The Anti-Kappa Monoclonal Antibody MDX-1097 Synergizes with Immunomodulatory Drugs to Enhance Antibody-Dependent Cell Cytotoxicity of Multiple Myeloma Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4012-4012
Author(s):  
Andrew R Cuddihy ◽  
Parisa Asvadi ◽  
Rosanne Dunn ◽  
Tiffany T. Khong ◽  
Andrew Spencer
Keyword(s):  
Cell Death ◽  
Plasma Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Immune Effector ◽  
Effector Cells ◽  
Dependent Cell ◽  
In Vitro Treatment

Abstract Abstract 4012 Multiple Myeloma (MM) is a cancer caused by the proliferation of malignant clonal plasma cells in the bone marrow and accounts for 10% of all hematologic malignancies. Recent advances have been made in the treatment and management of MM, however, despite these advances the majority of patients will ultimately relapse and die from their disease within 3–5 years from diagnosis. Several novel therapeutic approaches, including the use of antibody-based therapies, are being investigated to further improve the treatment of MM. MDX-1097 is a chimeric monoclonal antibody being assessed as a single agent in a Phase 2 clinical trial for the treatment of kappa light-chain restricted (κ-type) MM. MDX-1097 binds to the kappa myeloma antigen (KMA), a tumor-specific membrane-associated protein expressed on malignant plasma cells from patients with K-type MM. Previously we have demonstrated that MDX-1097 exerts its anti-tumour effects through multiple mechanisms, including antibody-dependent cell cytotoxicity (ADCC) in the presence of either normal human peripheral blood mononuclear cells (PBMCs) or purified natural killer (NK cells). The immunomodulatory drugs (IMiDs) lenalidomide (Revlimid) and pomalidomide (Actimid) are currently in use or being assessed for the treatment of MM. These IMiDs have been shown to exert their anti-tumor effects both directly, via apoptotic mechanisms, and indirectly via a number of different mechanisms including the augmentation of NK-dependent cellular cytotoxicity. In this study we report that IMiDs and MDX-1097 co-operate to promote enhanced ADCC of MM cells. In vitro treatment of normal PBMCs with IMiDs led to a 1.4-fold higher level of ADCC-mediated cell death of MDX-1097 spiked JJN3 cells (a κ-type MM cell line) compared with vehicle-treated PBMCs from the same donor. Similarly, in vivo lenalidomide exposed PBMCs isolated from a MM patient were, on average, 1.8-fold more effective in killing MDX-1097 spiked JJN3 cells in vitro compared to PBMC obtained from the same patient prior to lenalidomide treatment. Treatment of JJN3 cells with IMiDs resulted in significantly increased cell surface expression of KMA (lenalidomide: 1.9-fold, p < 0.001; pomalidomide: 2.3-fold, p < 0.01). These IMiD-treated JJN3 cells, when spiked with MDX-1097 were 1.7-fold more susceptible to ADCC-mediated cell death in the presence of untreated PBMCs, compared to JJN3 cells treated with vehicle alone. This difference in sensitivity to ADCC mediated cell death is presumably due to increased KMA expression resulting in more binding sites for MDX-1097, therefore facilitating recruitment of PB immune effector cells. Furthermore, combining IMiD-treated PBMCs with IMiD-treated, MDX-1097 spiked JJN3 cells resulted in a further increment in ADCC-mediated JJN3 cell death. This study demonstrates that in vivo and in vitro treatment of PBMCs with IMiDs engages the PB immune effector cells, leading to increased ADCC-induced κ-type MM cell death in vitro in the presence of MDX-1097. IMiDs also increase cell surface expression of KMA, leading to increased MDX-1097 binding and in turn also enhancing ADCC-induced MM cell killing. Our data provides a rationale for the clinical evaluation of a combination therapy involving both IMiDs and MDX-1097 for the treatment of k-type MM. Disclosures: Cuddihy: Immune System Therapeutics Ltd: Research Funding. Asvadi:Immune System Therapeutics Ltd: Employment. Dunn:Immune System Therapeutics Ltd: Employment, Equity Ownership. Spencer:Immune System Therapeutics Ltd: Research Funding.

