732 Chemerin reactivates PTEN and suppresses PD-L1 in tumor cells via a novel CMKRL1-mediated pathway

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A776-A776
Author(s):  
Russell Pachynski ◽  
Keith Rennier ◽  
Woo Jae Shin ◽  
Ethan Krug ◽  
Gurpal Virdi ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Human Tumor ◽  
In Vivo Studies ◽  
Cell Phenotype ◽  
Immune Effector ◽  
First Time

BackgroundChemokines and chemoattractants play critical roles in trafficking that help regulate leukocyte infiltrates in the tumor microenvironment. Chemokines/chemoattractants can also modulate tumor cell phenotype and function, as tumor cells express functional receptors for these agents. Chemerin (retinoic acid receptor responder 2, RARRES2) is an endogenous leukocyte chemoattractant that recruits innate immune cells through its receptor, CMKLR1. RARRES2 is widely expressed in nonhematopoietic tissues and often downregulated across multiple tumor types compared with normal tissue. We and others have shown that augmenting chemerin in the tumor microenvironment significantly suppresses tumor growth, in part, by immune effector cell recruitment. As chemerin has various roles outside of leukocyte trafficking (eg adipocyte differentiation and metabolic processes), we hypothesized that it may have additional, tumor-intrinsic effects.MethodsWe investigated the effect of exogenous chemerin on human prostate and sarcoma tumor lines. Key signaling pathway components were elucidated using qPCR, Western blotting, siRNA knockdown, and specific inhibitors. Functional consequences of chemerin treatment were evaluated using in vitro and in vivo studies.ResultsWe show for the first time that human tumors exposed to exogenous chemerin significantly upregulate PTEN expression/activity, and concomitantly suppress programmed death ligand-1 (PD-L1) expression. CMKLR1 knockdown abrogated chemerin- induced PTEN and PD-L1 modulation, revealing a novel CMKLR1/PTEN/PD-L1 signaling cascade. Targeted inhibitors suggest that signaling occurs through the PI3K/AKT/mTOR pathway. We found that chemerin treatment significantly reduced tumor migration, while significantly increasing T-cell–mediated cytotoxicity. Chemerin treatment was as effective as both PD-L1 knockdown and the anti–PD-L1 antibody atezolizumab in augmenting T cell mediated tumor lysis. Forced expression of chemerin in human DU145 prostate tumors significantly suppressed in vivo tumor growth, significantly increasing PTEN and decreasing PD-L1 expression. Primary prostate tumor cultures that were treated with recombinant chemerin showed significant increases in PTEN and decreases in PD-L1 expression compared to controls. Lastly, analyses of clinical trial data from human metastatic prostate cancer patients receiving treatment with ipilimumab (NCT02113657) showed higher tumoral levels of RARRES2 expression correlated with higher levels of PTEN, higher effector immune cell (eg cytotoxic T cells, NK cells) signatures, and improved clinical outcomes, suggesting a strategy to augment chemerin/RARRES2 levels in tumors may improve responses to immunotherapy.ConclusionsCollectively, our data show for the first time a novel link between chemerin, PTEN, and PD-L1 in human tumor lines. These results show that chemerin – in addition to its ability to suppress tumor growth by recruitment of immune effector cells, may also have a role in improving T-cell–mediated immunotherapies through favorable modulation of PTEN and PD-L1.

ANTITUMOR EFFECT OF COLLOIDAL SILVER BISILICATE IN EXPERIMENTAL IN VITRO AND IN VIVO STUDIES

2019 ◽  
Vol 65 (5) ◽  
pp. 760-765
Author(s):  
Margarita Tyndyk ◽  
Irina Popovich ◽  
A. Malek ◽  
R. Samsonov ◽  
N. Germanov ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Human Tumor ◽  
Significant Inhibition ◽  
In Vivo Studies ◽  
Ehrlich Carcinoma ◽  
In Vivo Experiments

The paper presents the results of the research on the antitumor activity of a new drug - atomic clusters of silver (ACS), the colloidal solution of nanostructured silver bisilicate Ag6Si2O7 with particles size of 1-2 nm in deionized water. In vitro studies to evaluate the effect of various ACS concentrations in human tumor cells cultures (breast cancer, colon carcinoma and prostate cancer) were conducted. The highest antitumor activity of ACS was observed in dilutions from 2.7 mg/l to 5.1 mg/l, resulting in the death of tumor cells in all studied cell cultures. In vivo experiments on transplanted Ehrlich carcinoma model in mice consuming 0.75 mg/kg ACS with drinking water revealed significant inhibition of tumor growth since the 14th day of experiment (maximally by 52% on the 28th day, p < 0.05) in comparison with control. Subcutaneous injections of 2.5 mg/kg ACS inhibited Ehrlich's tumor growth on the 7th and 10th days of the experiment (p < 0.05) as compared to control.

