591 Comparison of two oHSV Vectors for the treatment of glioblastoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A626-A626
Author(s):  
Joseph Jackson ◽  
Bonnie Hall ◽  
Lisa Bailey ◽  
E Chiocca ◽  
Justus Cohen
Keyword(s):  
Tumor Microenvironment ◽  
Immune Cell ◽  
Polymorphonuclear Cells ◽  
List Type ◽  
Cell Composition ◽  
Animal Survival ◽  
Strain Derivative ◽  
Anti Tumor Activity

BackgroundGlioblastoma multiforme (GBM) is the most common human brain cancer. Despite a well-established standard of care, the 5-year mortality rate of GBM patients is 95%, highlighting the need for innovative therapeutic interventions. A variety of oncolytic viruses, including those derived from herpes simplex virus (oHSV), have been designed for GBM therapy, but early-phase clinical trials have reported few complete responses and no evidence of durable anti-tumor immunity. Potential reasons for the lack of efficacy are limited vector potency (i.e., virulence) and the presence of a highly immunosuppressive tumor microenvironment (TME) comprised of few activated lymphocytes, large numbers of immunosuppressive myeloid cells (macrophages, myeloid derived suppressor cells [MDSCs], microglia), and an agglomerate of immunosuppressive cytokines (IL-10, VEGF, MIF, etc.).1 Herein we explore these obstacles by comparing the anti-tumor activity two different oHSV designs, an HSV-1 KOS strain derivative designated KG4:T124, and an F strain derivative designated rQNestin34.5v.1 (a similar oHSV, rQNestin34.5v.2, is currently in a phase I clinical trial for GBM).2MethodsUsing the murine syngeneic GBM models, GL261N4 and CT2A, we compared the anti-tumor activity of KG4:T124 and rQNestin34.5v1. In vitro, we evaluated the viral entry, replication capacity, and cytotoxicity of both oHSVs. In vivo, we measured the impact of both vectors on tumor progression, TME immune cell composition, and animal survival.ResultsVirus entry into cancer cells of KG4:T124 or rQNestin34.5v1 was relatively similar, but rQNestin34.5v.1 replicated more effectively and generally induced greater viral mediated cytotoxicity. In syngeneic mice, rQNestin34.5v.1 reduced orthotopic GL261N4 tumor burden and enhanced animal survival compared to KG4:T124. However, preliminary data indicate that multiple injections of KG4:T124 but not rQNestin34.5 enhance GL261N4 survival outcome. Neither oHSV impacted survival outcomes in the more pernicious CT2A model. Analysis, of either the GL261N4 or CT2A TME two days post virus administration revealed that both viruses had reduced microglia cell frequency, induced the influx of tumor associated macrophages and polymorphonuclear cells, but did not alter the frequency of monocytic MDSCs, natural killer cells, CD8+ or CD4+ T-cells.ConclusionsrQNestin34.5 had greater oncolytic activity In vivo and in vitro, but did not benefit from multiple oHSV injections. Both viruses induced similar changes in the TME immune cell composition. However, the presence of vital adaptive immune cell types within the TME was not observed at 2 days post oHSV treatment.AcknowledgementsResearch reported in this abstract was supported by the National Institutes of Health grants P01CA163205 (EAC & JCG) and 5T32CA082084-19 (JWJ).ReferencesMuller S, Kohanbash G, Liu SJ, Alvarado B, Carrera D, Bhaduri A, Watchmaker PB, Yagnik G, Di Lullo E, Malatesta M, Amankulor NM, Kriegstein AR, Lim DA, Aghi M, Okada H, Diaz A. Single-cell profiling of human gliomas reveals macrophage ontogeny as a basis for regional differences in macrophage activation in the tumor microenvironment. Genome Biol. 2017;18(1):234. Epub 2017/12/22. doi: 10.1186/s13059-017-1362-4. PubMed PMID: 29262845; PMCID: PMC5738907.Chiocca EA, Nakashima H, Kasai K, Fernandez SA, Oglesbee M. Preclinical Toxicology of rQNestin34.5v.2: An Oncolytic Herpes Virus with Transcriptional Regulation of the ICP34.5 Neurovirulence Gene. Mol Ther Methods Clin Dev 2020;17:871–93. Epub 2020/05/07. doi: 10.1016/j.omtm.2020.03.028. PubMed PMID: 32373649; PMCID: PMC7195500.

