698 Targeting HLA-G-mediated immunosuppression with a first-in-class antagonist antibody

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A740-A740
Author(s):  
Douglas Hodges ◽  
Christina Kochel ◽  
Michael Totagrande ◽  
Jeffrey Jones ◽  
Megan Welch ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Solid Tumors ◽  
Immune Cells ◽  
Target Cells ◽  
Human Antibody ◽  
Advanced Solid Tumors ◽  
Tumor Types

BackgroundHuman leukocyte antigen-G (HLA-G) is an immune checkpoint molecule that belongs to the non-classical HLA-class I family of receptors. HLA-G restrains immune cell activation and effector function by engaging with inhibitory receptors ILT2 and ILT4. While expression of HLA-G is highly restricted under normal healthy conditions, we have demonstrated that its expression in cancer is aberrantly upregulated and broadly detected across a variety of tumor types. Tizona Therapeutics has generated a novel, fully human antibody that specifically targets HLA-G and reverses HLA-G-mediated immunosuppression. Here we present in vitro and in vivo data demonstrating the functional impact of HLA-G blockade on immune cells and evidence to support the use of TTX-080 in the clinic to treat patients with advanced solid tumors.MethodsEvaluation of HLA-G expression in cancer was performed using immunohistochemistry, flow cytometry, and gene profiling. Expression of ILT2 and ILT4 was assessed on tumor infiltrating leukocytes by flow cytometry. To demonstrate the suppressive function of HLA-G, primary human NK cells, T cells, and monocyte-derived macrophages were cultured with target cells expressing HLA-G. TTX-080 was then evaluated for its ability to reverse this suppression. In addition, TTX-080 was investigated in vivo using a disseminated xenograft tumor model.ResultsExpression of HLA-G was detected on tumor cells and tumor infiltrating leukocytes across a variety of solid tumor types. TTX-080 blocked interaction of HLA-G with both ILT2 and ILT4 and restored cytotoxicity in multiple assays using either primary NK cells or NKL cell lines. Monocyte-derived macrophages expressing ILT2 and ILT4 exhibited decreased phagocytosis of HLA-G+ target cells; this inhibition was reversed with an antigen-binding fragment of TTX-080. TTX-080 was also able to reverse HLA-G-mediated suppression of ILT2+ CD8+ T cells as assessed by degranulation and proinflammatory cytokine secretion. Notably, mice with disseminated tumors had extended median survival when treated with a single dose of TTX-080.ConclusionsTTX-080 reverses HLA-G-mediated suppression of ILT2+ and ILT4+ immune cells that are found within the tumor microenvironment. Blockade of HLA-G using TTX-080 therefore has the potential to reverse broad immune suppression in patients with advanced solid tumors by reinvigorating CD8+ T cells, enhancing NK cytolytic activity, and increasing macrophage phagocytosis.

scholarly journals PD-1 expression on NK cells can be related to cytokine stimulation and tissue residency

2021 ◽  
Author(s):  
Arnika K Wagner ◽  
Nadir Kadri ◽  
Chris Tibbitt ◽  
Koen van de Ven ◽  
Sunitha Bagawath-Singh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Mhc Class I ◽  
Sequencing Analysis ◽  
Single Cell Rna Sequencing ◽  
Cytokine Stimulation ◽  
Deficient Mice

ABSTRACTAlthough PD-1 was shown to be a hallmark of T cells exhaustion, controversial studies have been reported on the role of PD-1 on NK cells. Here, we found by flow cytometry and single cell RNA sequencing analysis that PD-1 can be expressed on MHC class I-deficient tumor-infiltrating NK cells in vivo. We also demonstrate distinct alterations in the phenotype of PD-1-deficient NK cells which in part could be attributed to a decrease in tumor-infiltrating NK cells in PD-1-deficient mice. NK cells from PD-1-deficient mice exhibited a more mature phenotype which might reduce their capacity to migrate and kill in vivo. Finally, our results demonstrate that PD-L1 molecules in membranes of PD-1-deficient NK cells migrate faster than in NK cells from wildtype mice, suggesting that PD-1 and PD-L1 form cis interactions with each other on NK cells.

scholarly journals Nicotinamide combined with gemcitabine is an immunomodulatory therapy that restrains pancreatic cancer in mice

2020 ◽  
Vol 8 (2) ◽  
pp. e001250
Author(s):  
Benson Chellakkan Selvanesan ◽  
Kiran Meena ◽  
Amanda Beck ◽  
Lydie Meheus ◽  
Olaya Lara ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Immune Cells ◽  
Mouse Tumor ◽  
Epitope Spreading ◽  
Stromal Compartment ◽  
Positive Effects

