Mapping the Impact of Proteasome Inhibitor Therapy on the Immunopeptidome of Multiple Myeloma: Mass Spectrometry Identifies Robust Targets for T Cell Immunotherapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3001-3001
Author(s):  
Daniel Johannes Kowalewski ◽  
Simon D. Walz ◽  
Linus Backert ◽  
Heiko Schuster ◽  
Susanne M. Rittig ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Multiple Myeloma ◽  
T Cell ◽  
Proteasome Inhibitor ◽  
Research Funding ◽  
Inhibitor Therapy ◽  
Target Cells ◽  
Myeloma Cells ◽  
Hla Ligands ◽  
Support Research

Abstract Recent studies underscore that multiple myeloma is an immunogenic disease and suggest that it can be effectively treated by T cell based immunotherapy via immunomodulation. This strategy might be synergistically complemented by therapeutic vaccination, which may help induce and guide specific anti-cancer T cell responses. We have recently conducted a study which directly characterized the antigenic landscape of myeloma by mass spectrometric analysis of naturally presented HLA ligands and identified a panel of T cell epitopes characterized by exquisite myeloma-association (Walz, Stickel et. al., Blood 2015). As standard of care in myeloma includes proteasome inhibitor therapy and the proteasome plays a central role in the generation of MHC-presented peptides, it is of great importance to thoroughly characterize and take into account the effects of this treatment on the antigenic landscape of myeloma cells and implement only robustly presented targets for peptide vaccine design. This is even more important Here we present a mass spectrometry-based study, which longitudinally and semi-quantitatively maps the effects of treatment with the 2nd generation proteasome inhibitor carfilzomib in an in vitro model of multiple myeloma. We observed considerable plasticity of the HLA class I ligandome of MM.1S cells after treatment with carfilzomib with 17.9±1.1.% (mean of 3 biological replicates ± SD) of HLA ligands showing significant modulation (fold change ≥ 4, P ≤ 0.01) at t24h compared to mock-treated controls (down-modulated: 11.5±1.1%, up-modulated: 6.3±0.6%). We were able to longitudinally tracke the abundance of 28 previously defined myeloma antigens, confirming robust (16/28, 57.1%) or even increased presentation (8/28, 28.6%) under treatment for the majority of these peptides. However, - importantly - we observed highly distortive effects of carfilzomib treatment on the HLA allotype distribution of target cells, which manifested as a marked reduction of HLA ligands restricted by HLA-A*23:01 and A*24:02 (-62.5±1.8% and -57.0±0.6%, respectively, at t=24h after treatment). These findings indicate strong allotype-specific effects of carfilzomib on the antigenic landscape of myeloma cells, which we interpret to be a direct reflection of the mechanism of action of this drug. As a significant proportion of the U.S. population are carriers of the affected alleles (A*23:01: 8.2%; A*24:02: 22.6%), these findings could have broad implications for the design or implementation of antigen-specific therapies in patients under proteasome inhibitor treatment. Furthermore, these findings might indicate the possibility of altered cancer immunosurveillance as a consequence of proteasome inhibitor therapy. Disclosures Weisel: Amgen: Consultancy, Honoraria, Other: Travel Support; Onyx: Consultancy, Honoraria; Novartis: Other: Travel Support; Janssen Pharmaceuticals: Consultancy, Honoraria, Other: Travel Support, Research Funding; Noxxon: Consultancy; Celgene: Consultancy, Honoraria, Other: Travel Support, Research Funding; BMS: Consultancy, Honoraria, Other: Travel Support.

The Proteasome Inhibitor Bortezomib Stimulates Osteoblastic Differentiation of Human Osteoblast Precursor Cells Via Upregulation of Vitamin D Receptor Signaling

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1899-1899
Author(s):  
Martin Kaiser ◽  
Maren Mieth ◽  
Orhan Sezer ◽  
Ulrike Heider
Keyword(s):  
Multiple Myeloma ◽  
Vitamin D ◽  
Proteasome Inhibitor ◽  
Research Funding ◽  
Fold Increase ◽  
Osteoblastic Differentiation ◽  
Luciferase Reporter ◽  
Human Osteoblast ◽  
Myeloma Cells ◽  
Osteoblast Precursors