scholarly journals Potent in vitro and in vivo activity of an Fc-engineered humanized anti-HM1.24 antibody against multiple myeloma via augmented effector function

Blood ◽  
2012 ◽  
Vol 119 (9) ◽  
pp. 2074-2082 ◽  
Author(s):  
Yu-Tzu Tai ◽  
Holly M. Horton ◽  
Sun-Young Kong ◽  
Erik Pong ◽  
Hsing Chen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Immune Effector ◽  
Bone Marrow Plasma ◽  
Therapeutic Monoclonal Antibodies

Abstract HM1.24, an immunologic target for multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). In this study, we investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 mAb with Fc-domain engineered to significantly enhance FcγR binding and associated immune effector functions. XmAb5592 increased antibody-dependent cellular cytotoxicity (ADCC) several fold relative to the anti-HM1.24 IgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody dependent cellular phagocytosis (ADCP) by macrophages. Natural killer (NK) cells became more activated by XmAb5592 than the IgG1 analog, evidenced by increased cell surface expression of granzyme B–dependent CD107a and MM cell lysis, even in the presence of bone marrow stromal cells. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via FcγR-dependent mechanisms, and was significantly more effective than the IgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20 mg/kg XmAb5592 effectively depleted both blood and bone marrow plasma cells in cynomolgus monkeys. These results support clinical development of XmAb5592, both as a monotherapy and in combination with lenalidomide, to improve patient outcome of MM.

Investigating macrophage function as a mechanism of resistance to daratumumab in relapsed refractory multiple myeloma patients.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e20547-e20547
Author(s):  
Swetha Kambhampati ◽  
Kwun Wah Wen ◽  
Victoria Sung ◽  
Sandy Wai Kuan Wong ◽  
Thomas G. Martin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Plasma Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Refractory Multiple Myeloma ◽  
Macrophage Activity ◽  
Cell Counts ◽  
Significant Change

e20547 Background: Daratumumab (Dara), an anti-CD38 antibody, has a variety of Fc-dependent immune effector mechanisms of action including macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). However, it remains unclear which mechanism, when disrupted, drives in vivo resistance. Given widespread use of this agent for treatment of multiple myeloma, identifying how to overcome Dara resistance has become an urgent clinical need. We hypothesized that inhibiting macrophage activity, via increased plasma cell surface expression of the CD47 “don’t eat me” signal, could be a mechanism of acquired Dara resistance. Methods: We retrospectively identified 11 patients with relapsed refractory multiple myeloma who progressed on Dara and had sequential bone marrow core biopsies before and after Dara with both samples containing > 20% plasma cells. Nine patients had sufficient material for analysis. Core biopsy slides were stained and analyzed for expression of CD47 (H-scoring for total intensity on plasma cells, as well as annotation of membrane vs. cytosolic localization), CD68 (cell counts of macrophages), and CD4/CD8/FOXP3 triple stain (cell counts of CD4 and CD8 effector and regulatory T-cells). Results: Median age was 57 years. Median R-ISS stage was 2. Two patients had high-risk cytogenetics at diagnosis. Median time from diagnosis to progression on Dara was 56.3 months (95% CI 37.43 – 88.92). Median overall survival was 69.5 months (95% CI: 46.5 – 97.1). Plasma cells in all samples expressed CD47 (H-score median = 96, range 43 - 290). However, in contrast to our hypothesis, there was no significant change in CD47 H-score at progression on Dara (H-score median = 100, range 29 – 140). Furthermore, we observed no significant change in CD68+ macrophage counts nor CD68+ localization at Dara progression. We did note a trend toward CD47 expression shifting from membrane to cytosol at Dara resistance ( p = 0.14). Consistent with prior studies, in our cohort we observed no influx in Tregs but a markedly increased CD8/CD4 ratio at resistance ( p = 0.01). Conclusions: In vitro and murine studies have proposed ADCP as an important mechanism of Dara efficacy. However, we did not observe signatures of resistance in patients being driven by this pathway. Our work suggests that anti-CD47 therapy, leading to activation of macrophages to eliminate tumor cells, will potentially be efficacious for both the Dara-naïve and Dara-refractory settings.

