scholarly journals CLL B Cells Develop Resistance to Ibrutinib By Reinvigorating the IL-4R - IL-4 Axis Blocked By Bruton's Tyrosine Kinase Inhibitors Including Acalabrutinib and Zanubrutinib

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 477-477
Author(s):  
Shih-Shih Chen ◽  
Constantine S. Tam ◽  
Alan G. Ramsay ◽  
Priyadarshini Ravichandran ◽  
Natalia C. Couto-Francisco ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Subset ◽  
Advisory Committees

Bruton's tyrosine kinases inhibitors (BTKis) represent major advances in CLL therapy. However resistance to this form of therapy is emerging, and such patients often progress more rapidly. Hence there is an important need for therapies that address resistance. Microenvironmental input like IL-4 is critical for CLL disease progression. Compared with normal B cells, CLL cells exhibit significantly higher levels of surface membrane (sm) IL-4 receptor (IL4-R) and contain increased amounts of pSTAT6, a downstream mediator of IL-4R signaling. IL-4 stimulation of CLL B cells suppresses smCXCR4 and increases smIgM, thus promotes CLL cell retention and expansion. In this study, we aimed to examine if smIL-4R expression, IL4R signaling, and IL-4-producing cells are altered in patients sensitive or resistant to BTKis. To do so, T and B cell subset changes were studied overtime in 12 acalabrutinib-treated CLL patients, 6 zanubrutinib-treated CLL patients, 30 ibrutinib-sensitive and 5 ibrutinib-resistant CLL patients, 4 of which exhibited BTK mutations. Consistent with only ibrutinib inhibiting T-cell kinase (ITK), T-cell subset analyses revealed no changes in Th1, Th2, Th17, Th9, and Th22 cells after zanubrutinib or acalabrutinib treatment. In contrast, a Th1-biased T-cell immunity was observed in patients responsive to ibrutinib. In patients progressing on ibrutinib, significantly reduced Th2 T cells were found during the resistant as well as sensitive periods. In an in vitro T-cell function assay using T cells collected before and after the treatment with each BTKi, only ibrutinib treated patients exhibited a reduced ability of T cells to support CLL B cell survival. We next studied changes in CLL B cells, including numbers of IL-4, -10 and -13 producing B cells after BTKi treatment. IL-13 producing CLL B cells were not changed. IL-10 producing CLL B cells were reduced in both ibrutinib sensitive and resistant patients, but not in zanubrutinib or acalabrutinib treated patients. Importantly, IL-4 producing CLL B cells were significantly decreased in patients treated with all 3 BTKi. Significantly reduced smIL-4R levels, impaired IL-4R signaling, decreased smIgM and increased smCXCR4 were also seen in patients treated with each BTKi. To understand the mechanism responsible for inhibition of IL-4 production in CLL cells treated with BTKis, we stimulated CLL cells through IgM, Toll-like receptor and CD40L, finding that only anti-IgM stimulation significantly increased IL-4 production and p-STAT6 induction. We then explored the function of IL-4. IL-4 enhanced CLL B cell survival in vitro and this action was blocked by all 3 BTKis. Moreover, adhesion of CLL B cells to smIL-4R expressing stromal cells was decreased by IL-4 and IL-4R neutralizing antibodies, especially in M-CLL cases. In in vivo studies transferring autologous T cells and CLL PBMCs into alymphoid mice, we found less CLL B cells in mouse spleens post ibrutinib than zanubrutinib or acalabrutinib treatment. This might be due to the suppressed Th2 cells found only in ibrutinib, while IL-4 producing B cells were reduced in all 3 BTKi treated mice. These results support the idea that IL-4 promotes CLL B cell adhesion and growth in tissues. Finally, we investigated the IL-4/IL-4R axis in ibrutinib-resistant patients. Although IL-4 producing T cells remain reduced during the sensitive and resistant phases, CLL B cell production of IL-4 and expression of and signaling through smIL-4R returned when patients developed ibrutinib-resistance. When comparing paired ibrutinib-sensitive and -resistant CLL B cells collected from 3 patients in a xenograft model that requires T cell help, we found ibrutinib-resistant CLL B cells grew in vivo with only minimal (~15%) numbers of autologous T cells compared to B cells collected from ibrutinib-sensitive phase; this suggested a reduced requirement for T-cell help for growth of ibrutinib-resistant CLL cells. In summary, we found IL-4 is a key survival factor in the CLL microenvironment that also improves leukemia cell adhesion to stromal cells expressing smIL-4R. IL-4 production and signaling can be stimulated in CLL B cells through the B-cell receptor, and are consistently blocked by BTKis. Moreover, the recovered ability of ibrutinib-resistant CLL B cells to produce and respond to IL-4 leads to disease progression, suggesting blocking the IL-4/IL-4R axis is a potential treatment for ibrutinib-resistant CLL patients. Disclosures Chen: Pharmacyclics: Research Funding; Beigene: Research Funding; Verastem: Research Funding; ArgenX: Research Funding. Tam:Abbvie, Janssen: Research Funding; Abbvie, Janssen, Beigene, Roche, Novartis: Honoraria. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding. Kolitz:Boeringer-Ingelheim: Research Funding; Roche: Research Funding; Astellas: Research Funding. Zhou:BeiGene: Employment. Barrientos:Genentech: Consultancy; Gilead: Consultancy; Janssen: Consultancy; Abbvie: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Rai:Pharmacyctics: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Venetoclax Enhances Anti-Leukemia Activity of CD123-Specific BiTE-Secreting T-Cells in AML