scholarly journals P09.05 Plasma CD27, a surrogate of intratumoral CD27-CD70 interaction, correlates with immunotherapy resistance in renal cancer

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A30.1-A30
Author(s):  
N Benhamouda ◽  
I Sam ◽  
N Epaillard ◽  
A Gey ◽  
A Saldmann ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Single Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Principal Investigator ◽  
Cell Phenotype ◽  
Metastatic Rcc ◽  
Research Grant

BackgroundCD70, a costimulatory molecule on antigen presenting cells, is known to activate CD27-expressing T cells. CD27-CD70 interaction leads to the release of soluble CD27 (sCD27). However, persistent interaction of CD27 and CD70 such as in chronic infection may exhaust the T cell pool and promote apoptosis. Surprisingly, our analysis based on TCGA database show that clear cell renal cell carcinoma (ccRCC) expresses the highest levels of CD70 among all solid tumors. Despite the important clinical efficacy of immunotherapy by anti-PD-1 in RCC patients, the overall response to anti-PD1 remains modest. The relationship between the CD27-CD70 interaction in the RCC and the response to immunotherapy is still unclear.Materials and MethodsTo study the CD27 and CD70 expression in the tumor microenvironment (TME), FFPE tumor tissues from 25 RCC patients were analysed using multiplex in situ immunofluorescence. 10 fresh RCC tumor samples were collected to analyse the phenotype of CD27+ T cells by flow cytometry and 4 samples were proceeded for single-cell RNA-seq analysis. A cohort of metastatic RCC patients (n = 35) treated by anti-PD-1 were enrolled for the measurement of plasma sCD27 by ELISA and the survival analysis is also realized.ResultsIn the TME, we demonstrated that CD27+ T cells interact with CD70-expressing tumor cells. In fresh tumors from RCC patients, CD27+ T cells express higher levels of cleaved caspase 3 (a classical marker of apoptosis) than CD27- T cells. We confirmed the apoptotic signature (BAX, FASLG, BCL2L11, CYCS, FBXO32, LGALS1, PIK3R1, TERF1, TXNIP, CDKN2A) of CD27+ T cells by single-cell RNAseq analysis. CD27+T cells also had a tissue resident memory T cell phenotype with enriched gene expression of ITGAE, PRDM1, RBPJ and ZNF683. Moreover, CD27+T cells display an exhaustion phenotype with the expression of multiple inhibitory receptors gene signature (PDCD1, CTLA4, HAVCR2, LAG3, etc). Besides, intratumoral CD27-CD70 interaction significantly correlates with plasma sCD27 concentration in RCC (p = 0.0017). In metastatic RCC patients treated with anti-PD-1, higher levels of sCD27 predict poor overall survival (p = 0.037), while it did not correlate with inflammatory markers or clinical prognostic criteria.ConclusionsIn conclusion, we demonstrated that sCD27, a surrogate of T cell dysfunction in tumors likely induced by persistent interactions of CD27+T cells and CD70-expressing tumor cells, is a predictive biomarker of resistance to immunotherapy in mRCC. To our knowledge, this is the first report showing that a peripheral blood biomarker may reflect certain aspects of the tumor-host interaction in the tumor microenvironment. Given the frequent expression of CD70 and CD27 in solid tumors, our findings may be further extended to other types of tumors. CD70-CD27 interaction could thus be considered as a mechanism of tumor escape, but also a novel therapeutic target in cancers.Disclosure InformationN. Benhamouda: None. I. Sam: None. N. Epaillard: None. A. Gey: None. A. Saldmann: None. J. Pineau: None. M. Hasan: None. V. Verkarre: None. V. Libri: None. S. Mella: None. C. Granier: None. C. Broudin: None. P. Ravel: None. B. Jabla: None. N. Chaput: None. L. Albiges: None. Y. Vano: None. O. Adotevi: None. S. Oudard: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; SIRIC CARPEM, FONCER. E. Tartour: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Fondation ARC, INCA PLBio, Labex Immuno-Oncology, SIRIC CARPEM, FONCER, IDEX université de Paris, Inserm Transfert.