scholarly journals Mitochondrion-Dependent Cell Death in TDP-43 Proteinopathies

Biomedicines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 376
Author(s):  
Chantal B. Lucini ◽  
Ralf J. Braun
Keyword(s):  
Cell Death ◽  
Oxygen Species ◽  
In Vivo Models ◽  
Dependent Cell ◽  
Cell Death Mechanisms ◽  
Sting Pathway ◽  
Mitochondrial Membrane Permeabilization

In the last decade, pieces of evidence for TDP-43-mediated mitochondrial dysfunction in neurodegenerative diseases have accumulated. In patient samples, in vitro and in vivo models have shown mitochondrial accumulation of TDP-43, concomitantly with hallmarks of mitochondrial destabilization, such as increased production of reactive oxygen species (ROS), reduced level of oxidative phosphorylation (OXPHOS), and mitochondrial membrane permeabilization. Incidences of TDP-43-dependent cell death, which depends on mitochondrial DNA (mtDNA) content, is increased upon ageing. However, the molecular pathways behind mitochondrion-dependent cell death in TDP-43 proteinopathies remained unclear. In this review, we discuss the role of TDP-43 in mitochondria, as well as in mitochondrion-dependent cell death. This review includes the recent discovery of the TDP-43-dependent activation of the innate immunity cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway. Unravelling cell death mechanisms upon TDP-43 accumulation in mitochondria may open up new opportunities in TDP-43 proteinopathy research.

scholarly journals PD-L1hi plasmablasts limit the T cell response during the acute phase of parasite infections

2020 ◽  
Author(s):  
Melisa Gorosito Serrán ◽  
Facundo Fiocca Vernengo ◽  
Laura Almada ◽  
Cristian G Beccaria ◽  
Pablo F Canete ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Response ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Chronic Phase ◽  
Surface Expression ◽  
T Cell Response

ABSTRACTDuring infections with protozoan parasites or virus, T cell immunosuppression is generated simultaneously with a high B cell activation. Here, we show that in T. cruzi infection, all plasmablasts detected had higher surface expression of PD-L1, than other mononuclear cells. PD-L1hi plasmablasts were induced in vivo in an antigen-specific manner and required help from Bcl-6+CD4+T cells. PD-L1hi expression was not a characteristic of all antibody-secreting cells since plasma cells found during the chronic phase of infection express PD-L1 but at lower levels. PD-L1hi plasmablasts were also present in mice infected with Plasmodium or with lymphocytic choriomeningitis virus, but not in mice with autoimmune disorders or immunized with T cell-dependent antigens. PD-L1hi plasmablasts suppressed T cell response, via PD-L1, in vitro and in vivo. Thus, this study reveals that extrafollicular PD-L1hi plasmablasts, which precede the germinal center (CG) response, are a suppressive population in infections that may influence T cell response.Brief summaryPathogens develop different strategies to settle in the host. We identified a plasmablats population induced by pathogens in acute infections which suppress T cell response.

scholarly journals L1 endocytosis is controlled by a phosphorylation-dephosphorylation cycle stimulated by outside-in signaling by L1

2002 ◽  
Vol 157 (7) ◽  
pp. 1223-1232 ◽  
Author(s):  
Andrew W. Schaefer ◽  
Yoshimasa Kamei ◽  
Hiroyuki Kamiguchi ◽  
Eric V. Wong ◽  
Iris Rapoport ◽  
...  
Keyword(s):  
Surface Expression ◽  
Cell Surface Expression ◽  
Cross Linking ◽  
Cell Contact ◽  
Homophilic Binding ◽  
Dynamic Regulation ◽  
Nonreceptor Tyrosine Kinase ◽  
Neuronal Growth Cone

