scholarly journals Combined Blockage of E-Selectin and CXCR4 (GMI-1359) Enhances Anti-Leukemia Effect of FLT3 Inhibition (Sorafenib) and Protects Hematopoiesis in Pre-Clinical AML Models

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-18
Author(s):  
Weiguo Zhang ◽  
Kyung Hee Chang ◽  
Mahesh Basyal ◽  
Yannan Jia ◽  
Lauren B Ostermann ◽  
...  
Keyword(s):  
Stem Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Underlying Mechanism ◽  
Leukemia Cells ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Pdx Model ◽  
Selectin Ligands

Acute myelogenous leukemia (AML) is characterized by an accumulation of abnormal white blood cells. Internal tandem duplications in the fms-like tyrosine kinase 3 (FLT3-ITD) account for 30% of adult AML cases and confer poor prognosis (Nakao et al., Leukemia 1996). FLT3 inhibitors like sorafenib efficiently eliminate circulating leukemia blasts, but frequently not in the bone marrow (BM), which suggests a protective effect of the BM niche for leukemic stem cell survival (Zhang et al., JNCI 2008). The homing of AML cells in BM is mediated chiefly by the adhesion to E-selectin on endothelial cells (ECs) and by CXCR4-directed cellular migration to stromal CXCL12 (SDF1) sources (Chien et al., Blood 2013; Peled and Tavor, Theranostics 2013). In many respects, BM homing signals are shared between leukemia and hematopoietic stem cells (HSCs). Our previous study demonstrated that targeting E-selectin/CXCR4 with the dual E-selectin/CXCR4 antagonist GMI-1359 markedly reduced leukemia cell adhesion to ECs and mesenchymal stem cells, reduced the BM-mediated protection of leukemic cells during FLT3-targeted therapy in vitro, and effectively reduced leukemia cellularity in the BM in vivo (Zhang et al., Can Res suppl 2016). Further, GMI-1359 combined with cytarabine/daunorubicin provided a profound survival benefit in mice with FLT3-mutated leukemia (Zhang et al., Blood suppl 2015). In the present study, we sought to evaluate dual E-selectin/CXCR4 blockage in the context of FLT3 inhibition by sorafenib in vivo, and to better understand the underlying mechanism. We compared expression levels of E-selectin ligands and CXCR4 in FLT3 inhibitor-sensitive Ba/F3-FLT3-ITD cells and their inhibitor-resistant counterparts Ba/F3-FLT3-ITD+D835Y and Ba/F3-FLT3-ITD+F691L. Resistant cells expressed 1.7 to ~5.6-fold higher levels of total E-selectin ligand detected by a soluble E-selectin reagent, and 10-fold higher levels of CXCR4. In addition, BM-mimetic hypoxia culture profoundly upregulated the cell surface expression of E-selectin ligands and CXCR4 on leukemia cells. We evaluated anti-leukemia effects of co-targeting E-selectin/CXCR4 and FLT3 with GMI-1359 and sorafenib in a patient-derived AML xenograft (PDX) model harboring FLT3-ITD and WT1 mutations. We observed that addition of GMI-1359 to sorafenib greatly reduced leukemia cellularity compared to sorafenib alone, and as much as by 92%, 82%, 69% and 45% in, respectively, liver, lung, spleen and BM (Fig. 1) as compared with vehicle-treated mice (p < 0.05). As expected, the number of circulating leukemia cells transiently increased. The GMI-1359/sorafenib combination improved mouse survival (median survival 138.5 versus 109, 87 and 126 days for the GMI-1359/sorafenib versus vehicle, GMI-1359 and sorafenib, respectively, p < 0.001). Using intravital 2-photon microscopy, we observed AML cell behavior in calvarial BM and their response to acute GMI-1359 bolus infusion. Remarkably, AML cell mobility began to increase in the BM microenvironment as soon as 20 min after treatment (Fig. 2), followed by intravasation and cellular outflow through the BM capillary vasculature over the next 2-4 hours. Moreover, although BM homing signals are thought to be shared between leukemia and HSCs, the combination therapy improved hematopoiesis parameters compared to sorafenib alone. In particular, this important effect was associated with increased numbers of megakaryocytes (2.1-fold), myelocytes (2.1-fold), and erythrocytes (7.1-fold) in BM (p < 0.01). The underlying mechanism(s) of hematopoiesis protection by GMI-1359 are under investigation. Conclusion: Co-inhibition of E-selectin/CXCR4 enhances the anti-leukemia efficacy of FLT3 inhibition and preserves hematopoiesis in the BM in a PDX model of AML. Disclosures Fogler: GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Zal:Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding. Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

scholarly journals Targeting E-Selectin with GMI-1271 Overcomes Microenvironment-Mediated Resistance to Venetoclax/HMA Therapy

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 10-11
Author(s):  
Kyung Hee Chang ◽  
Muharrem Muftuoglu ◽  
Weiguo Zhang ◽  
Mahesh Basyal ◽  
Lauren B Ostermann ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Board Of Directors ◽  
Research Funding ◽  
Vehicle Control ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Pdx Model ◽  
Bm Niche ◽  
Combinatorial Treatment