scholarly journals 888 An integrative approach to optimize a synthetic EphA2/CD137 agonist: balancing potency, physiochemical properties, and pharmacokinetics to achieve robust anti-tumor activity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A931-A931
Author(s):  
Punit Upadhyaya ◽  
Gemma Mudd ◽  
Kristen Hurov ◽  
Johanna Lahdenranta ◽  
Elizabeth Repash ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Cell ◽  
Immune Cell ◽  
List Type ◽  
Continuous Exposure ◽  
Target Engagement ◽  
In Vivo Models ◽  
Anti Tumor Activity

BackgroundCD137 (4-1BB) is a resurging target in immunotherapy after the first generation of monoclonal antibodies were limited by hepatotoxicity1 or lack of efficacy.2 A new generation of CD137 agonists are now in clinical development but they exclusively utilize large molecules derived from recombinant technology and are associated with long circulating terminal half-lives.3–6 Unlike checkpoint inhibition where complete saturation of the receptors drives the reversal of immunosuppression, intermittent target engagement that reflects the physiological context of T cell co-stimulation may be more appropriate for a CD137 agonist.7 Bicyclic peptides or Bicycles are a class of small (MW~2kDa), highly constrained peptides characterized by formation of two loops cyclized around a symmetric scaffold. To develop a differentiated tumor antigen dependent CD137 agonist for treating EphA2 expressing solid tumors, we integrated structure activity relationship (SAR) data from biochemical binding studies and in-vitro and in-vivo models to understand the relationship between exposure, target engagement and preclinical efficacy.MethodsOver 150 different EphA2/CD137 tumor-targeted immune cell agonists (Bicycle TICAs) were synthesized by linking Bicycle® binders to EphA2 to those binding CD137.8 The molecules were assessed in vitro using a CD137 reporter assay and by measuring cytokine production from primary human PBMC in tumor cell co-cultures. The pharmacokinetics were evaluated in rodents using Phoenix WinNonlin. The in vivo activity was determined in syngeneic mouse tumor models by measuring tumor growth kinetics and using tumor immune cell and transcriptional profiling by IHC and NanoString.ResultsEvaluation of the Bicycle TICAs in co-culture assays with EphA2-expressing tumor cell lines and Jurkat reporter cells overexpressing CD137 or human PBMCs demonstrated that constructs bearing two CD137 binding Bicycles to one EphA2 binding Bicycle (1:2 format) were more potent than the 1:1 format.8 Several Bicycle TICAs with amino acid substitutions to the EphA2 binding Bicycle maintained sub-nanomolar potency in-vitro and exhibited a plasma terminal half-life (t1/2) in rodents ranging from 0.4 and 4.0 h. Modifications that conferred aqueous solubility of greater than 10 mg/mL were considered suitable for further development. Treatment of MC38 tumors in immunocompetent mice with this series of molecules demonstrated that low MW Bicycle TICAs with sub-nanomolar potency and a t½ of ~1 h in mouse maintained target coverages necessary to produce robust modulation of the tumor immune microenvironment and tumor regression.ConclusionsA differentiated EphA2-dependent CD137 agonist was developed that exploits intermittent rather than continuous exposure for robust anti-tumor activity.ReferencesSegal NH, Logan TF, Hodi FS, et al. Results from an integrated safety analysis of urelumab, an agonist anti-CD137 monoclonal antibody. Clin Cancer Res 2017;23(8):1929–1936.Segal NH, Aiwu RH, Toshihiko D, 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):1816–1823.Chester C, Sanmamed MF, Wang J, Melero I. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood 2018;131(1):49–57.Hinner MJ, Aiba RSB, Jaquin TJ, et al. Tumor-localized costimulatory T-cell engagement by the 4-1BB/HER2 bispecific antibody-anticalin fusion PRS-343. Clin Cancer Res 2019;25(19):5878–5889.Claus C, Ferrara, C, Xu W, et al. Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy. Sci Transl Med 2019;11(496):eaav5989.Eskiocak U, Guzman W, Wolf B, et al. Differentiated agonistic antibody targeting CD137 eradicates large tumors without hepatotoxicity. JCI Insight 2020;5(5):e133647.Mayes PA, Hance KW, Hoos A. The promise and challenges of immune agonist antibody development in cancer. Nat Rev Drug Discov 2018;17:509–27.Upadhyaya P, Lahdenranta J, Hurov K, et al. Anticancer immunity induced by a synthetic tumor-targeted CD137 agonist. J Immunother Cancer 2021;9:e001762.