Abstract Abstract 1899 Introduction In multiple myeloma (MM), interactions of the malignant plasma cell clone with the bone marrow microenvironment lead to an enhanced osteoclast recruitment and impaired osteoblast activity. The proteasome inhibitor bortezomib has been shown to suppress osteoclast activity, and there is recent evidence that bortezomib enhances osteoblast differentiation. The aim of this study was to investigate the effects of bortezomib on human osteoblast precursors, focusing on vitamin D (VD) dependent osteoblastic differentiation. Since vitamin D receptor (VDR) is degraded by the proteasome, we hypothesized that bortezomib could influence its signaling and hence vitamin D induced osteoblastic differentiation. This might be of clinical importance, since an increased rate of vitamin D deficiency has recently been reported in patients with MM. Methods Primary human mesenchymal stem cells (hMSC) and primary human osteoblasts (hOB) were isolated from bone marrow aspirates or from bone fragments of healthy donors undergoing orthopedic surgery, respectively. Ascorbic acid and β-glycerolphosphate were used for osteoblastic stimulation (OS), either in combination with or without vitamin D. In order to analyze the effects of proteasome inhibition on osteoblastic differentiation and activity, hMSC and hOB were incubated with bortezomib at subapoptotic doses (1 - 5 nM). In addition, coculture experiments of hMSC, hOB and myeloma cells were performed. Expression of osteocalcin and osteopontin (OPN) were quantified by real-time PCR as markers of osteoblastic lineage differentiation. Expression of VDR was analyzed by western blot in subcellular fractions and VDR signaling was investigated using luciferase reporter assays. Results In coculture experiments, myeloma cells inhibited the vitamin D dependent differentiation and activity of osteoblast precursors, e.g. coculture of hMSC with the myeloma cell line LP-1 for 4 days decreased their osteocalcin expression by 58%. Treatment with bortezomib led to an increased osteoblastic differentiation of hMSC and hOB by OS, represented by an enhanced expression of osteoblast markers osteocalcin and OPN. Importantly, this effect could be further increased, when vitamin D was added. In hMSC stimulated with OS only, addition of 5 nM bortezomib led to an 18.3 fold increase in OPN mRNA expression. In comparison, hMSC stimulated with OS + vitamin D showed a 27.5 fold increase in OPN mRNA with the addition of bortezomib. Osteocalcin expression was increased 1.9 fold by bortezomib in the presence of OS and vitamin D, but not with OS alone. Similar results were obtained with osteoblasts: Incubation with bortezomib slightly increased osteocalcin and OPN mRNA expression in cells stimulated with OS only (1.3 fold and 2.4 fold, respectively). In comparison, in cells stimulated with OS and vitamin D, bortezomib elevated osteocalcin and OPN expression 2.9 fold and 5.5 fold, respectively. Bortezomib led to an increase in nuclear VDR levels in hMSC in western blot analyses. Primary hMSC transfected with a VDR luciferase reporter construct showed a 3.7 fold increase in VDR signaling with bortezomib. Conclusion Our data show that bortezomib stimulates osteoblastic differentiation of hMSCs and hOBs and acts, at least in part, through VDR signaling. Substitution of vitamin D in multiple myeloma patients treated with bortezomib may be beneficial for bone turnover and needs clinical evaluation. Disclosures: Kaiser: Johnson & Johnson: Research Funding. Mieth:Johnson & Johnson: Research Funding. Sezer:Johnson & Johnson: Research Funding. Heider:Johnson & Johnson: Research Funding.

scholarly journals Safety and Efficacy of Letetresgene Autoleucel (lete-cel; GSK3377794) Alone or in Combination with Pembrolizumab in Relapsed and Refractory Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3865-3865
Author(s):  
Taiga Nishihori ◽  
James E. Hoffman ◽  
Jonathan L. Kaufman ◽  
Anne Huff ◽  
Charlotte Snape ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cell ◽  
Cancer Therapy ◽  
Research Funding ◽  
Cell Kinetics ◽  
Publicly Traded ◽  
Myeloma Cells ◽  
Safety And Efficacy ◽  
Anti Cancer ◽  
Grade 3