BackgroundTreatments for pancreatic ductal adenocarcinoma are poorly effective, at least partly due to the tumor’s immune-suppressive stromal compartment. New evidence of positive effects on immune responses in the tumor microenvironment (TME), compelled us to test the combination of gemcitabine (GEM), a standard chemotherapeutic for pancreatic cancer, with nicotinamide (NAM), the amide form of niacin (vitamin B3), in mice with pancreatic cancer.MethodsVarious mouse tumor models of pancreatic cancer, that is, orthotopic Panc-02 and KPC (KrasG12D, p53R172H, Pdx1-Cre) grafts, were treated alternately with NAM and GEM for 2 weeks, and the effects on efficacy, survival, stromal architecture and tumor-infiltrating immune cells was examined by immunohistochemistry (IHC), flow cytometry, Enzyme-linked immunospot (ELISPOT), T cell depletions in vivo, Nanostring analysis and RNAscope.ResultsA significant reduction in tumor weight and number of metastases was found, as well as a significant improved survival of the NAM+GEM group compared with all control groups. IHC and flow cytometry showed a significant decrease in tumor-associated macrophages and myeloid-derived suppressor cells in the tumors of NAM+GEM-treated mice. This correlated with a significant increase in the number of CD4 and CD8 T cells of NAM+GEM-treated tumors, and CD4 and CD8 T cell responses to tumor-associated antigen survivin, most likely through epitope spreading. In vivo depletions of T cells demonstrated the involvement of CD4 T cells in the eradication of the tumor by NAM+GEM treatment. In addition, remodeling of the tumor stroma was observed with decreased collagen I and lower expression of hyaluronic acid binding protein, reorganization of the immune cells into lymph node like structures and CD31 positive vessels. Expression profiling for a panel of immuno-oncology genes revealed significant changes in genes involved in migration and activation of T cells, attraction of dendritic cells and epitope spreading.ConclusionThis study highlights the potential of NAM+GEM as immunotherapy for advanced pancreatic cancer.

A phase Ia/Ib trial of the anti-PD-L1 human monoclonal antibody (mAb), CS1001, in patients (pts) with advanced solid tumors or lymphomas.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 2526-2526
Author(s):  
Lin Shen ◽  
Junning Cao ◽  
Jin Li ◽  
Hongming Pan ◽  
Nong Xu ◽  
...  
Keyword(s):  
Disease Progression ◽  
Solid Tumors ◽  
Dose Escalation ◽  
Hepatic Function ◽  
Human Antibody ◽  
Advanced Solid Tumors ◽  
Phase Ib ◽  
Flat Dose ◽  
Tumor Types ◽  
Anti Tumor Activity

2526 Background: CS1001 is the first full-length, fully human anti-PD-L1 mAb developed by the OMT transgenic rat platform, which mirrors natural IgG4 human antibody with expected PK profiles, and may potentially reduce the risk of immunogenicity and toxicity in pts. This first-in-human Phase Ia/Ib study of CS1001 was conducted to evaluate the safety, tolerability, PK profile, and anti-tumor activity of CS1001 in pts with advanced solid tumors or lymphomas. Methods: Pts with advanced solid tumors or lymphomas were enrolled in the dose escalation Phase Ia, receiving CS1001, Q3W, IV, at escalating doses from 3, to 10, 20, 40 mg/kg and 1200 mg. Dose escalation was aided by a 3+3 dose escalation scheme. DLT was evaluated within 3 weeks after the initial dose. Pts with various tumor types were enrolled in the dose expansion Phase Ib to assess anti-tumor activity and safety, including NSCLC, esophageal carcinoma, GC, HCC, cholangiocarcinoma, etc. Safety was assessed by monitoring AEs and the associated grades per NCI CTCAE v4.03, tumor assessed per RECIST v 1.1 (solid tumors) or Lugano 2014 (lymphomas). Results: As of 30 Nov 2018, 29 pts, median age of 53 (23-75) yrs, were enrolled in Phase Ia, 3 mg/kg (N = 3); 10 mg/kg (4); 20 mg/kg (3); 40 mg/kg (3) and 1200 mg flat dose (16). A total of 20 pts discontinued treatment due to disease progression (14), death (2), withdrawal by pts (2) and AEs (2; Grade [G] 4 hepatic function abnormal and G3 pulmonary tuberculosis, both were not related to treatment). 9 pts remain on treatment. Median treatment duration was 126 (21-408+) days. No DLTs were observed. 27 of 29 pts had TRAEs with the most frequent TRAEs including anaemia (14), proteinuria (13) and blood bilirubin increased (8). G3 TRAEs include anaemia (2) and platelet count decreased (1). SAEs were reported in 6 pts and they were TRAEs. Three G4 AEs were reported: anaemia (1), hypokalaemia (1) and hepatic function abnormal (1), they were not TRAEs as determined by the investigators. irAEs occurred in 7 pts (24%). Among the 29 evaluable pts, 7 pts had PR and 8 had SD, mDoR was not reached. In Phase Ib, 97 pts were enrolled, with 65 pts on treatment and 32 pts discontinued from treatment. The most frequent reason on the discontinuation was disease progression (21). Phase Ib enrollment is still ongoing. Conclusions: CS1001 is well tolerated without DLT across tested dose levels. Evidence of anti-tumor activities was observed. Currently, 1200 mg flat dose Q3W is being explored in various tumor types in Phase Ib, and safety and efficacy results will be displayed in the presentation. Clinical trial information: NCT03312842.