705 Anti-tumor activity of CB-668, a potent, selective and orally bioavailable small-molecule inhibitor of the immuno-suppressive enzyme Interleukin 4 (IL-4)-Induced Gene 1 (IL4I1)

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A747-A747
Author(s):  
Andrew MacKinnon ◽  
Deepthi Bhupathi ◽  
Jason Chen ◽  
Tony Huang ◽  
Weiqun Li ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Tumor Microenvironment ◽  
Immune Cell ◽  
Interleukin 4 ◽  
Mouse Tumor ◽  
Syngeneic Mouse ◽  
Molecule Inhibitor ◽  
Anti Tumor Activity ◽  
Mouse Tumor Models

BackgroundTumors evade destruction by the immune system through multiple mechanisms including altering metabolism in the tumor microenvironment. Metabolic control of immune responses occurs through depletion of essential nutrients or accumulation of toxic metabolites that impair immune cell function and promote tumor growth. The secreted enzyme interleukin 4 (IL-4)-induced gene 1 (IL4I1) is an L-phenylalanine oxidase that catabolizes phenylalanine and produces phenyl-pyruvate and hydrogen peroxide. IL4I1 regulates several aspects of adaptive immunity in mice, including inhibition of cytotoxic T cells through its production of hydrogen peroxide (reviewed in1). In human tumors, IL4I1 expression is significantly elevated relative to normal tissues and is notably high in ovarian tumors and B cell lymphomas. Motivated by the hypothesis that IL4I1 is an immuno-metabolic enzyme that suppresses anti-tumor immunity, we discovered CB-668, the first known small-molecule inhibitor of IL4I1.MethodsIL4I1 enzymatic activity was measured using an HRP-coupled enzyme assay. RNA in-situ hybridization was carried out on the RNAScope platform. Syngeneic mouse tumor models were used to evaluate the anti-tumor activity of CB-668. The level of phenyl-pyruvate in tumor homogenates was measured by LC/MS.ResultsOur clinical candidate, CB-668 is a potent and selective non-competitive inhibitor of IL4I1 (IC50 = 15 nM). CB-668 has favorable in vitro ADME properties and showed low clearance and high oral bioavailability in rodents. Twice-daily oral administration of CB-668 was well-tolerated in mice and resulted in single-agent anti-tumor activity in the syngeneic mouse tumor models B16-F10, A20, and EG7. Oral CB-668 administration reduced the levels of phenyl-pyruvate in the tumor, consistent with inhibition of IL4I1 enzymatic activity. Anti-tumor activity of CB-668 was immune cell-mediated since efficacy was abrogated in CD8-depleted mice, and CB-668 treatment caused increased expression of pro-inflammatory immune genes in the tumor. Moreover, CB-668 had no direct anti-proliferative activity on tumor cells grown in vitro (IC50 > 50 µM). CB-668 also favorably combined with anti-PD-L1 therapy to reduce tumor growth in the B16-F10 tumor model.ConclusionsThese data support an immune-mediated anti-tumor effect of IL4I1 inhibition by CB-668, and suggest inhibition of IL4I1 represents a novel strategy for cancer immuno-therapy.ReferencesMolinier-Frenkel V, Prévost-Blondel A, and Castellano F. The IL4I1 Enzyme: A New Player in the Immunosuppressive Tumor Microenvironment. Cells 2019;8:1–9.

In Vivo Evaluation of Immunomodulating Effects of Lenalidomide (L) on Tumor Cell Microenvironment as a Possible Underlying Mechanism of the Antitumor Effects Observed in Patients (pts) with Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2975-2975 ◽  
Author(s):  
Asher Alban Chanan-Khan ◽  
Swaminathan Padmanabhan ◽  
Kena C. Miller ◽  
Paula Pera ◽  
Laurie DiMiceli ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Clinical Study ◽  
Tumor Microenvironment ◽  
Antitumor Activity ◽  
Immune Cell ◽  
Underlying Mechanism ◽  
Antitumor Effects ◽  
Absolute Increase

Abstract Introduction: L is a more potent analogue of thalidomide with antitumor activity reported in MDS and multiple myeloma. Clinical anti-leukemic activity of L is reported for the first time by our group in pts with CLL. The underlying mechanism of its antitumor activity remains undetermined. We investigated the effect of L on the tumor microenvironment and studied the modulation of soluble cytokines and immune cells (T and NK cells) in pts receiving L. Patients and Methods: CLL pts enrolled on the clinical study with L were eligible. Pre and post (day 7) samples were obtained for evaluation of changes in serum cytokine and immune cell environment. Malignant cells were also obtained to investigate the in vitro antitumor activity of L prior to initiating treatment on clinical trial. Results: With Anexin V staining for evaluation of apoptosis induction, in vitro testing of pts samples (n=10) showed only a modest increase in apoptosis at 200mg of L - levels clinically not achievable. Yet same pts treated with L on clinical study showed significant antitumor response, suggesting the mechanism to be possibly related to modulation of the tumor microenvironment. In evaluation of the tumor cytokine microenvironment (n= 10) we noted significant L induced increase in IL-10 (n=6), IL-8 (n=8), IP-10 (n=10), IL-8 (n=8), TNF-alpha (n=4) and decrease in PDGF (n=5) and RANTES (n=5). While evaluation of the immune cell repertoire we observed an absolute increase in T-cell as well as NK-cell after treatment with L. Conclusion: Our in vitro evaluation does not suggest a direct apoptotic effect of L on the malignant CLL cells and thus support the hypothesis that the anti-leukemic effect noted in the clinical trial (reported separately) is most likely from in vivo modulation of the tumor microenvironment as is demonstrated from changes in the cytokine milieu and the cellular immune response. Collectively these changes may be responsible for the immune modulating properties of L and the resultant anti-CLL activity in pts.