Acute myeloid leukemia (AML) is an aggressive heterogeneous hematologic disease with high mortality in patients older than 60 years. Clinical studies have proven that combinations of FDA-approved Bcl-2 inhibitor, venetoclax and hypomethylating agents (Ven/HMA) are highly effective in elderly patients with AML (DiNardo et al., 2019). Despite improved remission rates, the duration of response is still inadequate. Adhesion to the bone marrow (BM) niche is critical for AML initiation, progression and leukemic stem cell (LSC) survival after induction therapy. The vascular adhesion molecule, E-selectin (E-sel) is responsible for the tethering and rolling of leukocytes on perivascular endothelial BM niche cells (EC). In leukemia, E-sel has crucial roles in BM homing and engraftment (Krause et al, 2006). Through patient-derived AML xenograft (PDX) models and single cell proteomics, we have elucidated the roles of E-sel in AML survival and drug resistance. A PDX model derived from a patient who had developed resistance to Ven/HMA were treated with the E-sel antagonist, GMI-1271 (uproleselan; GlycoMimetic, Inc). We found that targeting E-sel mobilized human AML cells and sensitized them to Ven/HMA. The number of circulating leukemic cells was significantly reduced by combinatorial treatment of GMI-1271 with Ven/HMA in comparison to Ven/HMA alone (p < 0.05). The synergistic effects of the combinatorial treatment on AML-PDX mouse survival were determined by Kaplan-Meier analysis. The combination of GMI-1271 and Ven/HMA significantly prolonged the survival of mice compared to vehicle control (p = 0.015) as well as the Ven/HMA (p = 0.0009) and GMI-1271 groups (p = 0.03). The median survival of the vehicle control, GMI-1271, Ven/HMA, and combination-treated groups of mice was 86, 91, 81.5, and 106.5 days, respectively. Histological analysis of BM, spleen, lung and liver demonstrated differences in leukemia cell infiltration, confirming enhanced anti-leukemia efficacy of the combination treatment. To delineate the mechanism of E-sel at the onset of drug mediated changes in AML signaling signatures, we employed another PDX model (Flt3-ITD and WT1 mutations, sorafenib-resistant). PDX mice with advanced AML (more than 20% human AML cells circulation in peripheral blood) were administered Ven/HMA, GMI-1271, or combination for 2 days. Single cell proteomics analysis by CyTOF determined that combinatorial treatment diminished levels of Ki67, IDU, and pRb compared to vehicle control or Ven/HMA alone, resulting in decreased proliferation of AML blasts. Activation of eNOS to produce nitric oxide (NO) through PI3K/AKT kinase, maintains clonogenic cell growth in malignant cells. A recent publication has demonstrated that introduction of NOS blockers in combination with chemotherapy led to slower leukemia progression and longer remissions in contrast to chemotherapy alone (Passaro et al, 2017). Interestingly, we observed reduced activation of PI3K and AKT in AML blasts as well as in BM CD31+EC cells in the GMI-1271 treated PDX model. eNOS phosphorylation was subsequently decreased in EC, suggesting that inhibition of E-sel may protect BM vasculature by blocking the production of NO. In addition, targeting E-sel showed signaling alterations in AML MSC. Administration of E-sel antagonist increased mTOR expression in MSC from AML-PDX. Combination treatment induced higher Ki67 positivity, and hyperactivation of pRb and p-S6 in MSC in vivo. Our group and others have recently reported that Ven-resistant AML cells exhibit an increased dependence on alternate anti-apoptotic proteins, Mcl-1 and Bcl-xl (Konopleva et al., 2016). We found that concomitant treatment in vivo with GMI-1271 and Ven/HMA further decreased the expression of Bcl-xl and Mcl-1 in AML blasts compared to Ven/HMA alone, suggesting a critical role for E-sel antagonists in overcoming drug resistance. E-selectin binding potential and focal adhesion kinase activity in AML blasts were decreased upon acute administration of pharmacological E-sel inhibitor. Other oncogenic signaling pathways including MAPK, p-S6, and STAT3, were all inhibited by the addition of GMI-1271 to Ven/HMA. Collectively, our results provide first evidence that an E-sel targeting strategy with GMI-1271 can overcome microenvironmental resistance to Ven/HMA-based therapy in AML by cancer cell autonomous and non-cell autonomous mechanisms in the BM vascular niche. Figure Disclosures Fogler: GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

Inhibition of Chronic Myeloid Leukemia Stem Cells by the Combination of the Hedgehog Pathway Inhibitor LDE225 with Nilotinib

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 514-514 ◽  
Author(s):  
Bin Zhang ◽  
David Irvine ◽  
Yin Wei Ho ◽  
Silvia Buonamici ◽  
Paul Manley ◽  
...  
Keyword(s):  
Stem Cells ◽  
Board Of Directors ◽  
Colony Formation ◽  
Research Funding ◽  
Leukemia Stem Cells ◽  
Advisory Committees ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Hh Signaling

Abstract Abstract 514 Background: Tyrosine kinase inhibitors (TKI), although effective in inducing remissions and improving survival in CML patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse on stopping treatment. Additional strategies to enhance elimination of LSC in TKI-treated CML patients are required. The Hedgehog (Hh) pathway, important for developmental hematopoiesis, has been shown to be activated in BCR-ABL-expressing LSC, in association with upregulation of Smoothened (SMO), and contributes to maintenance of BCR-ABL+ LSC. However the role of Hh signaling in chronic phase (CP) CML LSC is not clear. LDE225 (LDE, Novartis Pharma) is a small molecule SMO antagonist which is being clinically evaluated in patients with solid tumors. We have reported that LDE does not significantly affect proliferation and apoptosis of primary CP CML CD34+ cells, or reduce colony growth in CFC assays, but results in significant reduction in CML CFC replating efficiency and secondary colony formation. Treatment with LDE + Nilotinib resulted in significant reduction in colony formation from CD34+ CML cells in LTCIC assays compared to Nilotinib alone or untreated controls. These observations suggest that LDE may preferentially inhibit growth of primitive CML progenitors and progenitor self-renewal. We therefore further investigated the effect of LDE on growth of primitive CML LSC in vivo. Methods and Results: 1) CP CML CD34+ cells were treated with LDE (10nM), Nilotinib (5μ M) or LDE + Nilotinib for 72 hours followed by transplantation into NOD-SCID γ-chain- (NSG) mice. Treatment with LDE + Nilotinib resulted in reduced engraftment of CML CD45+ cells (p=0.06) and CD34+ cells (p=0.02) compared with controls, and significantly reduced engraftment of CML cells with CFC capacity (p=0.005). In contrast LDE or Nilotinib alone did not reduce CML cell engraftment in the bone marrow (BM) compared with untreated controls. LDE, Nilotinib, or LDE + Nilotinib treatment did not significantly inhibit engraftment of normal human CD34+ cells in NSG mice compared to controls. 2) We also used the transgenic Scl-tTa-BCR-ABL mouse model of CP CML to investigate the effect of in vivo treatment with LDE on CML LSC. BM cells from GFP-SCL-tTA/BCR-ABL mice were transplanted into wild type congenic recipients to establish a cohort of mice with CML-like disease. Recipient mice developed CML-like disease 3–4 weeks after transplantation. Transplanted CML cells were identifiable through GFP expression. Mice were treated with LDE225 (80mg/kg/d by gavage), Nilotinib (50 mg/kg/d by gavage), LDE + Nilotinib, or vehicle alone (control) for 3 weeks. Treatment with Nilotinib, LDE, and LDE + Nilotinib resulted in normalization of WBC and neutrophil counts in peripheral blood. LDE + Nilotinib treatment significantly reduced the number of splenic long term hematopoietic stem cells (LT-HSC, Lin-Sca-1+Kit+Flt3-CD150+CD48-, p<0.01) and granulocyte-macrophage progenitors (GMP) compared to controls, but did not significantly alter LT-HSC numbers in the BM. LDE alone reduced splenic LT-HSC but not GMP, whereas Nilotinib alone did not reduce LT-HSC numbers in spleen or BM but significantly reduced splenic GMP numbers. The mechanisms underlying enhanced targeting of LSC in the spleen compared to the BM are not clear but could reflect greater dependence on Hh signaling in the context of the splenic microenvironment and/or relocalization of LDE treated LT-HSC to BM. Experiments in which BM and spleen cells from treated mice were transplanted into secondary recipients to determine functional stem cell capacity of remaining LT-HSC are ongoing. Importantly mice treated with LDE + Nilotinib demonstrated enhanced survival on follow up after discontinuation of treatment compared with control mice or mice treated with LDE or Nilotinib alone. Conclusions: We conclude that LDE225 can target LSC from CP CML patients and in a transgenic BCR-ABL model of CP CML, and that LDE + Nilotinib treatment may represent a promising strategy to enhance elimination of residual LSC in TKI-treated CML patients. Disclosures: Buonamici: Novartis: Employment. Manley:Novartis: Employment. Holyoake:Novartis: Consultancy, Research Funding. Copland:Novartis Pharma: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bhatia:Novartis: Consultancy, Honoraria.