Abstract Background: Outcomes remain poor for patients with relapsed and refractory multiple myeloma (RRMM) progressing on conventional therapy (proteasome inhibitors, IMiDs, anti-CD38 antibodies, BCMA targeting agents, corticosteroids). Lete-cel, an autologous T-cell therapy, targets NY-ESO-1/LAGE-1a+ tumors using a genetically modified, high-affinity T-cell receptor. NY-ESO-1 and LAGE-1a are immunogenic cancer/testis antigens frequently overexpressed in MM and are linked to poor clinical outcomes. Additionally, PD-1 expression, which may limit adaptive immune response, has been observed previously in RRMM patients following treatment with lete-cel (Stadtmauer et al. Blood Adv, 2019; 3: 2022-2034). This open-label, pilot study evaluated the safety and efficacy of lete-cel +/- pembrolizumab (pembro) in patients with RRMM. Study design and methods: Key eligibility criteria include: age ≥18 yr; HLA-A*02:01; A*02:05, and/or A*02:06; NY-ESO-1+ and/or LAGE-1a+; protocol-required prior regimens; specified washouts from prior therapy; no active/chronic/intercurrent illness. Following lymphodepletion (LD), patients received lete-cel (Arm 1) or lete-cel + pembro (Arm 2). Planned pembro dosing was 200 mg/dose Q3 weeks (wks) starting at Wk 3. Primary endpoint was safety and tolerability. Key efficacy endpoint was investigator-assessed overall response rate (ORR) by International Myeloma Working Group uniform response criteria for MM (2016); response was assessed Q3 wks from Wk 3 to Wk 24, then Q6 wks to Wk 72, then every 3 months (mo) to disease progression/death/withdrawal. NY-ESO-1/LAGE-1a expression was assessed by qRT-PCR on myeloma cells. Transduced cell kinetics were assessed by qPCR of transgene vector copies in DNA from peripheral blood mononuclear cells. Results: Six patients (all male; median age 63 yr) were enrolled; 3 per arm. All had prior systemic anti-cancer therapy; 3 patients had received ≥5 prior regimens. Five of 6 patients received systemic anti-cancer therapy between leukapheresis and LD. All received reduced LD due to age and, in some patients, renal impairment. Patients in each arm received similar numbers of transduced T cells. Each of the 3 patients in Arm 2 received a median of 3 pembro doses (range: 3-4 doses). Start of pembro dosing was delayed to Wk 6 in 2 patients due to ongoing toxicities. There were no Grade 5 AEs. Grade 3/4 T-cell related AEs were reported in 3 patients, and all patients had Grade 3/4 LD-related AEs. Hematopoietic cytopenias were the most common treatment-emergent and treatment-related Grade 3/4 AE, occurring in all patients. All cytopenias were reported to have resolved for 4 patients or to have improved to Grade 1 at final patient follow-up for 2 patients. Three patients had cytokine release syndrome (Arm 1: 1 patient, Grade 2; Arm 2: 2 patients; 1 Grade 1 and 1 Grade 2); all patients recovered. There was 1 event of graft vs host disease (GvHD skin; Grade 1) and, in a separate patient, 1 event of Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS) (Grade 1). Both events resolved. All patients had reduction in tumor burden. Arm 1 (lete-cel alone) had an ORR of 33.3% (1 CR, 2 SD) and median progression-free survival (PFS) of 2.79 mo, while Arm 2 (combination) had an ORR of 66.7% (1 VGPR, 1 PR, 1 SD) and median PFS of 2.78 mo. Time to response for all responders was 3 weeks. Pembro dosing for the 2 Arm 2 responders began at Wk 6. Duration of response in each responder was 2.1 mo. Overall survival data are not mature. Two of 3 responders exhibited clearance of antigen positive myeloma cells in the bone marrow for up to 6 weeks after lete-cel infusion. T cell kinetics trended toward higher peak expansion (Cmax) and area under the curve (AUC) over the first 28 days post-dose (AUC0-28d) in responders vs. non-responders. Serum cytokine profiles in relation to response and CRS will be discussed. Conclusions: A single lete-cel infusion was associated with antitumor activity in 6/6 heavily pretreated RRMM patients, including 1 CR, 1 VGPR, 1PR. Both responses in Arm 2 occurred prior to pembro initiation. The associated safety profile was manageable and consistent with that seen in other lete-cel studies. Responders showed a trend toward higher Cmax and AUC0-28d as compared to non-responders. The study was closed in November 2020 due to protracted enrollment. This study (208470; NCT03168438) was funded by GlaxoSmithKline. Submission support was provided by Fishawack Health. Disclosures Nishihori: Novartis: Research Funding; Karyopharm: Research Funding. Kaufman: Janssen: Honoraria; Heidelberg Pharma: Research Funding; Fortis Therapeutics: Research Funding; Tecnofarma SAS, AbbVie: Honoraria; Amgen: Research Funding; BMS: Consultancy, Research Funding; Incyte, celgene: Consultancy; Incyte, TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Genentech, AbbVie, Janssen: Consultancy, Research Funding; Novartis: Research Funding; Roche/Genetech, Tecnopharma: Consultancy, Honoraria; Sutro, Takeda: Research Funding. Huff: GSK: Current Employment, Current equity holder in publicly-traded company. Snape: Veramed: Current Employment. Jain: Butterfly Network: Current equity holder in publicly-traded company; Marker Therapeutics: Current equity holder in publicly-traded company; 23 and Me: Current equity holder in publicly-traded company; Sema4 Holdings: Current equity holder in publicly-traded company; GSK: Current Employment, Current equity holder in publicly-traded company. Kapoor: GSK: Current equity holder in publicly-traded company. Zajic: GSK: Current Employment, Current equity holder in publicly-traded company. Suchindran: GSK: Current Employment, Current equity holder in publicly-traded company. Chisamore: Merck & Co. Inc: Current Employment, Current equity holder in publicly-traded company. Rapoport: GSK: Other: Support received as site principal investigator for this study.

scholarly journals Identification of Novel Regulatory Pathway for Immunoglobulin Production Provides Rational Treatment for Bortezomib-Resistant Multiple Myeloma Patients

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 40-42
Author(s):  
Alexander Vdovin ◽  
Michal Durech ◽  
Tomas Jelinek ◽  
Tereza Sevcikova ◽  
Juli R. Bago ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Multiple Myeloma ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Patient Survival ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Ubiquitinated Proteins ◽  
Ig Synthesis