A Novel T-Cell Redirecting Anti-CD19-F(ab)2/CD3scFv Bispecific Antibody Exhibits Potent Lymphoma Cytotoxicity.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2762-2762
Author(s):  
Diane L Rossi ◽  
Edmund A Rossi ◽  
Thomas M Cardillo ◽  
David M Goldenberg ◽  
Chien-Hsing Chang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Elimination Rate ◽  
Cell Killing ◽  
Antibody Engineering ◽  
In Vivo Studies ◽  
Target Cells ◽  
Daudi Cells

Abstract Abstract 2762 Background: The use of bispecific antibodies (bsAbs) to redirect effector T cells for the targeted killing of tumor cells is a very active area of antibody engineering. Various formats of such agents made recombinantly have shown considerable promise both pre-clinically and clinically. For example, one design termed Bispecific T-cell engager (BiTE) employs a single polypeptide containing 2 antigen-binding specificities (each contributed by a cognate VH and VL) linked in tandem via a flexible linker, and another design termed DART (Dual-Affinity Re-Targeting) utilizes a disulfide-stabilized diabody. Both BiTE and DART, however, exhibit fast blood clearance due to their small size (∼55 kDa). Herein, we describe, for the first time, the generation of a novel T-cell redirecting bsAb, (19)-3s, comprising an anti-CD3 scFv covalently conjugated to a stabilized anti-CD19 F(ab)2. The potential advantages of (19)-3s include bivalent binding to tumor cells, a larger size (∼130 kDa) to preclude rapid renal clearance, and potent T-cell mediated cytotoxicity. Methods and Results: The Dock-and-Lock (DNL) method was used to generate (19)-3s by combining a stabilized anti-CD19 F(ab)2 with an anti-CD3-scFv, resulting in a homogeneous covalent structure of the designed composition, as shown by SE-HPLC, ELISA, SDS-PAGE, and immunoblot analyses. Functionally, (19)-3s induced synapse formation between effector and target cells using freshly isolated human T cells mixed with Daudi Burkitt lymphoma cells. Using an E:T ratio of 2.5:1 and 1 μg/mL of (19)-3s, the cell mixture was stained with anti-CD20-APC (for Daudi) and anti-CD7-FITC (for T cells), and cobinding was measured by flow cytometry as the % of CD20+/CD7+ events. After treatment with (19)-3s, 45.5% of events were found to be CD20/CD7 dual-positive, indicating synapsed Daudi and T cells, compared with 2% measured for untreated cells. Gating of the Daudi cell population showed that >90% of Daudi cells were associated with T cells. To access the targeted T-cell killing of Daudi, isolated T cells and Daudi were mixed at an E:T ratio of 12.5:1 and treated with serial dilutions of (19)-3s. After 18-h incubation at 37°C, cytotoxicity was measured using a LDH-release assay. Potent (19)-3s-mediated T-cell killing of Daudi cells was observed at <1 pM, with maximal activity at 10 pM. Similar results were seen with both Ramos and Raji NHL cell lines. In vivo studies to determine Pk and efficacy are underway. Based on DNL constructs of similar design, we expect (19)-3s to have an elimination rate longer than that of MT103, a BiTE comprising scFvs derived from anti-CD19 and anti-CD3, thus perhaps avoiding continuous infusions with this new construct. Conclusions: (19)-3s can bind T cells and NHL cells simultaneously and induce T-cell-mediated killing at pM concentrations in an ex vivo setting. The modular nature of the DNL method will allow the rapid production of a large number of related conjugates for redirected T-cell killing of various malignancies, without the need for additional recombinant engineering and protein production. We are currently evaluating the in vivo activity of (19)-3s, as a prototype, to determine if this novel bsAb format offers additional advantages. Disclosures: Rossi: Immunomedics, Inc.: Employment. Rossi:Immunomedics, Inc.: Employment; IBC Pharmaceuticals Inc.: Employment. Cardillo:Immunomedics, Inc: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership. Chang:Immunomedics, Inc.: Employment.

Effect of cabazitaxel on the growth of human castration-refractory prostate cancer cells in vitro compared to docetaxel and on the anti-tumor properties of the angio-inhibitor PEDF in vivo.