Dynamic regulation of the cell surface expression of adhesion molecules is an important mechanism for controlling neuronal growth cone motility and guidance. Clathrin-mediated vesicular internalization of L1 via the tyrosine-based endocytosis motif YRSL regulates adhesion and signaling by this Ig superfamily molecule. Here, we present evidence that tyrosine-1176 (Y1176) of the YRSL motif is phosphorylated in vivo. The nonreceptor tyrosine kinase (p60src) is implicated in L1-mediated neurite outgrowth, and we find that p60src phosphorylates Y1176 in vitro. Phosphorylation of Y1176 prevents L1 binding to AP-2, an adaptor required for clathrin-mediated internalization of L1. mAb 74-5H7 recognizes the sequence immediately NH2-terminal to the tyrosine-based motif and binds L1 only when Y1176 is dephosphorylated. 74-5H7 identifies a subset of L1 present at points of cell–cell contact and in vesicle-like structures that colocalize with an endocytosis marker. L1–L1 binding or L1 cross-linking induces a rapid increase in 74-5H7 immunoreactivity. Our data suggest a model in which homophilic binding or L1 cross-linking triggers transient dephosphorylation of the YRSL motif that makes L1 available for endocytosis. Thus, the regulation of L1 endocytosis through dephosphorylation of Y1176 is a critical regulatory point of L1-mediated adhesion and signaling.

A Novel Agent SL-401 Triggers Anti-Myeloma Activity By Targeting Plasmacytoid Dendritic Cells: Implications for a Novel Immune-Associated Mechanism

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3000-3000 ◽  
Author(s):  
Arghya Ray ◽  
Deepika Sharma DAS ◽  
Yan Song ◽  
Vincent Macri ◽  
Christopher L. Brooks ◽  
...  
Keyword(s):  
Cell Growth ◽  
Board Of Directors ◽  
Xenograft Model ◽  
Advisory Committees ◽  
Immune Effector ◽  
Effector Cells ◽  
Murine Xenograft Model ◽  
Osteolytic Bone Disease