scholarly journals Gabarap Loss Mediates Immune Escape in High Risk Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 891-891
Author(s):  
Annamaria Gulla ◽  
Eugenio Morelli ◽  
Mehmet K. Samur ◽  
Cirino Botta ◽  
Megan Johnstone ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cell ◽  
Clinical Outcome ◽  
Board Of Directors ◽  
Research Funding ◽  
Immune Escape ◽  
Publicly Traded ◽  
Advisory Committees

Abstract Immune therapies including CAR T cells and bispecific T cell engagers are demonstrating remarkable efficacy in relapsed refractory myeloma (MM). In this context, we have recently shown that proteasome inhibitor bortezomib (BTZ) results in immunogenic cell death (ICD) and in a viral mimicry state in MM cells, allowing for immune recognition of tumor cells. Induction of a robust anti-MM immune response after BTZ was confirmed both in vitro and in vivo: treatment of 5TGM1 MM cells with BTZ induced tumor regression associated with memory immune response, confirmed by ELISPOT of mouse splenocytes. We have confirmed the obligate role of calreticulin (CALR) exposure in phagocytosis and the ICD process, since BTZ-induced ICD is impaired in CALR KO MM cells both in vitro and in vivo. We further showed that the therapeutic efficacy of BTZ in patients was correlated with ICD induction: BTZ-induced ICD signature was positively correlated with OS (p=0.01) in patients enrolled in the IFM/DFCI 2009 study. Together, these studies indicate that ICD is associated with long-term response after BTZ treatment. In this work, we reasoned that genomic or transcriptomic alterations associated with shorter survival of MM patients after BTZ treatment may impair activation of the ICD pathway. To this aim, we performed a transcriptomic analysis of purified CD138+ cells from 360 newly diagnosed, clinically-annotated MM patients enrolled in the IFM/DFCI 2009 study. By focusing on genes involved in the ICD process, we found that low levels of GABA Type A Receptor-Associated Protein (GABARAP) were associated with inferior clinical outcome (EFS, p=0.0055). GABARAP gene locus is located on chr17p13.1, a region deleted in high risk (HR) MM with unfavorable prognosis. Remarkably, we found that correlation of low GABARAP levels with shorter EFS was significant (p=0.018) even after excluding MM patients with del17p; and GABARAP is therefore an independent predictor of clinical outcome. GABARAP is a regulator of autophagy and vesicular trafficking, and a putative CALR binding partner. Interestingly, among a panel of MM cell lines (n=6), BTZ treatment failed to induce exposure of CALR and MM cell phagocytosis by DCs in KMS11 cells, which carry a monoallelic deletion of GABARAP. This effect was rescued by stable overexpression of GABARAP. Moreover, CRISPR/Cas9-mediated KO of GABARAP in 3 ICD-sensitive cell lines (AMO1, H929, 5TGM1) abrogated CALR exposure and ICD induction by BTZ. GABARAP add-back by stable overexpression in KO clones restored both CALR exposure and induction of ICD, confirming GABARAP on-target activity. Similarly, pre-treatment of GABARAP KO cells with recombinant CALR restored MM phagocytosis, further confirming that GABARAP impairs ICD via inhibition of CALR exposure. Based on these findings, we hypothesized that GABARAP loss may alter the ICD pathway via CALR trapping, resulting in the ICD resistant phenotype observed in GABARAP null and del17p cells. To this end, we explored the impact of GABARAP KO on the CALR protein interactome, in the presence or absence of BTZ. Importantly, GABARAP KO produced a significant increase of CALR binding to stanniocalcin 1 (STC1), a phagocytosis checkpoint that mediates the mitochondrial trapping of CALR, thereby minimizing its exposure upon ICD. Consistently, GABARAP KO also affected CALR interactome in BTZ-treated cells, which was significantly enriched in mitochondrial proteins. Importantly, co-IP experiments confirmed GABARAP interaction with STC1. These data indicate a molecular scenario whereby GABARAP interacts with STC1 to avoid STC1-mediated trapping of CALR, allowing for the induction of ICD after treatment with ICD inducers; on the other hand, this mechanism is compromised in GABARAP null or del17p cells, and the STC1-CALR complex remains trapped in the mitochondria, resulting in ICD resistance. To functionally validate our findings in the context of the immune microenvironment, we performed mass Cytometry after T cell co-culture with DCs primed by both WT and GABARAP KO AMO1 clones. And we confirmed that treatment of GABARAP KO clones with BTZ failed to activate an efficient T cell response. In conclusion, our work identifies a unique mechanism of immune escape which may contribute to the poor clinical outcome observed in del17p HR MM patients. It further suggests that novel therapies to restore GABARAP may allow for the induction of ICD and improved patient outcome in MM. Disclosures Bianchi: Jacob D. Fuchsberg Law Firm: Consultancy; MJH: Honoraria; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Richardson: AstraZeneca: Consultancy; Regeneron: Consultancy; Protocol Intelligence: Consultancy; Secura Bio: Consultancy; GlaxoSmithKline: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; Takeda: Consultancy, Research Funding; AbbVie: Consultancy; Karyopharm: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; Oncopeptides: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding. Chauhan: C4 Therapeutics: Current equity holder in publicly-traded company; Stemline Therapeutics, Inc: Consultancy. Munshi: Legend: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Inhibition of Sialylation Impairs Adhesion on Madcam-1 and E-Selectin and Sensitize Multiple Myeloma Cells to Bortezomib in a Xenograft Mouse Model

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3204-3204
Author(s):  
Alessandro Natoni ◽  
Mariah Farrell ◽  
Heather Fairfield ◽  
Lucy Kirkham-McCarthy ◽  
Matt Macauley ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Board Of Directors ◽  
Research Funding ◽  
Immune Cell ◽  
Sialyl Lewis X ◽  
Advisory Committees ◽  
Selectin Ligands