2015 ◽  
Vol 33 (7_suppl) ◽  
pp. 205-205
Author(s):  
Thomas Nelius ◽  
Courtney Jarvis ◽  
Dalia Martinez-Marin ◽  
Stephanie Filleur
Keyword(s):  
Prostate Cancer ◽  
Cell Proliferation ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Cell Lines ◽  
In Vitro Cytotoxicity ◽  
In Vivo Studies ◽  
Raw264.7 Macrophages

205 Background: Docetaxel/DTX and cabazitaxel/CBZ have shown promise in the treatment of metastatic Castration-Refractory Prostate Cancer/mCPRC however, comparative studies are missing. Toxicities of these drugs are significant, urging the need to modify taxane regimens. Recently, low-dose metronomic/LDM treatments using conventional chemotherapeutic drugs have shown benefits in CPRC in improving the effect of anti-angiogenic agents. Previously, we have demonstrated that LDM-DTX in combination with PEDF curbs significantly CRPC growth, limits metastases formation and prolongs survival in vivo. In this study, we intended to compare the cytotoxic effect of CBZ and DTX on CRPC cells in vitro and CL1 tumors in vivo. Methods: PC3, DU145 cell lines were from ATCC.CL1 cells were obtained from androgen-deprived LNCaP cells. Cell proliferation was assessed by crystal violet staining and cell cycle analyses. In vitro cytotoxicity assays were performed on CL1 cells/RAW264.7 macrophages co-cultures treated with PEDF and increasing doses of taxanes. For the in vivo studies, CL1 cells were engineered to stably express the DsRed Express protein +/- PEDF. PEDF anti-tumor effects were assessed on s.c. xenografts treated with DTX (5mg/kg ip ev. 4 day) as reference, CBZ (5mg/kg ip ev. 4 days, 1mg/kg for 10 days, 0.5mg/kg q.a.d. and 0.1mg/kg daily) or placebo. Results: CBZ limits cell proliferation with a greater efficacy than DTX in all CRPC cell lines tested. DU145 presented the largest difference. High doses of taxane blocked tumor cells in mitosis, whereas LDM increased the SubG1 population. This effect was significantly higher in DU145 cells treated with CBZ. In vivo, 5mg/kg CBZ delayed tumor growth more efficiently than 5mg/kg DTX. PEDF/5mg/kg CBZ markedly delayed tumor growth compared to all treatments. Finally, engulfment of tumor cells by macrophages was higher in combined treatments suggesting an inflammation-related process. Conclusions: CBZ is more efficient than DTX both in vitro and in vivo.The data also reinforce PEDF as a promising anti-neoplasic agent in combination with LDM taxane chemotherapies.

scholarly journals Neutrophil Extracellular Traps Promote T Cell Exhaustion in the Tumor Microenvironment

2021 ◽  
Vol 12 ◽  
Author(s):  
Christof Kaltenmeier ◽  
Hamza O. Yazdani ◽  
Kristin Morder ◽  
David A. Geller ◽  
Richard L. Simmons ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Cancer Metastasis ◽  
Neutrophil Extracellular Traps ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Extracellular Traps

While neutrophil extracellular traps (NETs) are important for directly promoting cancer growth, little is known about their impact on immune cells within the tumor microenvironment (TME). We hypothesize that NETs can directly interact with infiltrating T cells to promote an immunosuppressive TME. Herein, to induce a NET-rich TME, we performed liver Ischemia/Reperfusion (I/R) in an established cancer metastasis model or directly injected NETs in subcutaneous tumors. In this NET-rich TME, the majority of CD4+ and CD8+ tumor infiltrating lymphocytes expressed multiple inhibitory receptors, in addition these cells showed a functional and metabolic exhausted phenotype. Targeting of NETs in vivo by treating mice with DNAse lead to decreased tumor growth, decreased NET formation and higher levels of functioning T cells. In vitro, NETs contained the immunosuppressive ligand PD-L1 responsible for T cell exhaustion and dysfunction; an effect abrogated by using PD-L1 KO NETs or culturing NETs with PD-1 KO T cells. Furthermore, we found elevated levels of sPDL-1 and MPO-DNA, a NET marker, in the serum of patients undergoing surgery for colorectal liver metastases resection. Neutrophils isolated from patients after surgery were primed to form NETs and induced exhaustion and dysfunction of human CD4+ and CD8+ T cells. We next targeted PD-L1 in vivo by injecting a blocking antibody during liver I/R. A single dose of anti-PD-L1 during surgery lead to diminished tumors at 3 weeks and functional T cells in the TME. Our data thus reveal that NETs have the capability of suppressing T cell responses through metabolic and functional exhaustion and thereby promote tumor growth. Furthermore, targeting of PD-L1 containing NETs at time of surgery with DNAse or anti-PD-L1 lead to diminished tumor growth, which represents a novel and viable strategy for sustaining immune competence within the TME.