Introduction Monoclonal immunoglobulin (Ig) is a valuable diagnostic marker in patients with multiple myeloma (MM). An inevitable consequence of extensive Ig synthesis is overload of misfolded proteins that saturate proteasome capacity making the myeloma cells highly sensitive to proteasome inhibitors (PI). Even though PI are regularly used in the clinic, resistance often emerges leaving clinicians with limited treatment options. Therefore, there is a need for a robust marker selecting MM patients for precise PI-based combination therapy. Methods We performed a multiple database search for genes associated with Ig production and MM patients' survival. Additionally, we compared gene expression profiles (RNAseq) of primary MM cells with low and high Ig levels. Next, we validated the identified hits by shRNA knockdown and overexpression studies using myeloma cell lines, primary MM samples, and mouse models. We also applied mass spectrometry-based proteomic analysis, advanced biochemical approaches, and genetic models to reveal the Ig production pathway components and function. Finally, we performed a limited rational drug screening to select suitable compounds for combination treatment. Results RNAseq and database mining revealed a strong association between the expression of plasma cell-specific deubiquitinase OTUD1, Ig production, and MM patient survival. Suppression of OTUD1 with shRNAs in RPMI8226 and MM1.S cell lines reduced Ig levels, increased proliferation, and induced bortezomib resistance. Conversely, inducible OTUD1 overexpression enhanced Ig production, slowed down proliferation, and increased bortezomib sensitivity. In the xenografts mouse models cells with high OTUD1 levels synthesized more Ig and developed smaller tumors. Intriguingly, the transcription of Ig genes was not influenced by OTUD1 expression suggesting that OTUD1 functions as a posttranslational regulator of Ig assembly. To gain mechanistic insight into the Ig pathway regulation by OTUD1, we utilized the biotin proximity labeling method (Turbo-ID) combined with mass spectrometry analysis. We found several novel OTUD1 interaction partners including the E3 ubiquitin ligase KEAP1 and endoplasmic reticulum (ER) redox protein PRDX4. We demonstrated that KEAP1 acts upstream of OTUD1 by regulating OTUD1 ubiquitination and stability. Consistently, survival analysis revealed that MM patients with high KEAP1 expression (low OTUD1) had a worse prognosis than patients with low levels of KEAP1 (high OTUD1). PRDX4 regulates disulfite bonds formation during protein folding and is uniquely expressed in fully differentiated plasma cells. Here, we revealed that OTUD1 specifically deubiquitinates and thus stabilizes PRDX4 inside the ER. Additionally, we performed rescue genetic experiments and found a direct link between the OTUD1-PRDX4 axis and Ig production. The increase in OTUD1 expression (high Ig) led to a dramatic increase in the total pool of ubiquitinated proteins formed mainly by misfolded Ig, while OTUD1 knockdown (low Ig) had an opposite effect. We showed that changes in the level of ubiquitinated proteins correlated with PI sensitivity. Of note, OTUD1 did not affect the expression of proteasome subunits, either their enzymatic activity. Our mechanistic findings prompted us to propose a novel therapeutic opportunity in PI resistant MM patients. We hypothesize that the resensitization of Ig low MM cells to PI could be achieved by enhancing ER stress leading to an increase in misfolded proteins that would ultimately saturate proteasomes. Indeed, from clinically relevant drugs tested so far, the HSP-90 inhibitor (17-AAG) reverted the PI resistance in OTUD1 low (Ig low) myeloma cells. An in vivo validation of the combination treatment and testing of Ig involvement in PI sensitivity and proliferation of MM cells is ongoing. Conclusion Here we present the discovery of a novel regulatory mechanism for Ig production in plasma cells. Based on our results and previously published studies, we conclude that Ig synthesis is a clinically significant factor related to PI response and MM patient survival. Our findings suggest that the intracellular Ig level is an important biomarker to identify patients benefiting the most from PI-based therapies. Finally, we provide a rational solution for selective, combination therapy to overcome PI resistance in MM patients with a decreased capacity to synthesize Ig. Figure Disclosures Hajek: Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria; PharmaMar: Consultancy, Honoraria; Oncopeptides: Consultancy.

Biomarkers of Cardiotoxicity Among Multiple Myeloma Patients Subsequently Treated with Proteasome Inhibitor Therapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4257-4257 ◽  
Author(s):  
Nikoletta Lendvai ◽  
Sean Devlin ◽  
Minal Patel ◽  
Kristina Marie Knapp ◽  
Daniel Ekman ◽  
...  
Keyword(s):  
Heart Failure ◽  
Multiple Myeloma ◽  
Proteasome Inhibitor ◽  
Research Funding ◽  
Inhibitor Therapy ◽  
Matched Pair ◽  
P Value ◽  
High Dose ◽  
Underlying Mechanisms ◽  
Potential Biomarkers

Abstract BACKGROUND: Cardiovascular (CV) events are a known complication to proteasome inhibitor therapy in myeloma. Underlying mechanisms are unknown. We recently completed an investigator initiated, single institution Phase II study of high dose carfilzomib (56mg/m2) in patients with relapsed/refractory MM (NCT01351623). Among 42 response evaluable patients, 11 patients (25%) developed treatment-emergent heart failure of any grade, and 5 patients (11%) developed severe heart failure requiring mechanical ventilation. We undertook a study to identify potential biomarkers that may point to underlying mechanisms of CV events among multiple myeloma patients treated with carfilzomib therapy. METHODS: We performed a nested case-control study with 7 patients who experienced a CV event on our high dose carfilzomib study and had pre-treatment (baseline) plasma stored and 19 case matched controls treated on the same study who did not have a CV event. We screened for 90 proteins known to be associated with CV disease using O-linked glycosylation. We used the Proseek Multiplex CVD I 96x96 platform which is based on the Proximity Extension Assay (PEA) technique. PEA is a 96-plex immunoassay that allows high throughput detection of protein biomarkers in liquid samples. For each biomarker, a matched pair of antibodies linked to unique oligonucleotides (proximity probes) binds to the respective protein target. Upon binding, the unique proximity probes can hybridize to each other and subsequently be detected and quantified by real-time PCR. Mean biomarker levels were compared using a t-test. False discovery rate (FDR) was used for multiple comparisons adjustment. RESULTS: Using samples collected prior to initiation of carfilzomib therapy, in an agnostic statistical model we identified the following four proteins to have altered levels in myeloma patients who developed CV events (p=0.002-0.004, unadjusted; p=0.089, after FDR correction): matrix metalloproteinase-1 (MMP-1, heparin-binding EGF-like growth factor (HB-EGF), TNF-related apoptosis-inducing ligand (TRAIL), and myoglobin (MB). Myeloma patients who developed CV events had 37% lower MMP-1, 15% lower MB, and 4% lower HB-EGF, while TRAIL was 7% higher in patients who developed CV events. Matrix metalloproteinases are a family of proteolytic enzymes responsible, among other functions, for myocardial extracellular protein degradation. Interestingly, several MMP species, including MMP-1, have been identified within the human myocardium and are thought to be dysregulated in congestive heart failure. HB-EGF is a mitogenic and chemotactic glycoprotein that is essential for maintaining normal cardiac function and is known to play an important role in myocardial remodeling. CONCLUSIONS: We found that there was a trend towards lower MMP-1, HB-EGF, and MB levels and higher TRAIL levels in patients with CV events while receiving proteasome therapy. MMP-1 appears to be the most promising potential biomarker based on our data. Our study supports further investigation of these proteins as potential biomarkers for patients at risk of CV events when treated with carfilzomib. Table 1. CV event No CV event N=7 N=19 CKD Proteins1 Mean (SD) Mean(SD) Unadjusted P-value Adjusted P-value MMP_1 1.7 (0.5) 2.7 (0.9) 0.002 0.089 HB_EGF 6.9 (0.2) 7.2 (0.3) 0.004 0.089 TRAIL 8 (0.3) 7.5 (0.5) 0.004 0.089 MB 5 (0.5) 5.9 (0.8) 0.004 0.089 HSP_27 2.2 (0.3) 2.7 (0.8) 0.032 0.528 PDGF_subunit_B 4 (0.7) 5 (1.5) 0.036 0.528 CD40_L 3.4 (0.6) 4.2 (1.2) 0.042 0.533 EGF 3.7 (0.9) 4.7 (1.4) 0.053 0.592 CX3CL1 5.9 (0.2) 5.6 (0.6) 0.092 0.895 TRAIL_R2 4.2 (0.4) 4.6 (0.6) 0.101 0.895 1. Proteins are listed based on the p-value associated with the difference between patients who did and did not have CV events, with lowest p-value on the top. The top 10 biomarkers are shown. Disclosures Ekman: Olink Bioscience: Employment. Grundberg:Olink Bioscience: Employment. Hassoun:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Takeda: Research Funding; Novartis: Consultancy. Lesokhin:Aduro: Consultancy; Efranat: Consultancy; Genentech: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Landau:Janssen: Consultancy; Prothena: Consultancy, Honoraria; Janssen: Consultancy; Spectrum Pharmaceuticals: Honoraria; Onyx: Honoraria, Research Funding; Takeda: Research Funding. Giralt:TAKEDA: Consultancy, Honoraria, Research Funding; JAZZ: Consultancy, Honoraria, Research Funding, Speakers Bureau; AMGEN: Consultancy, Research Funding; SANOFI: Consultancy, Honoraria, Research Funding; CELGENE: Consultancy, Honoraria, Research Funding. Landgren:Onyx: Honoraria; Celgene: Honoraria; BMJ Publishing: Consultancy; International Myeloma Foundation: Research Funding; Bristol-Myers Squibb: Honoraria; Onyx: Research Funding; Medscape: Consultancy; Medscape: Honoraria; BMJ Publishing: Honoraria; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Onyx: Consultancy.