scholarly journals Single cell transcriptomics reveals the effect of PD-L1/TGF-β blockade on the tumor microenvironment

2020 ◽  
Author(s):  
Yoong Wearn Lim ◽  
Garry L. Coles ◽  
Savreet K. Sandhu ◽  
David S. Johnson ◽  
Adam S. Adler ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
Single Cell ◽  
Immune Cell ◽  
Single Cells ◽  
Cell Infiltration ◽  
Cytotoxic Lymphocytes ◽  
T Cell Infiltration ◽  
Anti Tumor Activity

AbstractBackgroundThe anti-tumor activity of anti-PD-1/PD-L1 therapies correlates with T cell infiltration in tumors. Thus, a major goal in oncology is to find strategies that enhance T cell infiltration and efficacy of anti-PD-1/PD-L1 therapy. TGF-β has been shown to contribute to T cell exclusion and anti-TGF-β improves anti-PD-L1 efficacy in vivo. However, TGF-β inhibition has frequently been shown to induce toxicity in the clinic, and the clinical efficacy of combination PD-L1 and TGF-β blockade has not yet been proven. To identify strategies to overcome resistance to PD-L1 blockade, the transcriptional programs associated with PD-L1 and/or TGF-β blockade in the tumor microenvironment should be further elucidated.ResultsFor the first time, we used single-cell RNA sequencing to characterize the transcriptomic effects of PD-L1 and/or TGF-β blockade on nearly 30,000 single cells in the tumor and surrounding microenvironment. Combination treatment led to upregulation of immune response genes, including multiple chemokine genes such as CCL5, in CD45+ cells, and down-regulation of extracellular matrix genes in CD45-cells. Analysis of publicly available tumor transcriptome profiles showed that the chemokine CCL5 was strongly associated with immune cell infiltration in various human cancers. Further investigation with in vivo models showed that intratumorally administered CCL5 enhanced cytotoxic lymphocytes and the anti-tumor activity of anti-PD-L1.ConclusionsTaken together, our data could be leveraged translationally to improve anti-PD-L1 plus anti-TGF-β combination therapy, for example through companion biomarkers, and/or to identify novel targets that could be modulated to overcome resistance.

scholarly journals Daratumumab, a Novel Anti-CD38 Monoclonal Antibody Shows Anti-Tumor Activity in CLL and hampers Leukemia-Microenvironment Interactions

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4680-4680 ◽  
Author(s):  
Alba Matas-Céspedes ◽  
Anna Vidal-Crespo ◽  
Vanina Rodriguez ◽  
Julio Delgado ◽  
Neus Villamor ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Migration ◽  
Cell Adhesion ◽  
Tumor Microenvironment ◽  
Primary Cells ◽  
Isotype Control ◽  
Mmp9 Expression ◽  
Anti Tumor Activity