scholarly journals 792 Creating an immune-favorable tumor microenvironment by a novel anti-CD39/TGFβ-Trap bispecific antibody

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A827-A827
Author(s):  
Hongtao Lu ◽  
Dawei Sun ◽  
Jun Sun ◽  
Yanan Geng ◽  
Jinhui Zhang ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
Tumor Growth ◽  
T Cell Activation ◽  
Specific Antibody ◽  
Cell Activation ◽  
Bispecific Antibody ◽  
Human Pbmc ◽  
Antibody Targeting

BackgroundAdenosine and TGFβ are two key immune suppressors in tumor microenvironment (“TME”) that cause broad immune suppression resulting in resistance to current CPI immunotherapies. Cancers frequently express transforming growth factor-β (TGFβ), which drives immune dysfunction in the tumor microenvironment by inducing regulatory T cells (Tregs), inhibiting CD8+ activation and infiltration into TME, and promoting epithelial–mesenchymal transition (EMT). We observed that TGFβ induces the expression of CD39, a critical enzyme that regulates adenosine generation. CD39 is highly expressed in Tregs within TME, it drives the production of adenosine, an immunoinhibitory molecule that partly mediates Treg inhibitory function. To inhibit CD39-Adenosine and TGFβ simultaneously to create an immune favorable tumor microenvironment, we designed a bi-specific antibody targeting both CD39 and TGFβ (ES014), which aims to inhibit the generation of adenosine and iTreg in TME. The immuno-stimulating effect of ES014 was demonstrated in a PD-1-unresponsive mouse model where tumor growth was significantly inhibited after the treatment of the bi-specific antibody.MethodsThe bifunctional antibody–ligand trap ES014 was created by fusing the TGFβ receptor II ectodomain to an antibody targeting CD39. ES014 molecule could simultaneously inhibit CD39 enzymatic function to prevent extracellular ATP from degradation and neutralize autocrine/paracrine TGFβ in the target cell microenvironment. The immunological function of ES014 was studied in an in vitro Elpiscience proprietary ImmunoShine platform which includes T cell activation and apoptosis assay, iTreg differentiation and suppression assay, NK cell activation assay and DC maturation activity. The in vivo efficacy of ES014 was investigated in a human PBMC engrafted cancer model.ResultsWe demonstrated that ES014 bispecific antibody can inhibit CD39 enzymatic activity and neutralizes TGFβ-induced effect, resulting in greater T cell activation and suppression of Treg differentiation. Interestingly, we found ES014 molecule demonstrated a unique mechanism by significantly protecting effector T cell from anti-Fas induced apoptosis or activation induced cell death (AICD) that is not observed in monotherapy or combo treatment. The ES014 molecule is more effective in inhibiting tumor growth as compared with anti-CD39 antibody or TGFβ-trap in a human PBMC engrafted in vivo model.ConclusionsWe find that by simultaneously targeting CD39 and TGFβ by a novel bi-specific molecule ES014, a more immune-favorable TME and synergistic anti-tumor effects can be achieved. Our pre-clinical data demonstrate that ES014 counteracts TGFβ-mediated inhibitory effect and adenosine induced immune tolerance and has a unique ability to protect T cell from apoptosis. ES014 demonstrated strong efficacy in in vivo tumor growth inhibition.

scholarly journals 230 Preclinical efficacy of CLEC-1 antagonist as novel myeloid immune checkpoint therapy for oncology

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A245-A245
Author(s):  
Vanessa Gauttier ◽  
Marion Drouin ◽  
Sabrina Pengam ◽  
Javier Saenz ◽  
Bérangère Evrard ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Cells ◽  
Immune Cell ◽  
Human Tumor ◽  
Myeloid Cell ◽  
Lectin Receptors ◽  
Knock Out ◽  
Cell Functions ◽  
Preclinical Efficacy