Abstract Introduction Multiple myeloma (MM) is a cancer of clonal plasma cells that hijack the bone marrow (BM) niche to create a drug resistant, incurable malignancy. Aberrant sialylation has been linked to immune cell evasion, drug resistance, and metastasis in cancer; indeed sialyltransferases, including ST3GAL1, ST3GAL4 and ST3GAL6, are aberrantly expressed in many cancers (Glavey et al., 2014). We have previously shown that targeting ST3GAL6 in MM cells inhibits their ability to extravasate and colonize the BM in mouse models (Glavey et al., 2014). Moreover, we also showed that a subpopulation of MM cells expresses functional E-Selectin ligands which, upon expansion, gives rise to a more aggressive disease and resistance to bortezomib in mice (Natoni et al., 2017). Based off these findings, we herein investigated whether inhibiting sialylation in E-selectin-enriched MM cells with 3Fax-Neu5Ac, a small molecule sialyltransferase inhibitor, could alter the ability of these cells to home in the BM and restore bortezomib sensitivity in vivo. We hypothesized that inhibiting homing of MM cells to the BM will improve survival and that co-treatment with bortezomib and 3Fax-Neu5Ac will have a synergistic effect. Methods E-selectin ligands enriched MM1S cells (either positive or negative for GFP/Luciferase) were derived from parental cells by cell sorting using the HECA-452 antibody, which recognize sialofucosylated E-selectin ligands. We then determined the 3Fax-Neu5Ac dose and exposure times needed to decrease sialylation on these MM cells without causing toxicity. HECA-452-enriched MM1S cells were pretreated with 3Fax-Neu5Ac or vehicle for 7 days before being injected into SCID-beige mice and then treated with vehicle or bortezomib (0.3 mg/kg twice a week). Mice were analyzed via bioluminescence imaging (BLI) to monitor tumor progression and weighed twice a week. Mice were euthanized when they began to show paralysis under our IACUC protocol. 3Fax-Neu5Ac pretreated HECA-452 MM1S cells were also tested in vitro for their ability to adhere and roll on VCAM-1, MAdCAM-1 and E-Selectin under shear stress and to respond to bortezomib in co-culture with HS5 cells. Results Treatment of HECA-452 MM1S cells with 3Fax-Neu5Ac, at 300 μM for 7 days significantly reduced sialylation on these cells. Importantly, reducing sialylation with 3Fax-Neu5AC reduced tumor burden and increased survival, although this did not reach significance for survival (Figure 1A). Both vehicle- and 3Fax-Neu5Ac-treated cells significantly responded to bortezomib in the first 5 weeks of the in vivo study (Figure 1B). However, the HECA-452 MM1S cells did not show increased survival when treated with bortezomib suggesting an acquired mechanism of resistance in vivo. Importantly, pretreatment of the HECA-452 MM1S with 3Fax-Neu5Ac could improve survival of these mice preventing bortezomib resistance. In vitro, the HS5 stromal cells protected the HECA-452 MM1S cells from bortezomib and pretreatment with 3Fax-Neu5Ac partially reverted this protection. Moreover, the HECA-452 MM1S cells pretreated with 3Fax-Neu5Ac displayed reduced adhesion on MAdCAM-1 and E-selectin. Conclusions Sialylation plays an instrumental role in bone homing, BM colonization, and drug resistance of MM cells. Pretreatment of HECA-452 MM1S cells with 3Fax-Neu5Ac decreased their sialylation, restored sensitivity to bortezomib in vivo and prolonged survival in mice. This is likely because 3Fax-Neu5Ac pretreatment has multiple effects on MM cells including reducing cell adhesion mediated-drug resistance and adhesion to key molecules involved in BM homing such as MAdCAM-1 and E-selectin. The reduced adhesion on E-selectin is most likely due to the disruption of E-selectin ligands on the surface of MM cells as they require Sialyl Lewis X to function. Notably, we also found that de-sialylation impairs adhesion on MAdCAM-1 (3Fax-Neu5Ac vs DMSO P=0.038) which, together with E-selectin, is another critical BM homing receptor. This data suggests for the first time that sialylation may controls the affinity of integrin α4β7 and its counter-receptor MAdCAM-1. In turn, this would reduce BM homing and increase MM cells in the circulation were they are more prone to the cytotoxic effects of bortezomib. This study supports the importance of targeting sialylation in MM and provides a strong rationale for further clinical translation of this novel approach. Disclosures O'Dwyer: Glycomimetics: Research Funding; Celgene: Research Funding; BMS: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Treatment with DC/AML Fusion Vaccine and CD3xCD123 Bi-Specific T-Cell Engager (CD123-CODV-TCE) for Treatment of Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 904-904
Author(s):  
Dina Stroopinsky ◽  
Anita G. Koshy ◽  
Jessica J. Liegel ◽  
Myrna Nahas ◽  
Giulia Cheloni ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Ex Vivo ◽  
Leukemia Cells ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Isotype Control ◽  
Ifn Γ

Abstract Introduction: Immunotherapy for AML holds promise in overcoming chemotherapy resistance and in preserving immunologic memory necessary for durable remissions.A bispecific T-cell engaging antibody targeting CD3 and CD123 (CD123-CODV-TCE) has been shown to stimulate T cells to target CD123-expressing leukemic cells in vitroand in mouse models 1. While the short-term immune stimulation mediated by the CD123TCE has the potential to result in clinical response, long-term disease control will require the development of immune memory. We have developed a personalized cancer vaccine in which patient's dendritic cells are fused with autologous leukemia cells resulting in presentation of a wide range of antigens to the immune system. Here, we describe a novel combination of CD123TCE with a DC/AML fusion vaccine ex vivo and in a xenograft murine model. We hypothesized that the CD123TCE will direct the vaccine-educated T cells to not only more effectively eradicate target leukemia cells but also evoke a repertoire of memory T cells and long-term response. Methods/Results: AML cells expressing CD123 were isolated from bone marrow mononuclear cells (BMMCs) from AML patients (n=3). DCs were generated from autologous adherent peripheral blood mononuclear cells (PBMCs) obtained at the time of disease remission as previously described 2,3. Concurrently, CD3+ T cells were isolated from the non-adherent fraction of PBMCs using magnetic bead separation. Fusion cells were generated by co-culturing the DC and tumor cells at a ratio of 3:1 in the presence of polyethylene glycol (PEG). Vaccine educated T cells were then generated by co-culture of T cells with the autologous fusion cells at a ratio of 10:1 for 5-7 days followed by T cell expansion via CD3/CD28 ligation. The capacity of the vaccine-educated T cells to target autologous leukemia cells with the addition of CD123TCE was assessed. The results demonstrated a statistically significant increase in Granzyme B activity in the target AML cells following co-culture with vaccine-educated T cells and the addition of the CD123TCE, compared to T cells + isotype control (n=3). Furthermore, vaccine stimulation in combination with CD123TCE led to a robust increase in induction of tumor specific activated T cells as detected by CD137 expression and intracellular IFN-γ production after co-culture of vaccine-educated T cells with autologous tumor cells in the presence of CD123TCE. The addition of CD123TCE to vaccine-educated T cells resulted in mean 25.4% and 9.6% intracellular IFN-γ expression for CD8 and CD4 T cells, respectively, compared to 8.5 and 3.1% IFN-γ expression following the addition of isotype control (n=3). Next, we examined the efficacy of the combined treatment with vaccine-educated T cells and CD123TCE in-vivo, in two independent xenograft experiments. NSG mice were irradiated with 300rads and challenged with 1x10 6 patient-derived CD123+ tumor cells via retro-orbital injections. After detection of human AML engraftment in the PB on day 76, the mice were inoculated IV with 1X10 6 resting, or ex vivo fusion vaccine educated autologous T cells IV. Subsequently, cohorts of mice were treated with CD123TCE or an appropriate isotype control every 3 days IP. A significant decrease in human leukemia burden was detected in the peripheral blood, spleen and bone marrows of analyzed animals after treatment with vaccine educated T cells and isotype control, or resting T cells and CD123TCE, compared to untreated mice (n=5). Strikingly, no detectable AML was found in peripheral blood, spleens and bone marrows of mice treated with vaccine educated T cells in combination with TCE (n=5). Of note, treatment with vaccine educated T cells led to an expansion of human CD3+ T cells in tissues obtained from the analyzed animals. These human T cells persisted in mice treated with the CD123TCE with a two-fold increase in tumor-specific CD8+ T cells, as assessed by intracellular IFN-γ secretion following ex vivo stimulation with autologous tumor lysate. Conclusions: We demonstrated that the combination of DC/AML fusion vaccine and CD123TCE led to increase in tumor specific T cell immunity, both ex-vivo and in a xenograft murine model when compared to uneducated T cells with CD123TCE or educated T cells with isotype control molecule. Most significantly, the combination treatment was shown to eradicate AML in this model with all animals remaining disease-free several months post inoculation. Disclosures Stroopinsky: The Blackstone Group: Consultancy. Nahas: Kite Pharma: Current Employment. Fraenkel: Sanofi: Current Employment. Yildirim: Sanofi: Current Employment. Bonnevaux: Sanofi: Current Employment. Guerif: Sanofi: Current Employment. Kufe: Genus Oncology: Current equity holder in publicly-traded company; Canbas: Consultancy; REATA: Consultancy, Current equity holder in publicly-traded company; Hillstream BioPharma: Current equity holder in publicly-traded company. Rosenblatt: Parexel: Consultancy; Wolters Kluwer Health: Consultancy, Patents & Royalties; Bristol-Myers Squibb: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imaging Endpoints: Consultancy; Attivare Therapeutics: Consultancy. Avigan: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Aviv MedTech Ltd: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Parexcel: Consultancy; Takeda: Consultancy; Sanofi: Consultancy.