Abstract Introduction Multiple myeloma (MM) remains incurable despite novel therapies, highlighting the need for further identification of factors mediating disease progression and resistance. Our studies have identified an integral role of bone marrow (BM) plasmacytoid dendritic cells (pDCs) in MM pathogenesis. The functional significance of increased numbers of pDCs in MM BM is evident from our observations that pDCs: are relatively resistant to novel and conventional therapies; protect tumor cells from therapy-induced cytotoxicity; promote tumor growth and survival; and suppress immune responses (Chauhan et al, Cancer Cell 2009, 16:309-323). Aberrant pDC function is evidenced in their interactions not only with MM cells, but also with other immune effector T cells and NK cells in the MM BM milieu (Ray et al, Leukemia 2015, 29:1441-1444). Directly targeting pDC interactions with MM and immune effector cells in the MM BM milieu will be required to enhance both anti-tumor immunity and cytotoxicity. However, therapies targeting pDCs are lacking. We found that IL-3R is highly expressed on pDCs, and that pDC-MM interactions trigger secretion of IL-3, which in turn, promotes both pDC survival and osteolytic bone disease. Recent efforts have led to the development of a novel therapeutic agent SL-401, which specifically targets IL-3R-expressing pDCs. Here we examined the effect of SL-401 on pDC-induced MM cell growth both in vitro and in vivo, as well as on IL-3R-expressing osteoclasts. Methods Patient MM cells, pDCs, and MNCs were obtained from normal donors or MM patients. Cell growth/viability was analyzed using MTT/WST assays. OCL function and bone resorption were measured using the OsteoAssays and TRAP staining. The RPMI-8226 cell line was used to isolate MM-SPs by flow-cytometry based Hoechst 33342 staining. SL-401 is a recombinant protein expressed in E. coli. The hybrid gene is comprised of human IL-3 fused to truncated diphtheria toxin (DT). The IL-3 domain of SL-401, which replaces the native binding domain of DT, targets SL-401 to cells that overexpress IL-3R. SL-401 was obtained from Stemline Therapeutics, NY; bortezomib, lenalidomide, pomalidomide, and melphalan were purchased from Selleck Chemicals. For animal model studies, SL-401 (16.5 μg/kg) was administered intravenously daily for 2 weeks. Results SL-401 triggered significant apoptosis in pDCs (>95%) at low picomolar concentrations that are well within clinically achievable doses.Higher concentrations of SL-401 trigger a modest apoptosis (30%± 1.3% apoptosis at 83 ng/ml or 1.3 nM) in MM cells due to lower IL-3R expression versus pDCs. Moreover, SL-401 did not significantly induce apoptosis of normal PBMCs (8% ± 0.5% apoptosis at 83 ng/ml), suggesting a favorable therapeutic index for SL-401. SL-401 inhibited pDC-induced growth of MM cell lines and patient MM cells in a dose-dependent manner. Moreover, 6 of 9 MM samples were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Combinations of SL-401 with melphalan, bortezomib, lenalidomide, or pomalidomide induced synergistic anti-MM activity (Combination index < 1). SL-401 blocked monocyte-derived osteoclast formation in a dose-dependent fashion, as well as restored MM patient BM-derived osteoblast formation. Having defined the efficacy of SL-401 in targeting pDCs and pDC-triggered MM cell growth in vitro, we validated these findings in vivo using our murine xenograft model of human MM, under auspices of protocols approved by our institutional animal protection committee. SL-401 inhibited pDC-induced MM cell growth in vivo and prolonged survival in a murine xenograft model of human MM. We also evaluated the efficacy of SL-401 in vivo using our SCID-human (SCID-hu) mouse model, which reflects the human BM milieu with human cytokines and extracellular matrix proteins. SL-401 significantly abrogated pDC-triggered MM cell growth in vivo in SCID-hu model. Conclusions Our data provide the basis for using SL-401 to directly target pDCs and inhibit the pDC-MM interaction as well as target osteolytic bone disease in novel therapeutic strategies in order to enhance MM cytotoxicity, overcome drug resistance, and improve patient outcome. The interactions of immune effector cells in the MM tumor microenvironment also provide a rationale for combining SL-401 with checkpoint inhibitors. Correspondence: Dharminder Chauhan Disclosures Macri: Stemline Therapeutics, Inc., New York, NY USA: Employment. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Rowinsky:Stemline Therapeutics: Employment, Equity Ownership. Richardson:Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Chauhan:Stemline Therapeutics: Consultancy.

scholarly journals Cholangiocyte expression of α2β1-integrin confers susceptibility to rotavirus-induced experimental biliary atresia

2008 ◽  
Vol 295 (1) ◽  
pp. G16-G26 ◽  
Author(s):  
Mubeen Jafri ◽  
Bryan Donnelly ◽  
Steven Allen ◽  
Alex Bondoc ◽  
Monica McNeal ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Biliary Atresia ◽  
Cell Lines ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Newborn Mice ◽  
A Cell

Inoculation of BALB/c mice with rhesus rotavirus (RRV) in the newborn period results in biliary epithelial cell (cholangiocyte) infection and the murine model of biliary atresia. Rotavirus infection of a cell requires attachment, which is governed in part by cell-surface expression of integrins such as α2β1. We hypothesized that cholangiocytes were susceptible to RRV infection because they express α2β1. RRV attachment and replication was measured in cell lines derived from cholangiocytes and hepatocytes. Flow cytometry was performed on these cell lines to determine whether α2β1 was present. Cholangiocytes were blocked with natural ligands, a monoclonal antibody, or small interfering RNA against the α2-subunit and were infected with RRV. The extrahepatic biliary tract of newborn mice was screened for the expression of the α2β1-integrin. Newborn mice were pretreated with a monoclonal antibody against the α2-subunit and were inoculated with RRV. RRV attached and replicated significantly better in cholangiocytes than in hepatocytes. Cholangiocytes, but not hepatocytes, expressed α2β1 in vitro and in vivo. Blocking assays led to a significant reduction in attachment and yield of virus in RRV-infected cholangiocytes. Pretreatment of newborn pups with an anti-α2 monoclonal antibody reduced the ability of RRV to cause biliary atresia in mice. Cell-surface expression of the α2β1-integrin plays a role in the mechanism that confers cholangiocyte susceptibility to RRV infection.