scholarly journals Fully human antibody VH domains to generate mono and bispecific CAR to target solid tumors

2021 ◽  
Vol 9 (4) ◽  
pp. e002173
Author(s):  
Guanmeng Wang ◽  
Xin Zhou ◽  
Giovanni Fucà ◽  
Elena Dukhovlinova ◽  
Peishun Shou ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Antigen Expression ◽  
Human Antibody ◽  
Single Chain ◽  
Car T Cells ◽  
Car T

BackgroundChimeric antigen receptor (CAR) T cells are effective in B-cell malignancies. However, heterogeneous antigen expression and antigen loss remain important limitations of targeted immunotherapy in solid tumors. Therefore, targeting multiple tumor-associated antigens simultaneously is expected to improve the outcome of CAR-T cell therapies. Due to the instability of single-chain variable fragments, it remains challenging to develop the simultaneous targeting of multiple antigens using traditional single-chain fragment variable (scFv)-based CARs.MethodsWe used Humabody VH domains derived from a transgenic mouse to obtain fully human prostate-specific membrane antigen (PSMA) VH and mesothelin (MSLN) VH sequences and redirect T cell with VH based-CAR. The antitumor activity and mode of action of PSMA VH and MSLN VH were evaluated in vitro and in vivo compared with the traditional scFv-based CARs.ResultsHuman VH domain-based CAR targeting PSMA and MSLN are stable and functional both in vitro and in vivo. VH modules in the bispecific format are capable of binding their specific target with similar affinity as their monovalent counterparts. Bispecific CARs generated by joining two human antibody VH domains can prevent tumor escape in tumor with heterogeneous antigen expression.ConclusionsFully human antibody VH domains can be used to generate functional CAR molecules, and redirected T cells elicit antitumoral responses in solid tumors at least as well as conventional scFv-based CARs. In addition, VH domains can be used to generate bispecific CAR-T cells to simultaneously target two different antigens expressed by tumor cells, and therefore, achieve better tumor control in solid tumors.

861 Development of FPA157, an anti-CCR8 depleting antibody engineered to preferentially eliminate tumor-infiltrating T regulatory cells

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A914-A914
Author(s):  
Andrew Rankin ◽  
Edwina Naik
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Regulatory T Cells ◽  
Tumor Growth ◽  
T Regulatory Cells ◽  
Target Cells ◽  
Regulatory Cells ◽  
Antibody Treatment ◽  
Tumor Types ◽  
Anti Tumor Activity