Abstract Daratumumab (DARA) is a anti-human CD38 antibody with Fc-mediated cell killing activity. DARA induces killing of tumor cells, mainly via complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers M. J Immunol 2011), and antibody-dependent cellular phagocytosis (ADCP) by macrophages (mΦ), both murine and human in multiple myeloma (MM) and Burkitt lymphoma cells. DARA is currently being evaluated in phase III clinical trials in patients with MM. We have previously reported that DARA induces cytotoxic activity in vitro via ADCC in primary cells and cell lines from Chronic Lymphoctic Leukemia (CLL), and significantly prolongs overall survival of animals in a systemic CLL mouse model. Here, we present additional data on in vivo mechanism of DARA and its effect on tumor-microenvironment interactions in CLL. We first evaluated whether ADCP contributes to DARA activity both in vitro and in vivo. For in vitro ADCP, mΦ were generated from monocytes of normal PBMCs and stimulated with GM-CSF (10ng/mL, 7 days). CLL cell lines and primary cells were labeled with calcein and incubated for 4h with mΦ at an effector:target ratio of 2:1 in the presence of a fixed mAb concentration of 1 μg/mL, followed by flow cytometric analysis. The amount of remaining CLL target cells (CD19+, CD11b-) was reduced by 3-16%. ADCP defined as percentage of mφ which had phagocytosed, referred to as double positive mΦ (CD11b+, calcein+, CD19-), ranged from 3-10%. To analyze ADCP in vivo, SCID beige mice, devoid of NK cells but with active macrophages, were inoculated intraperitoneally with CLL cells (20×106) and simultaneously treated with a single dose of DARA or isotype control (20mg/kg, n=3-5 per group). Forty-eight hours later, CLL cells were recovered from the intraperitoneal cavity and counted in a flow cytometer (identified as human CD45+/CD19+/CD5+cells). In DARA-treated mice the number of CLL cells recovered was reduced by 42% (n=2, p<0.05) compared to the isotype control group. Remarkably, the decrease in cell number was already detectable 2h after DARA administration. CLL pathogenesis relies on supportive tumor-microenvironment interactions both in the bone marrow (BM) and in the lymph node (LN), and CD38 constitutes a molecular hub integrating proliferative and migratory signals for CLL (Malavasi, F. Blood 2011). We evaluated the effect of DARA on migration and adhesion. In in vitro migrations assays, we have demonstrated that DARA (10-30 μg/mL) inhibited CXCL12/SDF1α-mediated migration up to 70% (n=5). In addition, DARA reduced up to 55% (n=2) of downstream pERK activation, that peaked after 5min of CXCL12/SDF1α stimulation. We analyzed the effect of DARA on primary CLL cell migration from Peripheral Blood (PB) to BM and spleen in vivo, using NOD/SCID/gamma (NSG) null mice (lacking NK cells and effective macrophages). In this system, NSG mice were pretreated (day 0) with DARA, control IgG or anti-CXCR4 as positive control for inhibition of cell homing, prior to injection of fresh primary CLL cells (50×106 cells/per mice) on day 1. PB, BM and spleen cells were isolated on day 2 and CLL cells were identified by staining for human CD45/CD19/CD5 and counted using a flow cytometer. Cell counting showed that CLL cells mainly migrate to the spleen, and that DARA significantly reduced this migration (55% inhibition on average, p<0.05). In addition to migration, CD38 also plays a key role in cell adhesion through interaction with integrins (CD49d/CD29) and with extracellular matrix proteins. We analyzed the effect of DARA on the adhesion of CLL cells to the extracellular matrix vascular-cell adhesion molecule-1 (VCAM-1) mediated by CD49d/CD29. DARA reduced adhesion of CLL cells (n=4), to VCAM-1 by 46±13% (range 27-57) compared to isotype control. By RT-PCR we observed an up-regulation of MMP9 transcripts (average 2 fold, n=2), and DARA abrogated both constitutive MMP9 expression (90% reduction) and VCAM-derived (94% reduction) MMP9 expression. In summary, DARA shows a positive effect on ADCP-mediated anti-tumor activity on CLL cells both in vitro and in vivo. In addition DARA exhibits a strong effect on CLL cell migration and adhesion. Based on these data, we hypothesize that DARA may exert unique and substantial effects on CLL tumor cell growth and contributes to potent therapeutic efficacy in a clinical setting. Disclosures Doshi: Janssen R&D: Employment. Parren:Genmab: Employment, Equity Ownership. Lammerts van Bueren:Genmab : Employment. Pérez-Galán:Genmab: Research Funding.

scholarly journals IMMU-28. TARGETING IMMUNOSUPPRESSIVE MYELOID DERIVED SUPPRESSOR CELLS VIA MIF/CD74 SIGNALING AXIS TO ATTENUATE GBM GROWTH

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi125-vi125
Author(s):  
Tyler Alban ◽  
Defne Bayik ◽  
Balint Otvos ◽  
Matthew Grabowski ◽  
Manmeet Ahluwalia ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Immune Cell ◽  
Suppressor Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Signaling Axis ◽  
Immunosuppressive Microenvironment ◽  
And Function