Effective Elimination of CML Progenitor and Stem Cells Through Combination of α-CD123 Antibody-Dependent Cell-Mediated Cytotoxicity and Tyrosine Kinase Inhibitor Treatment

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 32-32
Author(s):  
Eva Nievergall ◽  
Deborah L. White ◽  
Agnes S.M. Yong ◽  
Hayley S. Ramshaw ◽  
Samantha J. Busfield ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Target Cells ◽  
Molecular Response ◽  
Advisory Committees ◽  
Cd34 Cells

Abstract Abstract 32 Since the introduction of tyrosine kinase inhibitor (TKI) therapy overall survival and complete molecular response rates in chronic phase chronic myeloid leukemia (CP-CML) patients have significantly improved. However, leukemic stem cells (LSCs) and progenitor cells persist and are thought to be responsible for disease progression, development of TKI resistance and disease recurrence after stopping TKI therapy. Protection by cytokines, such as IL-3 and GM-CSF, provides a potential mechanism of LSC resistance. While in acute myeloid leukemia (AML) monoclonal antibody (mAb) targeting of IL-3 receptor α (CD123), a recognized marker for AML LSCs, has been studied in vitro and in vivo, similar investigations have not been undertaken in CML to date. CSL362 is a genetically-engineered form of the specific blocking mAb 7G3 optimized for Fc receptor binding to achieve maximal antibody-dependent cell-mediated cytotoxicity (ADCC) capacity. Here we investigate the expression of CD123 in CD34+ progenitors and CD34+CD38− LSCs, isolated from CP- and blast crisis (BC) - CML patients, and study the benefits of targeting those cells by CSL362 alone and in combination with TKIs. Flow cytometry analysis established significantly elevated expression of CD123 on CD34+CD38− cells from CP-CML (53.0 ± 5.8 %, n=16, p=0.003) and BC-CML (73.2 ± 6.7 %, n=18, p<0.001) patients compared to normal donors (20.3 ± 4.2 %, n=8), with clear increases in CD123 expression with disease progression in matched samples (n=2). Subsequent assessment of apoptosis, colony forming unit (CFU-GM) and long-term culture-initiating cell (LTC-IC) potential confirmed the ability of CSL362 to block IL-3-mediated rescue of TKI-induced cell death. However, in the presence of other cytokines, likely found in the physiological bone marrow microenvironment, this effect was lost. We also demonstrate by lactate dehyrogenase release and clonogenic assays that CML CD34+ cell numbers are significantly reduced, in a dose-dependent manner, by CSL362-induced ADCC employing NK cells from healthy donors (42.4 ± 8.1 % lysis, n=3, and CFU-GM decreased to 30 ± 10.8 % of controls, n=5, p=0.003). In keeping with this, flow cytometry analysis revealed specific elimination of CP- and BC-CML CD123+ CD34+CD38− cells (from 42.9 % to 18.6 %, n=5, p=0.004, and from 71 % to 35.3 %, n=3, p=0.044, respectively). Importantly, autologous CML patient NK cells, collected after achievement of major molecular response, also mediate CSL362-dependent cytotoxicity similar to allogeneic healthy donor NK cells as indicated by equivalent numbers of remaining CFUs (28 ± 6.7 % vs. 34.9 ± 3.4 %, n=5, Fig. A). We further have evidence to suggest preferential elimination of CML over normal LTC-ICs (30.3 ± 9.9 % vs. 62.6 ± 11.2 % remaining, n=3, p=0.096) in the autologous setting. Of clinical importance, the combination of Nilotinib and CSL362 resulted in a significantly greater reduction in CFUs (additive effect) when compared to either agent alone (Fig. B). Taken together these data suggest that selective ADCC-mediated lysis, likely the major mode of action of CSL362 in vivo, efficiently eliminates CML progenitor and stem cells. Promising results evaluating CSL362/TKI combination treatments, with the expectation to further enhance specificity for leukemic while sparing normal progenitor and stem cells as indicated from preliminary experiments, warrant further studies. A: Autologous NK cells are able to confer CSL362-induced ADCC against CML CD34+ cells. Cells were co-cultured at an effector to target cell ratio (E:T) of 10:1 in the absence and presence of CSL362 as indicated for 4 h and remaining CFU-GM were enumerated. Data is normalized to target cells alone (*** p<0.001). B: CSL362-mediated ADCC and TKI treatment show additive effects. CP-CML CD34+ cells were cultured with nilotinib at varying concentrations as indicated for 48 h before overnight exposure to CSL362 (1 μg/ml) with or without allogeneic NK cells (E:T 1:1). Mean ± SE of CFU-GM colony numbers is shown (n=3, * p<0.05, ** p<0.01). A: Autologous NK cells are able to confer CSL362-induced ADCC against CML CD34+ cells. Cells were co-cultured at an effector to target cell ratio (E:T) of 10:1 in the absence and presence of CSL362 as indicated for 4 h and remaining CFU-GM were enumerated. Data is normalized to target cells alone (*** p<0.001). . / B: CSL362-mediated ADCC and TKI treatment show additive effects. CP-CML CD34+ cells were cultured with nilotinib at varying concentrations as indicated for 48 h before overnight exposure to CSL362 (1 μg/ml) with or without allogeneic NK cells (E:T 1:1). Mean ± SE of CFU-GM colony numbers is shown (n=3, * p<0.05, ** p<0.01). Disclosures: Nievergall: CSL Ltd: Research Funding. White:BMS: Research Funding; CSL Ltd: Research Funding; Novartis Oncology: Honoraria, Research Funding. Ramshaw:CSL Ltd: Research Funding. Busfield:CSL Ltd: Employment. Vairo:CSL Ltd: Employment. Lopez:CSL Ltd: Research Funding. Hughes:Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Ltd: Research Funding. Hiwase:CSL Ltd: Research Funding.

scholarly journals Donor-Derived Adoptive T-Cell Therapy Targeting Multiple Tumor Associated Antigens to Prevent Post-Transplant Relapse in Patients with ALL