BackgroundThe clinical success of PD-1- and CTLA-4- immune checkpoint inhibitors highlights the key contribution of immunosuppression to limiting effective anti-tumor responses. However, as many patients do not respond to anti-PD1 or CTLA4 therapy1-3 novel therapeutics that target additional immune-suppressive mechanisms are needed. Regulatory T cells (Tregs) inhibit immune responses in the tumor microenvironment via multiple suppressive mechanisms.4 5 Existing Treg-targeting agents lack specificity for intratumoral Tregs and can also deplete effector cells, a property that has likely contributed to the lack of clinical activity observed to date. CCR8 (C-C chemokine receptor 8) is selectively expressed on highly activated intratumoral Tregs, its high expression correlates with poor prognosis in multiple human tumor types6 7 and depletion of CCR8+ Tregs in preclinical models elicited potent anti-tumor activity. These observations provided rationale for the development of a CCR8-specific human depleting antibody.MethodsHuman FOXP3 and CCR8 expression was correlated across multiple tumor types using TCGA datasets and expression of CCR8 evaluated in primary tumor explants and PBMCs by flow cytometry. The efficacy of anti-CCR8 antibody treatment was evaluated in the MC38 and CT26 murine tumor models. The depletion of Tregs following anti-CCR8 treatment was assessed by flow cytometry. Flow cytometric-based binding assays were performed using cell lines expressing human or cynomolgus CCR8. Purified human NK cells were co-cultured with CCR8+ target cells and flow cytometry used to evaluate antibody-dependent killing activity.ResultsCCR8 expression was highly correlated with FoxP3 across multiple cancer subtypes and was low to absent on effector T cells. Importantly, CCR8 was not detected on any peripheral human leukocyte subset. In murine tumor models, anti-CCR8 antibody treatment reduced tumor growth in a dose- and Fc-gamma-receptor-dependent manner and resulted in complete regressions and the development of memory. Tumor shrinkage was associated with a reduction in intratumoral Tregs and increased representation of intratumoral CD8 T cells. FPA157 is a highly specific human and cynomolgus crossreactive CCR8 antibody that does not bind closely related chemokine receptors. FPA157 was engineered to enhance antibody-dependent cell-mediated cytotoxicity (eADCC) and elicited potent NK-mediated killing of target cells expressing CCR8 at levels observed on human intratumoralTregs.ConclusionsFPA157 is a CCR8-specific monoclonal antibody with eADCC activity that is being developed for the treatment of cancer. Depletion of CCR8+ Tregs induced substantial anti-tumor activity in pre-clinical models, thus supporting the clinical evaluation of FPA157 as a novel approach to alleviate immune suppression in the microenvironment of human solid tumors.ReferencesHellmann MD, Ciuleanu TE, Pluzanski A, Lee JS, Otterson GA, Audigier-Valette C, Minenza E, Linardou H, Burgers S, Salman P, Borghaei H, Ramalingam SS, Brahmer J, Reck M, O’Byrne KJ, Geese WJ, Green G, Chang H, Szustakowski J, Bhagavatheeswaran P, Healey D, Fu Y, Nathan F, Paz-Ares L. Nivolumab plus Ipilimumab in lung cancer with a high tumor mutational burden. N Engl J Med 2018;378(22):2093-2104.Wolchok JD, Chiarion-Sileni V, Gonzalez R, Rutkowski P, Grob JJ, Cowey CL, Lao CD, Wagstaff J, Schadendorf D, Ferrucci PF, Smylie M, Dummer R, Hill A, Hogg D, Haanen J, Carlino MS, Bechter O, Maio M, Marquez-Rodas I, Guidoboni M, McArthur G, Lebbé C, Ascierto PA, Long GV, Cebon J, Sosman J, Postow MA, Callahan MK, Walker D, Rollin L, Bhore R, Hodi FS, Larkin J. Overall survival with combined nivolumab and ipilimumab in advanced melanoma. N Engl J Med 2017;377(14):1345-1356.Motzer RJ, Tannir NM, McDermott DF, Arén Frontera O, Melichar B, Choueiri TK, Plimack ER, Barthélémy P, Porta C, George S, Powles T, Donskov F, Neiman V, Kollmannsberger CK, Salman P, Gurney H, Hawkins R, Ravaud A, Grimm MO, Bracarda S, Barrios CH, Tomita Y, Castellano D, Rini BI, Chen AC, Mekan S, McHenry MB, Wind-Rotolo M, Doan J, Sharma P, Hammers HJ, Escudier B; CheckMate 214 Investigators. Nivolumab plus ipilimumab versus sunitinib in advanced renal-cell carcinoma. N Engl J Med 2018 Apr 5;378(14):1277-1290.Teng MW, Ngiow SF, von Scheidt B, McLaughlin N, Sparwasser T, Smyth MJ. Conditional regulatory T-cell depletion releases adaptive immunity preventing carcinogenesis and suppressing established tumor growth [published correction appears in Cancer Res. 2010; 70(23):10014]. Cancer Res 2010;70(20):7800-7809.Simpson TR, Li F, Montalvo-Ortiz W, Sepulveda MA, Bergerhoff K, Arce F, Roddie C, Henry JY, Yagita H, Wolchok JD, Peggs KS, Ravetch JV, Allison JP, Quezada SA. Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J Exp Med 2013;210(9):1695-710.Plitas G, Konopacki C, Wu K, Bos PD, Morrow M, Putintseva EV, Chudakov DM, Rudensky AY. Regulatory T cells exhibit distinct features in human breast cancer. Immunity 2016;45(5):1122-1134.De Simone M, Arrigoni A, Rossetti G, Gruarin P, Ranzani V, Politano C, Bonnal RJP, Provasi E, Sarnicola ML, Panzeri I, Moro M, Crosti M, Mazzara S, Vaira V, Bosari S, Palleschi A, Santambrogio L, Bovo G, Zucchini N, Totis M, Gianotti L, Cesana G, Perego RA, Maroni N, Pisani Ceretti A, Opocher E, De Francesco R, Geginat J, Stunnenberg HG, Abrignani S, Pagani M. Transcriptional landscape of human tissue lymphocytes unveils uniqueness of tumor-infiltrating T regulatory cells. Immunity 2016;45(5):1135-1147.

scholarly journals 105 4–1BB and optimized CD28 co-stimulation enhances function of human mono- and bi-specific third-generation CAR T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A115-A116
Author(s):  
Emiliano Roselli ◽  
Justin Boucher ◽  
Gongbo Li ◽  
Hiroshi Kotani ◽  
Kristen Spitler ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
The Other ◽  
Target Cells ◽  
Third Generation ◽  
Car T Cells ◽  
Nsg Mice ◽  
Car T