Abstract The immunosuppressive microenvironment in glioblastoma (GBM) enables persistent tumor growth and evasion from tumoricidal immune cell recognition. Despite a large accumulation of immune cells in the GBM microenvironment, tumor growth continues, and evidence for potent immunosuppression via myeloid derived suppressor cells (MDSCs) is now emerging. In agreement with these observations, we have recently established that increased MDSCs over time correlates with poor prognosis in GBM, making these cells of interest for therapeutic targeting. In seeking to reduce MDSCs in GBM, we previously identified the cytokine macrophage migration inhibitory factor (MIF) as a possible activator of MDSC function in GBM. Here, using a novel in vitro co-culture system to reproducibly and rapidly create GBM-educated MDSCs, we observed that MIF was essential in the generation of MDSCs and that MDSCs generated via this approach express a repertoire of MIF receptors. CD74 was the primary MIF receptor in monocytic MDSCs (M-MDSC), which penetrate the tumor microenvironment in preclinical models and patient samples. A screen of MIF/CD74 interaction inhibitors revealed that MN-166, a clinically relevant blood brain barrier penetrant drug, which is currently fast tracked for FDA approval, reduced MDSC generation and function in vitro. This effect was specific to M-MDSC subsets expressing CD74, and appeared as reduced downstream pERK signaling and MCP-1 secretion. In vivo, MN-166 was able reduce tumor-infiltrating MDSCs, while conferring a significant increase in survival in the syngeneic glioma model GL261. These data provide proof of concept that M-MDSCs can be targeted in the tumor microenvironment via MN-166 to reduce tumor growth and provide a rationale for future clinical assessment of MN-166 to reduce M-MDSCs in the tumor microenvironment. Ongoing studies are assessing the effects of MDSC inhibition in combination with immune activating approaches, in order to inhibit immune suppression while simultaneously activating the immune system.

scholarly journals CD146+CD107a+ Mesenchymal Stem/Stromal Cells with Signature Attributes Correlate to Therapeutic Potency as “First Responders” to Injury and Inflammation

2019 ◽  
Author(s):  
Annie C. Bowles ◽  
Dimitrios Kouroupis ◽  
Melissa A. Willman ◽  
Carlotta Perucca Orfei ◽  
Ashutosh Agarwal ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Immune Cell ◽  
First Responders ◽  
Infrapatellar Fat Pad ◽  
M2 Macrophage ◽  
List Type ◽  
Fat Pad ◽  
Secretory Capacity

ABSTRACTCD146+ bone marrow–derived Mesenchymal Stem/Stromal Cells (BM-MSC) play key roles in the perivascular niche, skeletogenesis and hematopoietic support, however elucidation of therapeutic potency has yet to be determined. Here, inflammatory challenge to crude BM-MSC captured a baseline of signatures including enriched expression of CD146+ with CD107a+, CXCR4+, and LepR+, transcriptional profile, enhanced secretory capacity, robust secretome and immunomodulatory function with stimulated target immune cells. These responses were significantly more pronounced in CD146+ (POS)-selected subpopulation than in the CD146- (NEG). Mechanistically, POS uniquely mediated robust immunosuppression while inducing significant frequencies of Naïve and Regulatory T cells in vitro. Moreover, POS promoted a pivotal M1-to-M2 macrophage shift in vivo, ameliorating inflammation/fibrosis of joint synovium and fat pad of the knee, failed by NEG. This study provides high-content evidence of CD146+CD107a+ BM-MSC, herein deemed ‘first responders’ to inflammation, as the underrepresented subpopulation within crude BM-MSC with innately higher secretory capacity and therapeutic potency.HIGHLIGHTSSignature phenotypic, transcriptional, and secretome profiles were identified and enriched in human CD146+ (POS)-selected subpopulation in response to inflammationInflammatory challenge consistently altered stemness (LIF) and differentiation master regulators (SOX9, RUNX2, PPARγ) in crude, POS, and NEG BM-MSC, and deduced unique expressions in POS compared to NEGPOS BM-MSC mediated the strongest immunomodulation, e.g. target immune cell suppression, Treg induction, diminished T cell differentiationPOS BM-MSC promoted the largest M1-to-M2 shift in vivo alleviating induced synovitis and infrapatellar fat pad fibrosis of the knee

scholarly journals Single-cell transcriptomics reveals the effect of PD-L1/TGF-β blockade on the tumor microenvironment

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yoong Wearn Lim ◽  
Garry L. Coles ◽  
Savreet K. Sandhu ◽  
David S. Johnson ◽  
Adam S. Adler ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
Single Cell ◽  
Immune Cell ◽  
Single Cells ◽  
Cell Infiltration ◽  
In Vivo Models ◽  
T Cell Infiltration ◽  
Anti Tumor Activity