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 471-471
Author(s):  
Swati Naik ◽  
Spyridoula Vasileiou ◽  
Ifigeneia Tzannou ◽  
Manik Kuvalekar ◽  
Ayumi Watanabe ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Management Support ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Post Infusion ◽  
T Cell Lines

Abstract Background: Hematopoietic stem cell transplant (HSCT) is a curative option for patients with high-risk Acute Lymphoblastic Leukemia (HR-ALL), but relapse remains a major cause of treatment failure. Strategies to enhance the graft-versus-leukemia (GVL) effect have been employed to prevent relapse, including modulating immune suppression post-HSCT to hasten immune reconstitution or with the use of donor lymphocyte infusions (DLIs). However, DLIs carry a significant risk of graft-versus-host disease (GVHD) due to the concurrent transfer of alloreactive T cells. To enhance the GVL effect while minimizing GVHD, we developed a protocol for the generation of ex vivo expanded, donor-derived T-cell lines targeting PRAME, WT1 and Survivin - tumor associated antigens that are frequently expressed in both B- and T-cell ALL. These multi-antigen-targeted T cells (multiTAAs) were adoptively transferred to pediatric and adult patients with HR-ALL who had undergone an allogeneic HSCT. Methods: Donor-derived multiTAA-specific T cells were generated by co-culturing PBMCs with autologous DCs loaded with pepmixes (15 mer peptides overlapping by 11 amino acids) spanning all 3 target antigens in the presence of a Th1-polarizing/pro-proliferative cytokine cocktail. Following 2-4 rounds of stimulation these multiTAA-specific T cells were infused to patients with ALL who had undergone an HSCT but remained at a high risk for disease relapse. Results: We have generated 15 clinical grade multiTAA-specific T cell lines comprising CD3+ T cells (mean 95.1±1.9%) with a mixture of CD4+ (mean 22.8±6.3%) and CD8+ (mean 52.5±5.3%) cells, which expressed central [CD45RO+/CD62L+: 13.5±2.8%] and effector memory markers [CD45RO+/CD62L-: 56.4±3.8%]. The expanded lines recognized the targeted antigens PRAME (range 0-370 SFC/2x10 5), WT1 (0-363 SFC/2x10 5), and Survivin (0-65 SFC/2x10 5) in an IFNg ELIspot. None of the lines reacted against non-malignant patient-derived cells (3.7±0.8% specific lysis; E: T 20:1) - a study release criterion indicating lack of alloreactivity. We have infused 11 HR-ALL patients (8 pediatric and 3 adult) with donor-derived multiTAA-specific T cells to prevent disease relapse (Table 1). Patients were administered with up to 4 infusions of cells at 3 escalating dose levels, ranging from 0.5 - 2x10 7 cells/m 2. Infusions were well tolerated with no dose-limiting toxicity, GVHD, cytokine release syndrome or other adverse events. Three patients were not evaluable per study criteria as they received &gt;0.5mg/kg of steroids (2 patients received stress doses for septic shock and 1 for elevated liver enzymes presumed to be GVHD that was later ruled out) within 4 weeks of infusion and were replaced. Six of the 8 remaining patients infused remain in CR on long-term follow up at a median of 46.5 months post-infusion (range 9-51 months). In patients who remained in long term CR we detected an expansion of tumor-reactive T cells in their peripheral blood post-infusion against both targeted (WT1, Survivin, PRAME) and non-targeted antigens (SSX2, MAGE-A4, -A1, -A2B, -C1, MART1, AFP and NYESO1) reflecting epitope and antigen spreading, which correlated temporally (within 4 weeks) with multiTAA infusions. By contrast in the two patients who relapsed we saw no evidence of in vivo T cell amplification within the first 4 weeks after infusion. Conclusion: The preparation and infusion of donor-derived multiTAA-specific T cells to patients with B- and T-ALL post allogeneic HSCT is feasible, safe and as evidenced by in vivo tumor-directed T cell expansion and antigen spreading in patients, may contribute to disease control. This strategy may present a promising addition to current immunotherapeutic approaches for prophylaxis for leukemic relapse in HSCT recipients. Figure 1 Figure 1. Disclosures Vasileiou: Allovir: Consultancy. Tzannou: Gileas: Honoraria; Allovir: Current equity holder in publicly-traded company. Kuvalekar: Allovir: Consultancy. Watanabe: Allovir: Consultancy. Grilley: QB Regulatory Consulting: Other: Ownership, project management support, Research Funding; Marker: Consultancy, Other: Regulatory and project management support; Allovir: Current equity holder in publicly-traded company, Other: Leadership. Hill: Incyte: Membership on an entity's Board of Directors or advisory committees. Omer: Allovir: Research Funding. Gottschalk: Tessa Therapeutics: Consultancy; Immatics: Membership on an entity's Board of Directors or advisory committees; Other: Other: patents and patent applications in the field of cancer cell and gene therapy ; Tidal: Consultancy; Novartis: Consultancy; Catamaran Bio: Consultancy. Heslop: Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Kiadis: Membership on an entity's Board of Directors or advisory committees; Kuur Therapeutics: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees; Allovir: Current equity holder in publicly-traded company; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Marker Therapeutics: Current equity holder in publicly-traded company; Fresh Wind Biotherapies: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Rooney: Allogene: Patents & Royalties; Bellicum: Patents & Royalties; Bluebird: Current equity holder in publicly-traded company; Allovir: Current equity holder in publicly-traded company; Alimera: Consultancy; Memgen: Consultancy; TScan Therapeutics: Consultancy; Takeda: Patents & Royalties; Marker: Current equity holder in publicly-traded company; Tessa: Consultancy, Other: Leadership, Research Funding. Vera: Allovir: Consultancy, Current equity holder in publicly-traded company, Other: Leadership, travel , accomodations, expenses, Patents & Royalties; Marker: Current Employment, Other: Travel, Accomodations, Expenses, Patents & Royalties, Research Funding. Leen: Allovir: Consultancy, Current equity holder in publicly-traded company; Marker: Current equity holder in publicly-traded company.

scholarly journals Bispecific Vγ9Vδ2-T and Type 1 NKT Cell Engager Lava-051 As First-in-Class Clinical Candidate to Target CD1d Expressing CLL, MM and AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2266-2266
Author(s):  
Roeland Lameris ◽  
Jurjen M Ruben ◽  
Iris de Weerdt ◽  
Rob Roovers ◽  
Niels W.C.J. van de Donk ◽  
...  
Keyword(s):  
T Cells ◽  
Antitumor Activity ◽  
Board Of Directors ◽  
Research Funding ◽  
Nkt Cells ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Vγ9vδ2 T Cells