BackgroundCo-stimulatory signals regulate the expansion, persistence, and function of chimeric antigen receptor (CAR) T cells. Most studies have focused on the co-stimulatory domains CD28 or 4-1BB. CAR T cell persistence is enhanced by 4-1BB co-stimulation leading to NF-κB signaling, while resistance to exhaustion is enhanced by mutations of the CD28 co-stimulatory domain.MethodsWe hypothesized that a third-generation CAR containing 4-1BB and CD28 with only PYAP signaling motif (mut06) would provide beneficial aspects of both. We designed CD19-specific CAR T cells with 4-1BB or mut06 together with the combination of both (BB06). We evaluated their immune-phenotype, cytokine secretion, real-time cytotoxic ability and polyfunctionality against CD19-expressing cells. We analyzed LCK recruitment by the different constructs by immunoblotting. We further determined their ability to control growth of Raji cells in NSG mice. Additionally, we engineered bi-specific CARs against CD20/CD19 combining 4-1BB and mut06 and performed repeated in vitro antigenic stimulation experiments to evaluate their expansion, memory phenotype and phenotypic (PD1+CD39+) and functional exhaustion. Bi-specific CAR T cells were transferred into Raji or Nalm6-bearing mice to study their ability to eradicate CD20/CD19-expressing tumors.ResultsCo-stimulatory domains combining 4-1BB and mut06 confers CAR T cells with an increased polyfunctionality and LCK recruitment to the CAR (figure 1A), after repeated-antigen stimulation these cells expanded significantly better than second-generation CAR T cells (figure 1B). A bi-specific CAR targeting CD20/CD19, incorporating 4-1BB and mut06 co-stimulation, showed enhanced antigen-dependent in vitro expansion with lower exhaustion-associated markers (figure 1C). Bi-specific CAR T cells exhibited improved in vivo anti-tumor activity with increased persistence and decreased exhaustion (figure 1D).Abstract 105 Figure 1A. pLCK is increased in h19BB06z CAR T cells and associated with the CAR. CAR T cells were stimulated with irradiated 3T3-hCD19 cells at a 10:1 E:T ratio for 24hr. Cells were lysed and CAR bound and unbound fractions were western blotted. A single-cell measure of polyfunctional strength index (PSI) of CAR T cells. B. h19BB06z CAR T cells have the highest proliferation after repeated antigen stimulations. 5x105 CAR T cells were stimulated with 1x105 irradiated 3T3-hCD19 cells. After 1 week, half of the cells were enumerated by flow cytometry and the other half was re-stimulated with 1x105 fresh irradiated 3T3-hCD19 cells. This was repeated for a total of 4 weeks. C. 5x105 CAR T cells were co-cultured with 5x105 target cells (Raji-CD19High). After 1 week half the cells were harvested enumerated and stained by flow cytometry while the other half was re-stimulated with 5x105 fresh target cells (indicated by arrows). This was repeated for a total of 4 weeks. Frequency of PD1+CD39+ cells within CD8+ CAR T cells. D. Raji-FFLuc-bearing NSG mice were treated with 1x106 CAR T cells 5 days after initial tumor cell injection. Tumor burden (average luminescence) of mice treated with bi-specific or monospecific CAR T cells, UT and tumor control. Each line represents an individual mouse. (n = 7 mice per group).ConclusionsThese results demonstrate that co-stimulation combining 4-1BB with an optimized form of CD28 is a valid approach to optimize CAR T cell function. Cells with both mono- and bi-specific versions of this design showed enhanced in vitro and in vivo features such as expansion, persistence and resistance to exhaustion. Our observations validate the approach and justify clinical studies to test the efficacy and safety of this CAR in patients.

scholarly journals Differential Effects of Iberdomide Versus Revlimid on Leukocyte Trafficking, Immune Activation and DLBCL Tumor Cell Killing

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 718-718
Author(s):  
Yumi Nakayama ◽  
Hsiling Chiu ◽  
Rama K. Narla ◽  
Arvind Shakya ◽  
Jim Gamez ◽  
...  
Keyword(s):  
Clinical Trials ◽  
T Cells ◽  
Mouse Model ◽  
Nk Cells ◽  
Immune Cells ◽  
Stock Options ◽  
Substrate Degradation ◽  
Privately Held