BackgroundC-type lectin receptors (CLRs) are powerful pattern recognition receptors shaping immune cell-mediated tissue damage by positively or negatively regulating myeloid cell functions and hence tumor elimination or evasion. We previously reported that the orphan CLR CLEC-1 expressed by dendritic cells (DCs) tempers T cell’s responses in vivo by limiting antigen cross-presentation by cDC1. Furthermore, we observed that CLEC-1 is highly expressed by myeloid cells purified from human tumor microenvironment, in particular tumor-associated macrophages.MethodsMacrophages were generated from monocytes of healthy volunteers for phagocytosis assays. MC38 and Hepa 1.6 murine tumor cells were implanted in Clec1a KO or KI mice for immunotherapeutic treatment evaluation.ResultsUsing newly developed anti-human CLEC-1 monoclonal antibodies (mAbs), we found that antagonist anti-CLEC-1 mAbs with the capacity to block CLEC-1/CLEC-1Ligand interaction, as opposed to non-antagonist CLEC-1 mAbs, increase the phagocytosis of CLEC-1Ligand-positive human tumor cells by human macrophages, in particular when opsonized by tumor-associated antigen mAbs (Rituximab, Cetuximab, Trastuzumab) or with anti-CD47 mAb (Magrolimab). In-vivo, CLEC-1 knock-out (KO) mice (n=19) display significant prolonged survival in monotherapy as compared to wild-type littermates (n=12) in an orthotopic hepatocellular carcinoma (HCC) model and anti-tumor memory responses was demonstrated by tumor rechallenge in cured mice. CLEC1 KO mice also illustrate significant eradication of MC38 colorectal tumors in combination with chemotherapy promoting CLEC-1Ligand expression by tumor cells (n=16 with Gemcitabine or n=11 with Cyclophosphamide). HCC tumor microenvironment analysis after 2 weeks of tumor implantation shows significantly higher number of CD8+ and memory CD8+ T cells with reduced PD1 expression in CLEC1 KO animals (n=16 versus n=12 for KO vs WT mice respectively). Finally, we recently generated human CLEC-1 knock-in mice expressing the extracellular human CLEC1 domain fused to the intracellular mouse CLEC1 tail and confirmed preclinical efficacy in vivo with anti-human CLEC1 antagonist mAb in monotherapy in the orthotopic HCC model.ConclusionsThese data illustrate that CLEC-1 inhibition represents a novel therapeutic target for immuno-oncology modifying T cell immune responses and tumor cell phagocytosis by macrophages.

CAR-T cells to deliver engineered peptide antigens and reprogram antigen specific T cell responses against solid tumors.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 2530-2530
Author(s):  
Daniel Lee ◽  
Andy J Minn ◽  
Lexus R Johnson
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
In Vivo Studies ◽  
Peptide Antigens ◽  
Car T Cells ◽  
Car T

2530 Background: Neoantigen depleted malignancies such as colorectal cancer demonstrate primary resistance to immune checkpoint blockade, and solid tumors in general have shown resistance to chimeric antigen receptor (CAR) T cell therapy. However, CAR-T cells have been shown to be capable of delivering various therapeutic molecules in a targeted fashion to the tumor microenvironment, in some cases through extracellular vesicles (EVs). In vivo studies have shown that the presentation of foreign viral peptides by solid tumors can reprogram bystander virus-specific cytotoxic T cells (CTLs) against tumor cells. In this study, we demonstrate that CAR-T cells can deliver engineered peptide antigens to solid tumors, leading to presentation on tumor cells and anti-tumor response. Methods: Second generation CAR-T cells (41BB endodomain) targeting human CD19 (19BBz) or human mesothelin (M5BBz) were generated via retroviral and lentiviral transduction respectively. CAR-T cells were engineered to co-express peptides such as SIINFEKL of ovalbumin and NLVPMVATV of CMV pp65 among others. Peptides were isolated from EVs via ultracentrifugation. For in vivo studies, C57BL/6 or NSG mice were injected on the flank with relevant tumors and treated with peptide-CAR-T cells. In vitro studies utilized flow cytometry and xCELLigence killing assays. Results: Murine 19BBz CAR-T cells expressing the SIINFEKL peptide of ovalbumin (ova-19BBz) were found to transfer SIINFEKL peptide to tumor cells via EVs in vitro and in vivo, leading to peptide presentation on MHC-I of tumor cells. This resulted in significantly delayed tumor growth in tumor bearing mice transfused with OT-I T cells to mimic an existing antigen specific T cell pool. We expanded on these findings by isolating EVs from human M5BBz CAR-T cells expressing CMV viral peptides. Peptide-CAR-T EVs were co-cultured with human ovarian cancer cells to assess presentation to Jurkat T cells. Finally, we utilized primary human T cells from CMV+ healthy donors to assess the clinical feasibility of our peptide delivery approach. Conclusions: CAR-T cells can be engineered to deliver peptides to tumor cells for presentation and subsequent targeting by antigen specific CTLs. This represents a novel strategy for the treatment of non-immunogenic tumors.