Abstract Background. Bispecific antibodies that target tumors by engaging innate-like T cell subsets with inherent antitumor activity, such as Vγ9Vδ2-T and type 1 natural killer T (NKT) cells, may combine high therapeutic efficacy with limited off-tumor toxicity. Type 1 NKT cells respond to self and foreign (glyco)lipid antigens presented in the context of the MHC class I like molecule CD1d which is expressed on various malignancies. Vγ9Vδ2-T cells respond to intracellular accumulation of phosphoantigens in cancer cells by sensing conformational alterations in the butyrophilin (BTN) 2A1-3A1 complex. CD1d is expressed by the majority of patients with CLL and MM, while expression in AML is most pronounced on (myelo)monocytic subtypes. Methods. LAVA-051 is a 27kD humanized bispecific single domain antibody (bsVHH) that directly engages CD1d and the Vδ2-TCR chain of Vγ9Vδ2-T cells. The anti-CD1d VHH specifically stabilizes the interaction between CD1d and the type 1 NKT cell TCR and thereby triggers strong activation of type 1 NKT cells (Nature Cancer 2020;1:1054-1065). Vγ9Vδ2-T and type 1 NKT effector cell activation, proliferation, cytokine production and target cell lysis were assessed in in vitro, ex vivo, and in vivo studies. Due to lack of cross reactivity of LAVA-051 with non-human primate (NHP) CD1d and Vγ9Vδ2-T cells, a cross-reactive surrogate bispecific engager was generated to assess tolerability, pharmacokinetic (PK) and pharmacodynamic (PD) parameters. Results. The CD1d-Vδ2 bsVHH LAVA-051 triggers activation of both Vγ9Vδ2-T and type 1 NKT cells (EC 50 4 pM for Vγ9Vδ2-T and 366 pM for type 1 NKT; induction of &gt; 80% degranulation in 4h assays) and mediates potent killing of CD1d expressing tumor cells by engagement of Vγ9Vδ2-T and/or type 1 NKT cells (EC 50 1 pM for Vγ9Vδ2-T and 216 pM for type 1 NKT; &gt; 85% target cell lysis in 16h assays at a low 1:2 E:T ratio). Further, LAVA-051 triggered pro-inflammatory cytokine production, proliferation of Vγ9Vδ2-T and type 1 NKT cells, and exerted substantial antitumor activity against patient AML, CLL and MM cells that express CD1d and improved survival in in vivo T-ALL, AML and MM mouse models. Multiple dose studies in NHP (7 daily doses up to 1 mg/kg iv) showed clear Vγ9Vδ2-T cell engagement and some cytokine release after the first administration, but no clinical, laboratory, or histopathological toxicity. Reflecting the low molecular size of this bispecific engager, PK studies revealed a short plasma half-life which was however compensated for by prolonged (up to 5 days) binding of the engager to peripheral blood Vγ9Vδ2-T cells allowing intermittent dosing. Conclusions. In this study, we demonstrate that the CD1d-Vδ2 bsVHH LAVA-051 triggers activation of both type 1 NKT and Vγ9Vδ2-T cells, which translates directly into antitumor activity. Based on the expression of CD1d in CLL, MM, and AML, the strong preclinical activity of LAVA-051 against CD1d-expresssing tumors, and the favorable tolerability profile of the surrogate engager in NHP, LAVA-051 is currently evaluated in a first-in-human clinical Phase 1/2a study in patients with CD1d-expressing CLL, MM, or AML refractory to prior therapy (NCT04887259). Disclosures Lameris: Lava Therapeutics: Honoraria, Patents & Royalties, Research Funding. Ruben: Lava Therapeutics: Current Employment, Honoraria, Research Funding. Weerdt: LAVA Therapeutics: Research Funding. Roovers: LAVA Therapeutics: Current Employment, Current equity holder in publicly-traded company. van de Donk: Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: 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; Roche: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Cellectis: Research Funding. Broyl: Amgen: Honoraria; Bristol-Meyer Squibb: Honoraria; Celgene: Honoraria; Janssen Pharmaceuticals: Honoraria; Sanofi: Honoraria. Kater: Abbvie: Honoraria, Other: Ad Board, Research Funding; Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; Genmab, LAVA: Other: Ad Board, Steering Committee; BMS, Roche/Genentech: Other: Ad Board, , Research Funding. Riedl: LAVA THerapeutics: Current Employment, Current equity holder in publicly-traded company; Genmab BV: Current equity holder in publicly-traded company. Iglesias: LAVA therapeutics: Current Employment. Winograd: LAVA therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Celgene: Ended employment in the past 24 months; BMS: Current equity holder in publicly-traded company. Adang: LAVA therapeutics: Current Employment, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees. de Gruijl: LAVA therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; DCPrime: Membership on an entity's Board of Directors or advisory committees; Macrophage Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Idera Pharmaceuticals: Research Funding; ORCA Therapeutics: Patents & Royalties. Parren: Lava Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Sparring Bioconsult BV: Membership on an entity's Board of Directors or advisory committees; Genmab: Patents & Royalties; Roche: Consultancy. Vliet: Lava Therapeutics: Current Employment, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Glycostem: Research Funding.

Combinatorial Antigen Targeting Strategy for Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-23
Author(s):  
Pinar Ataca Atilla ◽  
Mary K McKenna ◽  
Norihiro Watanabe ◽  
Maksim Mamonkin ◽  
Malcolm K. Brenner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Tumor Control ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Introduction: Efforts to safely and effectively treat acute myeloid leukemia (AML) by targeting a single leukemia associated antigen with chimeric antigen receptor T (CAR T) cells have had limited success. We determined whether combinatorial expression of chimeric antigen receptors directed to two different AML associated antigens would augment tumor eradication and prevent relapse in targets with heterogeneous expression of myeloid antigens. Methods: We generated CD123 and CD33 targeting CARs; each containing a 4-1BBz or CD28z endodomain. We analyzed the anti-tumor activity of T cells expressing each CAR alone or in co-transduction with a CLL-1 CAR with CD28z endodomain and CD8 hinge previously optimized for use in our open CAR-T cell trial for AML (NCT04219163). We analyzed CAR-T cell phenotype, expansion and transduction efficacy by flow cytometry and assessed function by in vitro and in vivo activity against AML cell lines expressing high, intermediate or low levels of the target antigens (Molm 13= CD123 high, CD33 high, CLL-1 intermediate, KG1a= CD123 low, CD33 low, CLL-1 low and HL60= CD123 low, CD33 intermediate, CLL-1 intermediate/high) For in vivo studies we used NOD.SCID IL-2Rg-/-3/GM/SF (NSGS) mice with established leukemia, determining antitumor activity by bioluminescence imaging. Results: We obtained high levels of gene transfer and expression with both single (CD33.4-1BBʓ, CD123.4-1BBʓ, CD33.CD28ʓ, CD123.CD28ʓ, CLL-1 CAR) and double transduction CD33/CD123.4-1BBʓ or CD33/CD123.CD28ʓ) although single-transductants had marginally higher total CAR expression of 70%-80% versus 60-70% after co-transduction. Constructs containing CD28 co-stimulatory domain exhibited rapid expansion with elevated peak levels compared to 41BB co-stim domain irrespective of the CAR specificity. (p&lt;0.001) (Fig 1a). In 72h co-culture assays, we found consistently improved anti-tumor activity by CAR Ts expressing CLL-1 in combination either with CD33 or with CD123 compared to T cells expressing CLL-1 CAR alone. The benefit of dual expression was most evident when the target cell line expressed low levels of one or both target antigens (e.g. KG1a) (Fig 1b) (P&lt;0.001). No antigen escape was detected in residual tumor. Mechanistically, dual expression was associated with higher pCD3ʓ levels compared to single CAR T cells on exposure to any given tumor (Fig 1c). Increased pCD3ʓ levels were in turn associated with augmented CAR-T degranulation (assessed by CD107a expression) in both CD4 and CD8 T cell populations and with increased TNFα and IFNɣ production (p&lt;0.001 Fig 1d). In vivo, combinatorial targeting with CD123/CD33.CD28ʓ and CLL-1 CAR T cells improved tumor control and animal survival in lines (KG1a, MOLM13 and HL60) expressing diverse levels of the target antigens (Fig 2). Conclusion: Combinatorial targeting of T cells with CD33 or CD123.CD28z CARs and CLL-1-CAR improves CAR T cell activation associated with superior recruitment/phosphorylation of CD3ʓ, producing enhanced effector function and tumor control. The events that lead to increased pCD3ʓ after antigen engagement in the dual transduced cells may in part be due to an overall increase in CAR expression but may also reflect superior CAR recruitment after antigen engagement. We are now comparing the formation, structure, and stability of immune synapses in single and dual targeting CARs for AML. Disclosures Brenner: Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Maker Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Memmgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Atilla:Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: founder; Marker Therapeuticsa: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Memgen: Membership on an entity's Board of Directors or advisory committees; KUUR: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Effect of Moderate and Severe Hemophilia a on Daily Life in Children and Their Caregivers: A CHESS Paediatrics Study Analysis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 43-45
Author(s):  
Kate Khair ◽  
Francis Nissen ◽  
Mariabeth Silkey ◽  
Tom Burke ◽  
Aijing Shang ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Disease Burden ◽  
Hemophilia A ◽  
Daily Life ◽  
Social Activity ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Lost Work ◽  
Hours Of Work