Abstract Background The anti-tumor activity of anti-PD-1/PD-L1 therapies correlates with T cell infiltration in tumors. Thus, a major goal in oncology is to find strategies that enhance T cell infiltration and efficacy of anti-PD-1/PD-L1 therapy. TGF-β has been shown to contribute to T cell exclusion, and anti-TGF-β improves anti-PD-L1 efficacy in vivo. However, TGF-β inhibition has frequently been shown to induce toxicity in the clinic, and the clinical efficacy of combination PD-L1 and TGF-β blockade has not yet been proven. To identify strategies to overcome resistance to PD-L1 blockade, the transcriptional programs associated with PD-L1 and/or TGF-β blockade in the tumor microenvironment should be further elucidated. Results We used single-cell RNA sequencing in a mouse model to characterize the transcriptomic effects of PD-L1 and/or TGF-β blockade on nearly 30,000 single cells in the tumor and surrounding microenvironment. Combination treatment led to upregulation of immune response genes, including multiple chemokine genes such as CCL5, in macrophages, and downregulation of extracellular matrix genes in fibroblasts. Analysis of publicly available tumor transcriptome profiles showed that the chemokine CCL5 was strongly associated with immune cell infiltration in various human cancers. Further investigation with in vivo models showed that intratumorally administered CCL5 enhanced cytotoxic lymphocytes and the anti-tumor activity of anti-PD-L1. Conclusions Taken together, our data could be leveraged translationally to complement or find alternatives to anti-PD-L1 plus anti-TGF-β combination therapy, for example through companion biomarkers, and/or to identify novel targets that could be modulated to overcome resistance.

561 DuoBody®-PD-L1×4–1BB (GEN1046) induces superior immune-cell activation, cytokine production and cytotoxicity by combining PD-L1 blockade with conditional 4–1BB co-stimulation

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A595-A595
Author(s):  
Alexander Muik ◽  
Isil Altintas ◽  
Rachelle Kosoff ◽  
Friederike Gieseke ◽  
Kristina Schödel ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Cytokine Production ◽  
Immune Cell ◽  
Anti Tumor Activity

BackgroundCheckpoint inhibitors targeting the PD-1/PD-L1 axis (CPI) have changed the treatment paradigm and prognosis for patients with advanced solid tumors; however, many patients experience limited benefit due to treatment resistance. 4-1BB co-stimulation can activate cytotoxic T-cell- and NK-cell-mediated anti-tumor immunity and has been shown to synergize with CPI in preclinical models. DuoBody-PD­L1×4-1BB is a first-in-class, Fc-silenced, bispecific next-generation checkpoint immunotherapy that activates T cells through PD-L1 blockade and simultaneous PD-L1-dependent 4-1BB co-stimulation. Here we present preclinical evidence for the mechanism of action of DuoBody-PD-L1×4-1BB, and proof-of-concept using mouse-reactive mbsAb-PD-L1×4-1BB in vivo.MethodsRNA sequencing analyses was performed on primary human CD8+ T cells that were co-cultured with PD-L1+ monocytes in the presence of anti-CD3/anti-CD28 and test compounds. T-cell proliferation and cytokine production were analyzed in primary human T-cell and mixed lymphocyte reaction (MLR) assays in vitro, and using patient-derived tumor-infiltrating lymphocytes (TILs). Cytotoxic activity was assessed in co-cultures of CLDN6+PD-L1+ MDA-MB-231 tumor cells and CLDN6-TCR+CD8+ T cells. Anti-tumor activity of mbsAb-PD-L1×4-1BB was tested in vivo using the CT26 mouse tumor model. Immunophenotyping of the tumor microenvironment (TME), tumor-draining lymph nodes (tdLNs) and peripheral blood was performed by flow cytometry.ResultsDuoBody-PD-L1×4-1BB significantly induced expression of genes associated with immune cell proliferation, migration and cytokine production in activated CD8+ T cells, which were not altered by CPI. DuoBody-PD-L1×4-1BB dose-dependently enhanced expansion of human TILs ex vivo. DuoBody-PD-L1×4-1BB dose-dependently enhanced T-cell proliferation and pro-inflammatory cytokine production in vitro (e.g. IFNγ and TNFα; in polyclonal and antigen-specific T-cell proliferation assays and MLR), which was dependent on crosslinking to PD-L1+ cells and superior to CPI or the combination of Fc-silenced PD-L1- and 4-1BB-specific antibodies. DuoBody-PD-L1x4-1BB induced upregulation of degranulation marker CD107a and granzyme B in CD8+ T cells, resulting in antigen-specific T-cell-mediated cytotoxicity of MDA-MB-231 tumor cells in vitro, superior to CPI. In mice bearing subcutaneous CT26 tumors, a model that was insensitive to PD-L1 blockade, mbsAb-PD-L1×4-1BB elicited tumor rejection in the majority of the mice at active dose levels and significantly improved survival. Dose-dependent anti-tumor activity was associated with expansion of tumor antigen-specific T cells in the blood and enhanced immune-cell activation in tdLNs and TME.ConclusionsCombining PD-L1 blockade with conditional 4-1BB co-stimulation using bispecific antibodies induced T-cell activation, expansion, and cytotoxic activity in vitro and potent anti-tumor activity in vivo superior to CPI. DuoBody-PD-L1×4-1BB is currently being evaluated in patients with advanced solid tumors in a first-in-human trial (NCT03917381).Ethics ApprovalAll mice studies were performed by BioNTech SE at its research facilities in Germany, and the mice were housed in accordance with German federal and state policies on animal research. All experiments were approved by the regulatory authorities for animal welfare in Germany. The use of tumor tissue resections was approved by BioNTech SE‘s Ethics Board, approval number 837.309.12 (8410-F).