scholarly journals Evolving Exhaustion of T Cells during the Course of the Disease in AML Can be Abrogated By CD33 BiTE ® Construct Mediated Cytotoxicity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1172-1172
Author(s):  
Maryam Kazerani Pasikhani ◽  
Anetta Marcinek ◽  
Bettina Brauchle ◽  
Jonathan Jonas Taylor ◽  
Helena Domínguez Moreno ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Cell Function ◽  
Target Cells ◽  
Cell Cytotoxicity ◽  
T Cell Cytotoxicity ◽  
Support Research

Abstract Novel immunotherapeutic strategies like BiTE ® (bispecific T cell engager) constructs aim to eradicate neoplastic cells by TCR-independent T-cell activation, and therefore rely on the function of autologous T cells. Currently, their efficacy is also evaluated in heavily pre-treated patients with relapsed/refractory acute myeloid leukemia (AML). Previous data demonstrated dysfunction in CD8 + T cells of AML patients (Knaus et al 2018). Thus, we aimed to characterize the progressive modulation of T-cell activity over the course of AML progression to improve the optimal application of T-cell based immunotherapeutic approaches. Bone marrow mononuclear cells (BMMCs) from AML patients at time of initial diagnosis (ID), complete remission (CR), relapse (RL), as well as of age-matched healthy donors (HD) were analyzed for T-cell subset distribution and expression of exhaustion markers by flow cytometry. Additionally, T-cell function was assessed after stimulation with 1) CD3/CD28 beads; 2) AMG 330, a CD33/CD3 specific BiTE ® construct, after incubation with OCI-AML3 target cells; or 3) AMG 330 in an autologous ex vivo long-term culture system after incubation with primary AML cells (pAML). After 6 days, T cell proliferation, expression of effector molecules and cytokines, and AMG 330-mediated T-cell cytotoxicity were assessed by flow cytometry. Lastly, we performed longitudinal bulk RNA-sequencing on 5000 sorted T cells from 7 matched ID-RL primary AML samples. Immunophenotypic analysis of BM T-cell subsets revealed a shift from T NAIVE toward central/effector memory subsets during AML progression. We observed lower percentages of T NAIVE in RL (n=3) compared to CR (n=3) CD8 + T cells(11.8 vs. 45.2%, p=0.07; RL vs. CR). Conversely, RL patients showed increased percentages of CD8 + memory T cells (T CM: 23.4 vs. 6.7%; T EM: 29.4 vs. 20.2%; T EMRA: 35.3 vs. 27.8%; RL vs. CR). Further characterization of exhaustion markers exhibited a significantly higher percentage of both CD4 + and CD8 + T cells expressing 2B4 (CD244) in ID (n=19) and RL (n=13) compared to HD (n=10, both p < 0.001). A higher percentage of PD-1 + CD8 + and TIM-3 + CD4 + T cells was detected in both ID and RL relative to HD (all p < 0.05). However, a significantly increased percentage of CD8 + T cells expressing TIM-3 and CD160 was detected in ID relative to HD (p < 0.05). Intriguingly, RL CD4 + T cells demonstrated a significantly higher level of LAG3 compared to ID (p < 0.01). In line with phenotypic exhaustion features, ID (n=4) and RL (n=5) CD8 + T cells showed reduced proliferation compared to HD (n=4) CD8 + T cells after CD3/CD28 bead stimulation (both p < 0.01). Correspondingly, we observed a marked reduction in the expression of Granzyme B (GZMB) by CD8 + T cells (both p < 0.05). Interestingly, when compared to ID, RL CD4 + T cells showed decreased TNF-α secretion (p < 0.05). In contrast to these findings, AMG 330-mediated T cell cytotoxicity against OCI-AML3 target cells was superior with RL T cells compared to ID T cells (p < 0.001). The percentage of GZMB + CD8 + T cells strikingly enhanced in RL relative to ID (p < 0.01). In an autologous setting with pAML samples, T cells from RL patients (n=6) showed higher AMG 330-mediated cytotoxicity compared to ID (n=9) T cells (67.7 vs. 35.2; RL vs. ID). In our longitudinal RNA-sequencing, differentially expressed genes analysis detected 61 up- and 30 downregulated genes (log2 FC > 1 or < -1; p < 0.01) in RL T cells compared to their matched ID counterparts. Among the significantly upregulated genes in RL, we identified genes associated with memory T cell function (TP53INP2, DUSP4) and exhaustion (NR4A1, TOX2). Moreover, Gene set enrichment analysis showed significant enrichment of gene signatures associated to memory and exhausted T cells (normalized enrichment score (NES)=1.2 and 1.3; p-value= 0.026 and 0.008, respectively), depletion of oxidative phosphorylation (NES=-2.05; p adj < 0.0001) and protein secretion (NES=-1.49; p adj < 0.05) gene signatures in RL vs. ID T cells. Taken together, our data show that patient T cells acquire an activated/exhausted phenotype upon AML progression. However, this is not reflected in the T-cell effector functions upon AMG 330 stimulation, in contrast to bead stimulation. These observations may highlight the significant role of the AML target cells in shaping a T-cell response. To this end, we will further analyze the longitudinal communication between T cells and their corresponding AML blasts. Disclosures Brauchle: Adivo: Current Employment. Kischel: Amgen GmbH Munich: Current Employment. Buecklein: BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; Miltenyi: Research Funding; Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau. Subklewe: Novartis: Consultancy, Research Funding, Speakers Bureau; MorphoSys: Research Funding; Roche: Research Funding; Miltenyi: Research Funding; Seattle Genetics: Consultancy, Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Klinikum der Universität München: Current Employment.