The Heparanase Inhibitor SST0001 Is a Potent Inhibitor of Myeloma Growth In Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 246-246 ◽  
Author(s):  
Yang Yang ◽  
Joseph P. Ritchie ◽  
Telisha Swain ◽  
Annamaria Naggi ◽  
Giangiacomo Torri ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Heparan Sulfate ◽  
Cell Line ◽  
Tumor Cells ◽  
Subcutaneous Injection ◽  
Scid Mice ◽  
Rpmi 8226

Abstract Heparanase (HPSE) is an enzyme that cleaves heparan sulfate (HS) chains of proteoglycans. Work by us and others has demonstrated that heparanase promotes the growth and metastasis of many types of tumors, including multiple myeloma (MM). Heparanase expression is rare in normal tissue but becomes evident in many human tumors, making it a viable target for cancer therapy. SST0001, a chemically modified heparin that is 100% N-acetylated and 25% glycol-split, dramatically inhibits heparanase activity. SST0001 lacks anticoagulant activity and thus can be administered at relatively high doses in vivo. We previously reported that delivery of SST0001 by Alzet osmotic pumps to SCID mice potently inhibited growth of subcutaneous tumors formed by CAG human myeloma cells. In the present studies, we further tested the effects of SST0001 against additional MM cell lines, using alternative routes of drug delivery in two different animal models. Ten days after subcutaneous injection of either MM.1S or RPMI 8226 tumor cells, mice were treated for 28 days using Alzet pumps delivering 30 mg/kg/day of SST0001. Results showed that, compared to PBS control, MM.1S and RPMI-8226 tumors in SST0001-treated mice were reduced by 50% and 51%, respectively. In a separate experiment, delivery of SST0001 by distant subcutaneous injection inhibited tumor growth by 77% in comparison to controls. In the SCID-hu model, in which CAG cells were implanted directly into human bones engrafted in SCID mice, SST0001 also significantly inhibited tumor growth as measured by human immunoglobulin kappa light chain in murine sera (1055 ± 295 ng/ml in PBS-treated mice vs 155 ± 295 ng/ml in SST0001- treated mice (P &lt;0.003)). These data demonstrate that SST0001 is a strong inhibitor of MM growth in vivo, even when tumors grow within the bone microenvironment and that the effect of SST0001 is not cell-line specific. We did not observe any adverse side effects in animals, even at doses as high as 120 mg/kg/day. To determine the mechanism of action of SST0001, we examined several pharmacodynamic parameters. Immunohistochemistry demonstrated that SST0001 treatment significantly reduced microvessel density of tumors as compared to controls (99% in CAG and 54% in RPMI-8226 tumors). In addition, SST0001 treatment blocked HGF expression (CAG, RPMI 8226 and MM.1S tumors) and inhibited VEGF expression in CAG tumors but not RPMI 8226 and MM.1S tumors. Moreover, a series of in vitro experiments, using the CAG MM cell line and human umbilical vein endothelial cells (HUVEC), were performed. Unlike its strong antitumor effect in vivo, SST0001 only slightly inhibited CAG cell proliferation, cell cycle and growth factor signaling in vitro, suggesting that the compound does not have a direct cytotoxic effect on tumor cells. Since blood vessels are an important element of the tumor microenvironment and angiogenic endothelium in tumors also expresses high levels of heparan sulfate proteoglycans and heparanase, we assessed the effects of SST0001 on HUVEC cells. In contrast with results on CAG MM cells, SST0001 treatment showed a strong inhibition on HUVEC proliferation (46%, MTT assay), dramatically blocked the phosphorylation of ERK stimulated by HS-binding growth factors (HGF, VEGF, HDGF and EGF), blocked the Akt pathway of HGF signaling in HUVECs and inhibited HUVEC tube formation, stimulated by HGF and VEGF. Based on these results, we conclude that SST0001 strongly inhibits the growth of myeloma tumors in vivo by targeting the tumor microenvironment, including a significant inhibition of tumor angiogenesis. Because of its unique target site in the tumor microenvironment, we predict that the combination of SST0001 with conventional tumor cell-targeting chemotherapeutic drugs will greatly improve patient outcome in MM.