Abstract Introduction: Revlimid (Rev), binds to CRL4 CRBN E3 ligase leading to recruitment and proteasomal degradation of transcription factors Aiolos and Ikaros. This inhibits proliferation of malignant B cells and stimulates activity of T, NK and macrophage cells, thereby providing clinical activity of Rev as a single agent and in combination with CD19/CD20 antibodies in DLBCL and FL. Iberdomide (Iber), a new CELMoD with enhanced substrate degradation compared to Rev, is currently being studied in clinical trials for B-NHL and MM. Presented here is extensive in vitro and in vivo characterization of immune enhancement and antitumor effects of Iber with direct comparison to Rev. Results: In a panel of DLBCL cell lines, comprising ABC and GCB-DLBCL models, Iber degraded Aiolos/Ikaros with faster kinetics and to a greater depth than Rev, which led to enhanced antiproliferative and cytotoxic effects. Iber acted in a cell of origin independent manner, whereas Rev is preferentially active in ABC-DLBCL. To examine the molecular effects of Iber and Rev in immune cells, we performed RNAseq and proteomic based analyses on Iber and Rev treated T, NK and monocyte cell populations. These experiments revealed a complex series of immunomodulatory activities including promotion of pro-inflammatory cytokine production, activation marker expressions and migratory machinery with a trend of Iber exhibiting greater enhancements. We confirmed these findings by demonstrating that secretion of chemoattractants for T cells, NK cells and monocytes including CXCL9, 10 and 11 (10-90% increase) and CCL8 (30% increase, p<0.01) were higher in PBMCs treated with Iber compared to Rev. Additionally, functional chemotaxis assays demonstrated that Iber and Rev increased the trafficking capacity of T-cells compared to DMSO alone, with Iber demonstrating a greater increase than Rev (46% vs 21%, p<0.01). Furthermore, Iber increased the proliferative capacity of CD8+ T and NK cells compared to Rev (10 and 3.6-fold vs 4 and 2.8-fold, respectively). Functional co-culture assays with DLBCL cells showed that Iber induced NK cell mediated killing of DLBCL cells to a greater extent than Rev and each molecule enhanced ADCC with Rituximab compared to vehicle controls. Translational data from clinical trials of a related CELMoD, Avadomide, revealed significant trafficking of immune cells such a T cells, NK cells and monocytes to the tumor microenvironment (TME). To examine the effects of Rev and Iber in an in vivo DLBCL GEMM model, we developed a humanized CRBN (hCRBN) mouse capable of facilitating proteasomal degradation of target substrates upon treatment with a CELMoD. The hCRBN mouse was then crossed with the Eμ-Myc DLBCL mouse model resulting in Eμ-Myc/hCRBN progeny that then developed disease. Splenocytes were collected and transplanted to recipient hCRBN mice. The tumor cells were allowed to engraft for 5 days upon which 3 daily doses of vehicle, Rev and Iber were given prior to the mice being sacrificed. Non-transplanted hCRBN mice served as controls. Similar to human disease, DLBCL cells remodeled the myofibroblast-immune network within lymph node and the splenic tissues including activated podoplanin (PDPN)-expressing fibroblastic reticular cells (FRCs) and diminished CD8+ T cells and CD11c+ DCs within the lymphoid TMEs. Treatment with Iber resulted in significantly enhanced infiltration of DCs and notably, cytolytic granzyme B positive T cells into the TME compared to Rev or vehicle treated mice (Figure 1). Additional characterization of the immune (T cell, NK and monocyte)-stroma TME is on-going and will be presented. Conclusion: Our data demonstrate that Iber is more potent in substrate degradation and functionality in anti-proliferative activity against DLBCL cell line models and at triggering immunostimulatory activities in multiple lymphoid and myeloid populations. Additionally, we generated a humanized CRBN mouse model that revealed the ability of CELMoDs in inducing immune-rich TMEs supporting rational combination strategies with immune focused agents being explored in lymphoma such as SIRPα blockade, CAR T and CD3xCD20 bispecifics. Figure 1 Figure 1. Disclosures Nakayama: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Chiu: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Narla: Bristol Myers Squibb: Current Employment. Shakya: Bristol Myers Squibb: Current Employment. Gamez: Bristol Myers Squibb: Current Employment. Hagner: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Gandhi: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

First Clinical Evidence of In Vivo Natural Killer (NK) Cell Modulation in Chronic Lymphocytic Leukemia (CLL) Patients (pts) Treated with Lenalidomide (L).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2109-2109 ◽  
Author(s):  
Swaminathan Padmanabhan ◽  
Noreen Ersing ◽  
Paul K. Wallace ◽  
Kena C. Miller ◽  
Laurie Musiel ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Nk Cells ◽  
Phase Ii ◽  
Nk Cell ◽  
Cytokine Profile ◽  
Lymphocytic Leukemia ◽  
Appreciable Change