scholarly journals 283 TIM-3 blockade modulates the tumor microenvironment improving the outcome of preclinical pediatric diffuse midline glioma models

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A307-A307
Author(s):  
Iker Ausejo-Mauleon ◽  
Sara Labiano ◽  
Virginia Laspidea ◽  
Marc Garcia-Moure ◽  
Daniel de la Nava ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
Nk Cells ◽  
Immune Cell ◽  
Animal Experiments ◽  
Cell Types ◽  
Immune Memory ◽  
List Type ◽  
Treg Population

BackgroundDiffuse Midline Gliomas (DMGs), encompassing Diffuse Intrinsic Pontine Gliomas (DIPGs), are the most aggressive pediatric brain tumors. Their meager survival has not changed despite the combination of radiotherapy with targeted therapies emphasizing the urgent need for effective treatments. Recent research suggested that the DIPG tumor microenvironment is neither highly immunosuppressive nor inflammatory.1 These analyses showed the lack of infiltrating lymphocytes and the abundance of CD11b+ cells. TIM-3 is a member of the T-cell immunoglobulin and mucin domain protein family expressed on multiple immune cell types, including T cells, Treg, NK cells, monocytes, dendritic cells, and microglia, where it potently regulates not only adaptive immunity but also innate immunity.2–3 Therefore, TIM-3 inhibitors could challenge several components in the tumor microenvironment, thereby providing potentially effective treatment for DMGs.MethodsNP53 and XFM murine DIPG cell lines were used for animal experiments in immunocompetent orthotopic models. The tumors were processed by mechanical and enzymatic digestion and immune populations were analyzed by a flow cytometry panel. Antibodies against NK cells (NK1.1), CD4 (GK1.5), CD8 (CD8β) were used for animal depletion experiments alone or in combination.ResultsIn silico assessment of TIM-3 expression in DIPG datasets showed a robust expression of this gene. Moreover, single-cell sequencing analyses of DIPG biopsies uncover its expression in the myeloid compartment (especially in microglia). In vivo efficacy studies showed that treatment with anti-TIM-3 antibody significantly increased the overall survival in two DIPG immunocompetent orthotopic animal models (doubling the median), lead to long-term survivors free of disease (50%) and showed immune memory. Analyses of CD45+ populations in the tumor microenvironment showed a significant increase in microglia, granulocytes, NK and CD8+ cells corresponding with a NK and T-cell activate phenotypes in treated-mice. In addition, we have a substantial decrease in the Treg population, which causes an increase in the CD8/Treg ratio. CD4 and CD8 T-cell depletion led to a significant but not total loss of treatment efficacy. NK cells depletion also reduced the effectiveness of this therapy, albeit to a lesser extent than CD4-CD8 depletion. We are currently investigating the role of microglia in the outcome of the treatment.ConclusionsOur data uncovered TIM-3 as a potential target for the treatment of DIPG tumors. Inhibition of this molecule led to a potent antitumor effect mediated by a profound tumor microenvironment remodelling.ReferencesLieberman NAP, DeGolier K, Kovar HM, et al. Characterization of the immune microenvironment of diffuse intrinsic pontine glioma: implications for development of immunotherapy. Neuro Oncol 2019;21(1):83–94. doi:10.1093/neuonc/noy145.Acharya N, Sabatos-Peyton C, Anderson AC. Tim-3 finds its place in the cancer immunotherapy landscape. J Immunother Cancer 2020;8(1):e000911. doi:10.1136/jitc-2020-000911.Wolf Y, Anderson AC, Kuchroo VK. TIM3 comes of age as an inhibitory receptor. Nat Rev Immunol 2020;20(3):173–185. doi:10.1038/s41577-019-0224-6

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