodies are not optimally designed to co-engage all FcγRIIIA variants. However, Fc-enhanced anti-TIGIT antibodies unlock a novel FcγR-dependent mechanism of action to enhance T and NK cell-dependent anti-tumor immunity and further improve therapeutic outcomes.ReferencesJohnston RJ, et al., The immunoreceptor TIGIT regulates antitumor and antiviral CD8(+) T cell effector function. Cancer Cell 2014; 26:923–37.Rodriguez-Abreu D, et al., Primary analysis of a randomized, double-blind, phase II study of the anti-TIGIT antibody tiragolumab (tira) plus atezolizumab (atezo) versus placebo plus atezo as first-line (1L) treatment in patients with PD-L1-selected NSCLC (CITYSCAPE). Journal of Clinical Oncology 2020; 38:15_suppl, 9503–9503.

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