Abstract Introduction: Pts with Chronic Lymphocytic Leukemia (CLL) are reported to have quantitative and qualitative T and NK cell dysfunction. While NK cells act through non-specific killing, T-cells are more specific. The 2 types of T-lymphocytes, CD4+ (Th; helper) and CD8+ (Ts; cytotolytic/suppressor) are subcategorized based on cytokine secretion profile upon activation. Release of different cytokines from these immune cells modulates the host response. T1 cells (Th1, Ts1) secrete IL-2 and interferon-g which initiate the Th1 response- mainly CD4+ activation along with B and T cells, leading to proliferation and differentiation of these cells. T2 (Th2, Ts2) cells initiate the Th2 response (release of TNF-a, IL-10) resulting in direct lysis of the target cell by production of cytokines such as IL-4, IL-5 and IL-10. Hypothesis: To decipher this antitumor mechanism of L in CLL pts we investigated its effect on the efferent arm of immune response by evaluating the T cell population and the afferent response by change in expression of co-stimulatory molecules on B-CLL cells and cytokine profile in these pts treated on a phase II clinical study. Methods: CLL pts treated with L were evaluated for absolute number of T (CD4+, CD8+) and NK (CD56+) cells by flow cytometry on day before (day0) start and on Day 8 of treatment with L. Peripheral blood was collected and ficolled to obtain enriched mononuclear cells. The serum was used to study the cytokines. Activation status was determined by co-expression of CD45+. Serum cytokine profile was measured by Flow cytometry using the Luminex system. B-CLL surface co-stimulatory molecules were detected by flow cytometry and analyzed by FACS. These responses were correlated with the tumor flare (TF) reaction that the patients developed during the first week of treatment with L. Results: Eighteen out of 45 pts have so far been evaluated for immunomodulatory activity of L. There were 2 complete responders (CRs) and 6 partial responders (PRs); while 4 had stable disease (SD), 4 were clinically unevaluable and 2 were too early for response in this group. Mean baseline (bl) NK cell count pretreatment was 251 (range 31–1510) vs. post treatment was 193 (range 6–13,482). Six out of 18 patients showed an increase, ranging from 20 −199% in the absolute NK (CD16+/CD56+/CD45+). While there was no appreciable change in CD4+ numbers there was a general trend in increase of CD8+ cells. No change in monocyte population was noted. Concurrent increase in the expression of co-stimulatory molecules such as CD95 and CD80 was noted. This response in co-stimulation was confirmed by in vitro experiments done on isolated B-CLL cells (n=4)treated with L. An increase in Th-2 cytokines such as IL-4, IL-5, IL-6 and IL-10 was noted in all eight responders, while VEGF levels were decreased in 6/18 patients. 99% of patients had a TF and the grade of TF correlated with the changes in T cells and cytokine profile. Conclusion: It appears that in vivo L is able to orchestrate an anti-tumor response in CLL by modulating the NK cells, changing the cytokine profile and up-regulating co-stimulatory molecules. This change in the immune effector cell repertoire and the Th2 skewing may explain the initial flare reaction noted in these L treated pts. Data from these correlative studies is being evaluated in the context of the phase II clinical trial to be reported at the 48th ASH annual meeting.

scholarly journals Reverse Signaling by MHC-I Molecules in Immune and Non-Immune Cell Types

2020 ◽  
Vol 11 ◽  
Author(s):  
Elke M. Muntjewerff ◽  
Luca D. Meesters ◽  
Geert van den Bogaart ◽  
Natalia H. Revelo
Keyword(s):  
T Cells ◽  
Immune Cells ◽  
Cell Types ◽  
Signaling Molecules ◽  
Target Cells ◽  
Class I Mhc ◽  
Mhc I ◽  
Reverse Signaling ◽  
Mhc Molecules

Major histocompatibility complex (MHC) molecules are well-known for their role in antigen (cross-) presentation, thereby functioning as key players in the communication between immune cells, for example dendritic cells (DCs) and T cells, or immune cells and their targets, such as T cells and virus-infected or tumor cells. However, much less appreciated is the fact that MHC molecules can also act as signaling receptors. In this process, here referred to as reverse MHC class I (MHC-I) signaling, ligation of MHC molecules can lead to signal-transduction and cell regulatory effects in the antigen presenting cell. In the case of MHC-I, reverse signaling can have several outcomes, including apoptosis, migration, induced or reduced proliferation and cytotoxicity towards target cells. Here, we provide an overview of studies showing the signaling pathways and cell outcomes upon MHC-I stimulation in various immune and non-immune cells. Signaling molecules like RAC-alpha serine/threonine-protein kinase (Akt1), extracellular signal-regulated kinases 1/2 (ERK1/2), and nuclear factor-κB (NF-κB) were common signaling molecules activated upon MHC-I ligation in multiple cell types. For endothelial and smooth muscle cells, the in vivo relevance of reverse MHC-I signaling has been established, namely in the context of adverse effects after tissue transplantation. For other cell types, the role of reverse MHC-I signaling is less clear, since aspects like the in vivo relevance, natural MHC-I ligands and the extended downstream pathways are not fully known.The existing evidence, however, suggests that reverse MHC-I signaling is involved in the regulation of the defense against bacterial and viral infections and against malignancies. Thereby, reverse MHC-I signaling is a potential target for therapies against viral and bacterial infections, cancer immunotherapies and management of organ transplantation outcomes.

scholarly journals Functional Characterization of Gamma Delta T Cells and Allogeneic Activated NK Cells As Effector Cells for Tafasitamab (MOR208)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3801-3801
Author(s):  
Jung Hyun Her ◽  
Dominik Pretscher ◽  
Sungyoo Cho ◽  
Yu-Kyeong Hwang ◽  
Timm Hoeres ◽  
...  
Keyword(s):  
T Cells ◽  
Combination Therapy ◽  
B Cell ◽  
Nk Cells ◽  
Effector Cell ◽  
Γδ T Cells ◽  
Target Cells ◽  
Effector Cells