HLA Ligandome Analysis in Multiple Myeloma Reveals Novel Naturally Presented Myeloma-Associated Antigens Showing Stable Expression Under Proteasome Inhibitor Therapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3438-3438
Author(s):  
Simon D. Walz ◽  
Daniel J. Kowalewski ◽  
Heiko Schuster ◽  
Katja Weisel ◽  
Helmut R. Salih ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cell ◽  
Proteasome Inhibitor ◽  
Plasma Cells ◽  
Hla Class I ◽  
Surface Expression ◽  
Myeloma Cell ◽  
Fold Change ◽  
Class I ◽  
Myeloma Cells

Abstract Results of allogeneic transplantation and reports that multiple myeloma (MM) cells can act as antigen presenting cells, but also the favorable immune effector-to-target cell ratio in the minimal residual disease (MRD) setting suggest that MM, which previously was considered to be weakly immunogenic, may be targeted effectively by T cell based immunotherapy. Following conventional therapy, peptide-based immunotherapy might offer a low side-effect opportunity to extend time to disease progression and possibly eradicate MRD. Therefore we aim to develop a multi-peptide vaccine for the immunotherapy of MM patients based on naturally processed and presented HLA ligands. In a first step we analyzed HLA surface expression on MM cells, which constitutes a prerequisite for effective T cell recognition. In MM patients (n=20), HLA class I expression was found to be heterogeneous with a mean of 415,000 ± 55,000 molecules on malignant cells. This expression was significantly (p<0.005, unpaired t-test) higher than that on autologous healthy B cells (200,000 ± 20,000), T cells (165,000 ± 15,000) and hematopoietic progenitor cells (205,000 ± 30,000). In addition, HLA class I expression on primary MM cells was also found to be significantly higher than that on plasma cells of healthy volunteers (HV, n=15) (290,000 ± 25,000, p<0.05). Mean HLA-DR expression on myeloma cells (25,000 ± 7,000) was significantly lower compared to HLA class I expression (p<0.0001) without significant difference compared to expression levels on autologous bone marrow cells as well as normal plasma cells of HVs (40,000 ± 5,000). Using the approach of direct isolation and identification of naturally presented HLA peptides by affinity chromatography and mass spectrometry, we identified more than 15,000 different HLA class I ligands from 8 primary MM samples, 2 plasma cell leukemia samples and 5 myeloma cell lines (MCLs). Comparative mapping of MM HLA ligandomes and the ligandomes of 40 HVs (30 blood and 10 bone marrow samples) revealed 15 ligandome-derived tumor-associated antigens (LiTAAs) represented by 36 corresponding HLA peptides (LiTAPs), showing exclusive representation in >30% of myeloma samples. Among them we identified 3 novel HLA peptides derived from Multiple Myeloma SET domain(MMSET), which is an established driver of myeloma cell proliferation, stimulating the expression of c-myc. We further studied the effect of the second-generation proteasome inhibitor carfilzomib on HLA surface expression and HLA ligandome composition. Notably, a slight albeit not significant increase in mean HLA class I expression was observed after 24h (t24) and 48h (t48) of carfilzomib treatment in MCL (n=5, fold change t24=1.4, pt24=0.37, fold change t48=1.6, pt48=0.25) as well as in primary myeloma cells in vitro (n=7, fold change t24=1.2, pt24=0.21, fold change t48=1.1, pt48=0.46). No significant loss or down regulation of HLA class I expression on MM cells was observed in ex vivo analysis of primary samples obtained from 2 myeloma patients before therapy and after 4 weeks of carfilzomib treatment. In order to analyze the impact of proteasome inhibition on the composition of the HLA class I ligandome we incubated MCLs (U266, MM.1S) with carfilzomib and performed quantitative mass spectrometry at baseline as well as at t24 and t48. On U266, 4.6% (4.0%) and 4.4% (8.6%) of total identified peptides (n=2444) were significantly up- or down regulated after 24h/48h of treatment with carfilzomib, respectively, whereas on MM.1S (n=1693) 1.6% (12.4%) and 10.8% (18.0%) were significantly regulated after 24h/48h. Focusing on the LiTAPs presented on these MCLs, 76.5% (88.2%) on U266 and 83.3% (66.7%) on MM.1S remained stably expressed after incubation with carfilzomib. Further analyses evaluating overall changes in peptide motifs and the occurrence of novel, cryptic peptides after carfilzomib treatment are ongoing. Taken together our data demonstrate that myeloma cells are valid targets for T cell based immunotherapy and provide a panel of novel, naturally presented myeloma-associated antigens, which exhibit stable presentation under proteasome inhibitor therapy. Disclosures No relevant conflicts of interest to declare.