Introduction: Hemophilia A (HA) is a congenital bleeding disorder, caused by a deficiency in clotting factor VIII (FVIII) and characterized by uncontrolled bleeding and progressive joint damage. This analysis assesses the impact of disease burden on the daily life of children with hemophilia A (CwHA) and their caregivers, addressing a deficit of current research on this topic. Methods: The Cost of Haemophilia in Europe: a Socioeconomic Survey in a Paediatric Population (CHESS Paediatrics) is a retrospective, burden-of-illness study in children with moderate and severe HA (defined by endogenous FVIII [IU/dL] relative to normal; moderate, 1-5%; severe, &lt;1%) across France, Germany, Italy, Spain and the UK. CwHA were recruited and stratified by both age group (0-5 years:6-11 years:12-17 years=1:1:1) and disease severity (severe:moderate=approximately 2:1, prioritizing children with severe HA [CwSHA]). Data for this analysis were captured from physicians, children, and their caregivers. Physicians completed online case report forms for treated children, and the child and/or their caregivers completed a paper-based questionnaire utilizing 5-point Likert scales. For CwHA aged 0-7, the questionnaire was completed by the caregiver, while for CwHA aged 8-17, children and caregivers completed different sections. Hours of care provided by the caregiver and work lost by the caregiver were reported as median values due to non-normal data distribution. Informed consent was obtained for all participants. Upon review, the study was approved by the University of Chester ethical committee. Results: Data from child/caregiver questionnaires were available for 196 CwHA (moderate, 25.5%; severe, 74.5%); the majority of these children, as expected, were receiving prophylaxis (72.4%), and did not have FVIII inhibitors (89.8%; Table 1). There was a direct impact of disease burden on CwHA, particularly with regard to physical and social activities (Figure 1). Overall, it was agreed or strongly agreed by the child or caregiver that 48.0% and 57.5% of children with moderate HA (CwMHA) and CwSHA respectively, have reduced physical activity due to HA, and 46.0% and 57.5%, respectively, have reduced social activity due to HA. A total of 36.0% and 61.0% of CwMHA and CwSHA, respectively, had adapted their treatment in anticipation of physical or social activity (Table 1). Furthermore, 34.0% of CwMHA and 55.4% of CwSHA were frustrated due to their disease, and many (CwMHA, 36.0%; CwSHA, 50.7%) felt that they had missed opportunities (Figure 1). For 66.0% of CwMHA and 76.0% of CwSHA, it was reported that their daily life was compromised due to their HA. Caregivers provided a median (interquartile range [IQR]) of 19.0 (10.0-59.5) and 12.0 (5.0-20.0) hours a week of care for the hemophilia-related needs of their CwMHA (n=30) or CwSHA (n=105), respectively. Of those who responded, 17.4% (n=4/23) and 25.0% (n=20/80) of caregivers to CwMHA or CwSHA, respectively, stated they have lost work due to their caregiving duty. This was more than twice as common for caregivers in families with multiple CwHA (42.9%, n=9/21 responses) compared with those in families with one CwHA (18.5%, n=15/81 responses). Median (IQR) hours of work per week estimated to be lost were 20.0 (17.0-22.0) for caregivers of CwMHA (n=4) and 12.5 (4.50-20.0) for caregivers of CwSHA (n=20). Conclusions: In conclusion, both children and caregivers make sacrifices in their daily lives due to HA; many CwHA reported reduced physical and social activities, fewer opportunities and feelings of frustration due to their HA. Caregivers reported spending a significant number of hours caring for their child and some reported losing work due to their caring responsibilities. However, some outcomes may be limited by the small number of respondents and narrow response options, particularly those regarding the caregiver burden. Responses on the hours of work lost may be subject to selection bias, as caregivers who have lost work may be more likely to respond to this question. Additionally, as this question is targeted at caregivers in employment, it is unknown if some caregivers have left employment due to their caregiving responsibilities. According to this analysis, children/caregivers are frequently required to adapt the child's treatment before the child engages in activities. Overall, the burden of disease was similar in children with moderate and severe HA. Disclosures Khair: Takeda: Honoraria, Speakers Bureau; Bayer: Consultancy, Honoraria, Speakers Bureau; Biomarin: Consultancy; HCD Economics: Consultancy; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medikhair: Membership on an entity's Board of Directors or advisory committees; Sobi: Consultancy, Honoraria, Research Funding, Speakers Bureau; CSL Behring: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Honoraria, Research Funding; Haemnet: Membership on an entity's Board of Directors or advisory committees. Nissen:GSK: Research Funding; Novartis: Research Funding; Actelion: Consultancy; F. Hoffmann-La Roche Ltd: Current Employment. Silkey:Aerotek AG: Current Employment; F. Hoffmann-La Roche Ltd: Consultancy. Burke:HCD Economics: Current Employment; University of Chester: Current Employment; F. Hoffmann-La Roche Ltd: Consultancy. Shang:F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company, Other: All authors received support for third party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Aizenas:F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company. Meier:F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company. O'Hara:HCD Economics: Current Employment, Current equity holder in private company; F. Hoffmann-La Roche Ltd: Consultancy. Noone:Research Investigator PROBE: Research Funding; Healthcare Decision Consultants: Membership on an entity's Board of Directors or advisory committees; European Haemophilia Consortium: Membership on an entity's Board of Directors or advisory committees.

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