Introduction Tafasitamab (MOR208) is an Fc-enhanced, humanized, monoclonal antibody that binds to the human B cell surface antigen CD19. CD19 is broadly and homogeneously expressed across different B cell malignancies, including diffuse large B cell lymphoma (DLBCL), and amplifies B cell receptor signaling and induces tumor cell proliferation. Tafasitamab is currently in clinical development in patients with relapsed or refractory DLBCL in combination with the immunomodulatory drug lenalidomide (L-MIND study) and the chemotherapeutic agent bendamustine (B-MIND study). The modes of action of tafasitamab comprise antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and direct cytotoxicity (apoptosis). Tafasitamab carries two amino acid exchanges in the Fc region to increase its affinity to Fcγ receptors, including FcγRIIIa. FcγRIIIa plays a key role in mediating ADCC and is expressed on the surface of natural killer (NK) cells, as well as the majority of γδ T cells. MG4101 (a novel therapeutic agent consisting of cryopreserved, ex vivo-expanded, highly activated NK cells) has demonstrated potent anticancer activity in preclinical in vitro and in vivo studies. Currently, MG4101 is in clinical development in patients with malignant lymphoma and advanced solid tumors. Here, we have characterized two FcγRIIIa receptor-expressing cell types, γδ T cells and NK cells (MG4101), as effector cells for tafasitamab in vitro and explored the concept of supplementing MG4101 during tafasitamab therapy using disseminated in vivo models of non-Hodgkin's lymphoma. Methods γδ T cells (CD3+/γδ T cell receptor+) were derived from different donors by stimulation of peripheral blood mononuclear cells with zoledronate/IL-2 for 9-10 days. These were applied as effector cells in in vitro ADCC assays with tafasitamab in Mino and Jeko-1 mantle cell lymphoma (MCL) cell lines, as well as primary patient-derived chronic lymphocytic lymphoma (CLL) and MCL cells. Further, effector cell activity of MG4101 was assessed using tafasitamab-mediated ADCC assays in Raji and Ramos Burkitt's lymphoma cells. The concept of combining tafasitamab with allogeneic effector cell therapy in vivo was studied in two therapeutic survival models of disseminated lymphoma in SCID mice. In the Raji model, tafasitamab (0.05 µg/mouse) was given on Day 1 after intravenous (IV) tumor inoculation, while MG4101 (2x107 cells/mouse) was given on Days 1, 3, 6, 8 and 10. In the Ramos model, tafasitamab (10 mg/kg) and MG4101 (2x107 cells/mouse) were applied twice weekly for 3 weeks starting on Days 3 and 4, respectively, after IV tumor inoculation. Results Tafasitamab in combination with γδ T cells showed distinctly increased ADCC in Mino and Jeko-1 target cells in vitro, compared with a negative control IgG1 antibody. ADCC assays with patient-derived CLL and MCL target cells confirmed tafasitamab-mediated cytotoxic activity and demonstrated a clear enhancement in activity compared with the non-Fc-enhanced version of tafasitamab that was unable to induce substantial cytotoxicity. In vitro ADCC assays with tafasitamab and MG4101 on Raji and Ramos cell lines confirmed potent effector cell activity of the ex vivo-expanded, cryopreserved, allogeneic NK cells. In the disseminated Raji survival model, combination therapy with a single low dose of tafasitamab (0.05 µg) and MG4101 resulted in a distinct increase in survival of the mice with an increased life span (ILS) of 100% compared with monotherapy (ILS of 57% for tafasitamab and 50% for MG4101). Of note, the combination demonstrated a substantial and more than additive enhancement in survival in the more therapeutic Ramos survival model (Figure 1). Combination therapy with tafasitamab (10 mg/kg) and MG4101 NK cells resulted in superior antitumor activity (ILS of 103%) compared with either tafasitamab monotherapy (ILS of 49%) or MG4101 alone (ILS of 25%). Conclusions FcγRIIIa-expressing immune cell types, including NK cells and γδ T cells, are potent effector cells for tafasitamab-mediated ADCC. Combination therapy with tafasitamab and allogeneic MG4101 NK cells in vivo demonstrated a more than additive survival benefit compared with tafasitamab or MG4101 monotherapy in a disseminated therapeutic lymphoma model. Combination of tafasitamab supplemented with immune effector cells could represent a promising new approach for lymphoma therapy. Disclosures Her: GC LabCell: Employment, Patents & Royalties. Cho:GC LabCell: Employment, Patents & Royalties. Hwang:GC LabCell: Employment, Equity Ownership, Patents & Royalties. Boxhammer:MorphoSys AG: Employment, Patents & Royalties. Steidl:MorphoSys AG: Employment. Patra:MorphoSys AG: Employment. Schanzer:MorphoSys AG: Employment. Endell:MorphoSys AG: Employment, Patents & Royalties.

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