Effects of INCB052793, a Selective JAK1 Inhibitor, in Combination with Anti-Myeloma Agents on Human Multiple Myeloma (MM) In Vitro and In Vivo

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3297-3297
Author(s):  
Eric Sanchez ◽  
Mingjie Li ◽  
Cathy S Wang ◽  
Puja Mehta ◽  
George Tang ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Cell Line ◽  
Broad Spectrum ◽  
Alkylating Agents ◽  
Single Agent ◽  
In Vivo Studies ◽  
Post Implantation

Abstract Introduction: Several studies have demonstrated constitutive activation of the JAK-STAT pathway in MM through dysregulated signaling of cytokines such as IL-6. In addition to its crucial role in promoting the growth, proliferation and survival of myeloma cells, IL-6 is also a potent stimulator of osteoclastogenesis and influences the tumor microenvironment in the bone marrow (BM) of MM patients by promoting an immunosuppressive milieu. Since JAK1 has been shown to be important for IL-6 signaling in MM, studies to assess the effect of JAK1 inhibition alone and in combination with other anti-MM agents were undertaken. Methods: The human MM cell lines RPMI8226 and U266 were obtained from ATCC and MM1S was kindly supplied by Steven Rosen, MD (Northwestern University, Chicago, IL). BM aspirates were obtained from patients with MM as approved by the Institutional Review Board (Western IRB BIO 001) and informed consent was obtained in accordance with the Declaration of Helsinki. BM mononuclear cells (MCs) were isolated using density-gradient centrifugation with Histopaque-1077 (Sigma-Aldrich, St. Louis, MO). All cells were maintained in RPMI1640 (Omega Scientific, Tarzana, CA) supplemented with 10% fetal bovine serum (FBS), 2mM l-glutamine, 100 IU/mL penicillin and 100 µg/mL streptomycin, in an atmosphere of 5% carbon dioxide at 37◦C. Primary MM BMMCs were cultured in the presence of the JAK1 selective inhibitor INCB052793 plus a panel of anti-MM agents including the alkylating agents cyclophosphamide (CY), melphalan (MEL), and bendamustine (BEN), the proteasome inhibitor carfilzomib (CAR), dexamethasone (DEX) or the immunomodulatory agents lenalidomide (LEN) and pomalidomide (POM). Cells from RPMI8226 or U266 MM cell lines were cultured in the presence of INCB052793 plus CY, MEL, BEN, CAR, DEX, LEN, or POM. After 48 hours, cell viability was assessed using the MTS assay. For the in vivo studies, mice were implanted with a piece of the human MM tumor LAGk-1A. Seven days post-implantation, mice were randomized into treatment groups, and tumor size was measured on a weekly basis. All in vivo studies were approved by the institutional animal care and use committee. Results: In vitro studies demonstrated that combinations of INCB052793 with a broad spectrum of anti-MM agents synergistically inhibited the viability of BMMCs from MM patients. INCB052793 plus the three alkylating agents or CAR synergistically inhibited the viability of these cells. INCB052793 plus CY or MEL also significantly decreased the viability of the MM1 cell line. In vivo, LAGk-1A-bearing mice had significantly smaller tumors when treated with INCB052793 alone when compared to vehicle control at day 35 post implantation. This was in contrast to mice treated with single agent DEX, LEN or POM. Although the combination of INCB052793 with DEX, LEN or POM did not synergistically inhibit MM cell line growth in vitro, mice receiving the doublets of INCB052793 and DEX, LEN or POM demonstrated an effect on tumor growth that was superior to the doublets of DEX with LEN or POM. Mice receiving the triple combination of INCB052793 + DEX with LEN or POM demonstrated the most significant reduction in tumor growth compared with all other combinations tested. The inhibition of tumor growth with these combinations was observed throughout the study (through day 70) and all combinations were well tolerated. Concomitant with effects on tumor growth, a significant reduction in serum human IgG levels was also observed. In a separate study also using the LAGk-1A model, we evaluated the combination of INCB052793 with CAR or bortezomib (BOR). Combinations of INCB052793 + CAR or BOR were superior at inhibiting tumor growth when compared to single agent INCB052793. Conclusion: These in vitro and in vivo preclinical studies demonstrate that the combination of the JAK1 inhibitor INCB052793 with a broad spectrum of anti-MM agents are effective, and provide further support for the clinical evaluation of these drug combinations for treating MM patients. Studies to further understand the mechanistic effects of these combinations on MM signaling and the tumor microenvironment are ongoing. Disclosures Berenson: Amgen Inc: Consultancy, Honoraria, Research Funding, Speakers Bureau.

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