Identification of Novel Immune-Checkpoint Molecules on Multiple Myeloma Cells Using a High-Throughput Discovery Platform

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 380-380
Author(s):  
Valentina Volpin ◽  
Till Michels ◽  
Antonio Sorrentino ◽  
Dirk Hose ◽  
Anthony D. Ho ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cell ◽  
Board Of Directors ◽  
High Throughput ◽  
Immune Checkpoint ◽  
Research Funding ◽  
Immune Checkpoints ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Immune Checkpoint Molecules

Abstract Introduction: Multiple myeloma (MM) is a B-cell malignancy, characterized by accumulation of plasma cell clones in the bone marrow. While novel therapeutic agents like immunomodulatory drugs and proteasome inhibitors have improved overall survival of MM patients, the disease remains incurable in most patients. Several studies showed that immune-checkpoint molecules are expressed by myeloma cells and induce tumor-related immune suppression. Despite the promising results achieved by blocking CTLA4 and the PD-1/PD-L1 axis in the treatment of various solid tumors and Hodgkin's lymphoma, targeting these checkpoints did not induce objective responses in Phase I/II trials in MM patients. Therefore, identification of novel immune-checkpoints and defining the subsequent molecular mechanisms of inhibition are essential for further improvement. Methods: Our main goal is to identify novel MM-related immune-checkpoint molecules by taking advantage of a high-throughput (HT) RNAi screen and sequentially validate the role of candidate molecules, whose blockade could potentially induce anti-tumor immunity in MM patients. Methods: High-throughput RNAi screens offer a possibility to systemically search for immune-checkpoint molecules. Therefore, we established a high-throughput screening system to discern candidate molecules and evaluate their use as potential targets for multiple myeloma immunotherapy. We established a luciferase based read-out system by generating a stable luciferase expressing MM cell line (KMM-1-luc). To test the effect of immune-checkpoint molecules, KMM-1-luc cells were transfected with a siRNAs library targeting 2514 genes encoding for cell surface proteins, kinases and GPCRs. Transfected tumor cells were subsequently co-cultured with patient-derived HLA-matched Myeloma Infiltrating T Lymphocytes (MILs) and the effect of gene knock-down on T-cell mediated tumor lysis was measured. Results: Based on our primary HT-screening, we have identified 132 candidate molecules (hits) whose knockdown increased T-cell mediated killing more efficiently than the established checkpoint genes CCR9. To confirm the hits and the robustness of the screening, we re-tested the identified candidates in a secondary screening. Among these potential immune-checkpoints we selected 10 hits for further validation. So far, we were able to confirm expression of the hits at mRNA level and to validate siRNAs on-target effect by qPCR and luciferase-based cytotoxicity assay. Detailed results will be presented at the meeting. Conclusion: Altogether we optimized a high-throughput RNAi screen to discover novel immune-checkpoints that are potential immunotherapeutic targets for the treatment of multiple myeloma. We are currently investigating the mode of action of the candidate hits in vitro. Further in vivo validation of these immune-checkpoint molecules is still required for clinical studies. Disclosures Goldschmidt: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: 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, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Witzens-Harig:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Bleeding and Thrombosis Are Associated with Endothelial Dysfunction in CAR-T Cell Therapy and Are Increased in Patients Experiencing Neurologic Toxicity

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-33
Author(s):  
Andrew Johnsrud ◽  
Juliana Craig ◽  
John H. Baird ◽  
Jay Y. Spiegel ◽  
Lori S. Muffly ◽  
...  
Keyword(s):  
T Cell ◽  
Research Funding ◽  
Bleeding Events ◽  
Private Company ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T ◽  
Thrombotic Complications ◽  
D Dimer ◽  
Support Research