scholarly journals Comprehensive Analysis of Nef Functions Selected in Simian Immunodeficiency Virus-Infected Macaques

2004 ◽  
Vol 78 (19) ◽  
pp. 10588-10597 ◽  
Author(s):  
Michael Schindler ◽  
Jan Münch ◽  
Matthias Brenner ◽  
Christiane Stahl-Hennig ◽  
Jacek Skowronski ◽  
...  
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Rhesus Macaques ◽  
Surface Expression ◽  
Viral Fitness ◽  
Cell Surface Expression ◽  
Mhc I ◽  
Mhc Ii ◽  
Immunodeficiency Virus

ABSTRACT A variety of simian immunodeficiency virus (SIVmac) nef mutants have been investigated to clarify which in vitro Nef functions contribute to efficient viral replication and pathogenicity in rhesus macaques. Most of these nef alleles, however, were only functionally characterized for their ability to down-modulate CD4 and class I major histocompatibility complex (MHC-I) cell surface expression and to enhance SIV replication and infectivity. To obtain information on the in vivo relevance of more recently established Nef functions, we examined the ability of a large panel of constructed SIVmac Nef mutants and of variants that emerged in infected macaques to down-regulate CD3, CD28, and MHC-II and to up-regulate the MHC-II-associated invariant chain (Ii). We found that all these four Nef functions were restored in SIV-infected macaques. In most cases, however, the initial mutations and the changes selected in vivo affected several in vitro Nef functions. For example, truncated Nef proteins that emerged in animals infected with SIVmac239 containing a 152-bp deletion in nef efficiently modulated both CD3 and Ii surface expression. Overall, our results suggest that the effect of Nef on each of the six cellular receptors investigated contributes to viral fitness in the infected host but also indicate that modulation of CD3, MHC-I, MHC-II, or Ii surface expression alone is insufficient for SIV virulence.

scholarly journals Granulocyte macrophage colony stimulating factor produced in lesioned peripheral nerves induces the up-regulation of cell surface expression of MAC-2 by macrophages and Schwann cells.

1996 ◽  
Vol 133 (1) ◽  
pp. 159-167 ◽  
Author(s):  
A Saada ◽  
F Reichert ◽  
S Rotshenker
Keyword(s):  
Cell Surface ◽  
Schwann Cells ◽  
Interleukin 1 ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Gm Csf ◽  
Molecular Events ◽  
Macrophage Colony

Peripheral nerve injury is followed by Wallerian degeneration which is characterized by cellular and molecular events that turn the degenerating nerve into a tissue that supports nerve regeneration. One of these is the removal, by phagocytosis, of myelin that contains molecules which inhibit regeneration. We have recently documented that the scavenger macrophage and Schwann cells express the galactose-specific lectin MAC-2 which is significant to myelin phagocytosis. In the present study we provide evidence for a mechanism leading to the augmented expression of cell surface MAC-2. Nerve lesion causes noneuronal cells, primarily fibroblasts, to produce the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF). In turn, GM-CSF induces Schwann cells and macrophages to up-regulate surface expression of MAC-2. The proposed mechanism is based on the following novel observations. GM-CSF mRNA was detected by PCR in in vitro and in vivo degenerating nerves, but not in intact nerves. The GM-CSF molecule was detected by ELISA in medium conditioned by in vitro and in vivo degenerating peripheral nerves as of the 4th h after injury. GM-CSF activity was demonstrated by two independent bioassays, and repressed by activity blocking antibodies. Significant levels of GM-CSF were produced by nerve derived fibroblasts, but neither by Schwann cells nor by nerve derived macrophages. Mouse rGM-CSF enhanced MAC-2 production in nerve explants, and up-regulated cell surface expression of MAC-2 by Schwann cells and macrophages. Interleukin-1 beta up-regulated GM-CSF production thus suggesting that injury induced GM-CSF production may be mediated by interleukin-1 beta. Our findings highlight the fact that fibroblasts, by producing GM-CSF and thereby affecting macrophage and Schwann function, play a significant role in the cascade of molecular events and cellular interactions of Wallerian degeneration.