Background Treatment with chimeric antigen receptor (CAR) T cell therapies have shown dramatic, often durable responses for relapsed/refractory B-cell malignancies. However, it can be associated with significant side effects such as cytokine release syndrome (CRS), immune effector-cell associated neurotoxicity syndrome (ICANS) and life-threatening consumptive coagulopathies. The underlying pathobiology of such hemostatic defects and their distinct clinical sequelae remains obscure. This retrospective study aims at quantifying CAR T therapy associated bleeding and thrombotic complications and their association with CRS, ICANS, and laboratory derangements. Methods 130 adult patients with DLBCL or B-ALL treated between 2017-2020 with CD19 CAR-T therapy axicabtagene ciloleucel (N=90) or a bispecific CD 19/22 CAR construct utilizing 4-1BB costimulatory domains (N=40) were analyzed to determine dynamics of coagulation parameters and platelet counts as well as incidences of bleeding or thrombosis in the first three months after CAR T infusion. Events were included if graded ≥ 2 or if intervention was required. Platelet counts and coagulation parameters were collected prior to lymphodepletion (pre-LD), day 0, 3, 7, 14, 21, 28, 60 and 90. Results 12 (9.2%) and 8 (6.2%) patients developed bleeding and thrombotic complications in the first three months after CAR-T infusion, respectively. Events are characterized in Figure 1. All bleeding events occurred between days 0-30 (median 17.5, range 8-30), while thrombotic events occurred between days 2-91 (median day 29, range, 2-91). Two (1.5%) patients experienced both bleeding and thrombosis. Bleeding events coincided with the onset of thrombocytopenia and hypofibrinogenemia, and patients who bled had lower platelet (median 22.5 vs. 47 K/uL; p=0.03) and fibrinogen (median 151 vs. 351 ug/mL; p=0.007) nadirs in the first 30 days compared to those without bleeding. Temporally, the lowest median platelet nadir occurred at day 7 in patients with bleeding events vs. day 21 in patients without bleeding, while timing of fibrinogen nadirs were at day 21 in both. Patients with bleeding episodes were more likely to be older (median age: 70 vs. 60 yrs, p=0.03), have thrombocytopenia prior to lymphodepletion therapy (median 117.5 vs. 174.5 K/uL, p=0.01), and have elevated LDH (lymphoma subgroup; p=0.07). Other lab derangements in the first 30 days seen more frequently in patients with bleeding included prolonged thrombin time (TT) (21% vs. 6%; p=0.02), PT (16% vs. 5%; p=0.06), and elevated d-dimer (16% vs. 3%; p=0.01) indicative of a consumptive process. Thrombotic events were not significantly associated with elevated or peak d-dimer values (median 4.97 vs. 2.37 ug/mL, p=0.20). Interestingly, occurrence or severity of CRS was not associated with bleeding or thrombotic events, nor was it associated with marked derangements in coagulation abnormalities. However, higher grade ICANS (grade &gt; 3) was associated with bleeding (42% vs. 15%; p=0.038), thrombosis (50% vs. 16%; p=0.03), and evidence of endothelial activation including PT prolongation (78% vs. 35%; p&lt;0.001), hypofibrinogenemia (57% vs. 20%; p=0.001), and trend towards elevated d-dimer (70% vs. 46%; p=0.06). 13 (10%) patients received anticoagulation for prophylaxis or therapeutic indications that predated CAR T infusion. Four started anticoagulation secondarily for thrombotic events after CAR-T infusion, and one received tissue plasminogen activator (tPA) for an acute stroke. In this group, no patients developed bleeding complications from anticoagulation. Conclusion Both bleeding (9.2%), and thrombotic (6.2%) events are observed after CAR T cell therapy, with bleeding limited to the first month in our cohort. Notably, ICANS was uniquely associated with PT prolongation, hypofibrinogenemia, and increased fibrin degradation, in addition to both bleeding and thrombosis. These results suggest that a systemic coagulopathy coincides with high grade ICANS and whether these neurologic events truly represent sequelae of widespread vascular dysfunction warrants further investigation. Anticoagulation was safe in the patients whom it was indicated. Risk factors for bleeding and thrombotic complications should be studied prospectively to develop risk-assessment models and clinical guidelines for management of bleeding and thrombosis (including prophylaxis) during CAR T therapy. Disclosures Muffly: Adaptive: Research Funding; Servier: Research Funding; Amgen: Consultancy. Negrin:BioEclipse Therapeutics: Current equity holder in private company; Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company; KUUR Therapeutics: Consultancy; Biosource: Current equity holder in private company; Amgen: Consultancy; UpToDate: Honoraria. Shizuru:Jasper Therapeutics, Inc: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Meyer:Orca Bio: Research Funding. Shiraz:Kite, a Gilead Company: Research Funding; ORCA BioSystems: Research Funding. Rezvani:Pharmacyclics: Research Funding. Mackall:Apricity Health: Consultancy, Current equity holder in private company; NeoImmune Tech: Consultancy; Nektar Therapeutics: Consultancy; Allogene: Current equity holder in publicly-traded company; BMS: Consultancy; Lyell Immunopharma: Consultancy, Current equity holder in private company. Miklos:Adaptive Biotech: Consultancy, Other: Travel support, Research Funding; Kite-Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Juno-Celgene-Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Allogene Therapeutics Inc.: Research Funding; Novartis: Consultancy, Other: Travel support, Research Funding; Pharmacyclics: Consultancy, Other: Travel support, Patents & Royalties, Research Funding; Janssen: Consultancy, Other: Travel support; Miltenyi Biotec: Research Funding. Sidana:Janssen: Consultancy.

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