scholarly journals Immunotherapeutic Targeting of TSLPR with Chimeric Antigen Receptor T Cells in AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2789-2789
Author(s):  
Lindsey F Call ◽  
Sommer Castro ◽  
Thao T. Tang ◽  
Cynthia Nourigat-Mckay ◽  
LaKeisha Perkins ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cell Surface ◽  
Peripheral Blood ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Car T Cells ◽  
Car T

Abstract Adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) has achieved impressive outcomes in the treatment of refractory/relapsed B-ALL, providing potentially curative treatment options for these patients. The use of CAR T in AML, however, is still in its infancy with limitations due to the innate heterogeneity associated with AML and the lack of AML-specific targets for therapeutic development. The CRLF2 gene encodes for thymic stromal lymphopoietin receptor (TSLPR) and has previously been shown to be highly upregulated in a subset of children and adults with B-ALL. Targeting TSLPR with CAR T cells demonstrates potent anti-leukemia activity against TSLPR-positive B-ALL (PMID 26041741). Through Target Pediatric AML (TpAML), we profiled the transcriptome of nearly 3000 children and young adults with AML and identified CRLF2 (TSLPR) to be highly expressed in a subset of AML, including the majority of AML harboring KM2TA (aka MLL) fusions. TSLPR cell surface expression was validated in primary patient samples using flow cytometry, which showed uniform expression of TSLPR on AML blasts. Given that TSLPR is expressed in AML with confirmed cell surface expression, we developed TSLPR-directed CAR T for preclinical evaluation in AML. We generated a TSLPR-directed CAR using the single-chain variable fragment (scFv) derived from an anti-TSLPR binder (clone 3G1, MD Anderson), IgG4 spacer and 41-BB/CD3zeta signaling domains. The in vitro cytotoxicity of TSLPR CAR T cells was evaluated against the REH-1 cell line and primary AML specimens. TSLPR CAR T cells demonstrated anti-leukemia activity against REH-1 as well as against primary AML specimens. To evaluate the in vivo efficacy of TSLPR CAR T cells, we developed a patient-derived xenograft (PDX) model using bone marrow cells from a TSLPR-positive patient. These cells provided a robust model system to evaluate the in vivo activity of TSLPR CAR T cells, as they produced an aggressive leukemia in humanized NSG-SGM3 mice. The PDX generated from these cells died within 2 months of transplant with significant leukemia infiltration into the bone marrow, liver, and spleen. In the in vivo study, the leukemia burden was assessed by flow cytometric analysis of AML cells in the peripheral blood and bone marrow aspirates following treatment with unmodified control or TSLPR CAR T cells given at 10x10 6 T cells per mouse. After CAR T treatment, we detected a significant decrease in leukemia infiltration into the peripheral blood and bone marrow in the CAR T-treated mice compared to mice that received unmodified T cells. In this study, we report that similar to B-ALL, CRLF2 (TSLPR) is overexpressed in a subset of AML, providing a strategy to eliminate AML cells with CAR T cell therapy. We validated the cell surface expression of TSLPR and showed that the expression is uniform across AML specimens. We further demonstrate that CAR T cells targeting TSLPR were effective in eliminating AML cells in vitro and in vivo. Given that TSLPR is highly expressed in the KMT2A-rearranged AML, a subtype that is associated with poor outcomes, TSLPR-directed CAR T cells represent a promising immunotherapy for this high-risk AML subset. Disclosures Pardo: Hematologics, Inc.: Current Employment.

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