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-13
Author(s):  
Hong Mu-Mosley ◽  
Lauren B Ostermann ◽  
Ran Zhao ◽  
Challice L. Bonifant ◽  
Stephen Gottschalk ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Vehicle Control ◽  
Advisory Committees ◽  
Cellular Therapies

Background: CD123 is frequently expressed in hematologic malignancies including AML. CD123 has been a potential immunotherapeutic target in AML due to its association with leukemic stem cells that play an essential role in disease progression and relapse. Our previous study using T-cells secreting CD123/CD3-bispecific T-cell engagers (BiTEs) (CD123-ENG T-cells) has shown activity in preclinical studies, recognizing and killing acute myeloid leukemia (AML) blasts in vitro and in vivo. CD123-ENG T-cells secrete bispecific molecules that recognize CD3 (T-cells) and CD123 (AML blasts), and are able to direct transduced T-cells and recruit bystander T-cells to kill CD123-positive blasts. Venetoclax is a BCL-2 inhibitor that can restore functional apoptosis signaling in AML cells, and has been FDA approved for the treatment of AML patients in combination with hypomethylating agents. To improve the efficacy of CD123-ENG T-cells we explored efficacy in AML by combining targeted immunotherapy (CD123-ENG T cells) with targeted inhibition of anti-apoptotic BCL-2 (venetoclax) in vitro and in vivo models of AML. Methods : CD123-ENG T-cells were generated by retroviral transduction and in vitro expansion. Non-transduced (NT) T-cells served as control. In vitro, GFP+ MOLM-13 AML cells were pretreated with venetoclax (0, 10µM, and 20µM) for 24 hours prior to co-culture with CD123-ENG or NT T-cells at an effector/target ratio of 1:10. After 16 hours, MOLM-13 AML cells were analyzed by flow cytometry and quantitated using counting beads; cytotoxicity was calculated relative to untreated MOLM-13 control. The anti-AML activity of the combination was further evaluated in a MOLM-13-luciferase xenograft AML mouse model. Leukemia progression was assessed by bioluminescence imaging. The frequency of MOLM13 AML and human T cells in periphera blod (PB) was determined by flow cytometry. Results: In vitro, we demonstrated that pretreatment of Molm13 AML cells with venetoclax enhanced the cytolytic activity of CD123-ENG T-cells compared to NT- or no T-cell controls. Interestingly, venetoclax sensitized Molm13 to CD123-ENG T-cell killing in a dose-dependent manner (Fig.1; 50%/31% killing by CD123-ENG T-cells versus 27%/14% of killing by NT T cells post pretreatment with 10µM or 20µM ventoclax, p<0.001). In the Molm13 luciferase xenograft model, NSGS mice were randomized into 5 groups after AML engraftment was confirmed: 1) vehicle control, 2) Venetoclax (Ven) only, 3) CD123-ENG T-cells only, 4) Ven+CD123-ENG T-cells, 5) Ven+CD123-ENG T-cells/2-day-off Ven post T-cell infusion (Ven[2-day-off]+CD123-ENG). Venetoclax treatment (100 µg/kg daily via oral gavage) was started on day 4 post Molm13 injection, and on day 7, mice received one i.v. dose of CD123-ENG T-cells (5x106 cells/mouse). Venetoclax or CD123-ENG T-cell monotherapy reduced leukemia burden compared to the control group, and combinational treatments further inhibited leukemia progression as judged by BLI and circulating AML cells (%GFP+mCD45-/total live cells) by flow cytometry on day 15 post MOLM-13 injection: vehicle control: 19.6%; Ven+: 3.4%; CD123-ENG T-cells:1.2 %; Ven+CD123-ENG T-cells: 0.3%; Ven[2-day-off]+CD123-ENG T-cells (p<0.01 Ven+ or CD123-ENG T-cells versus control; p<0.001 Ven+CD123-ENG or Ven[2-day-off]+CD123-ENG T cells versus CD123-ENG T cells, n=5). The enhanced anti-AML activity of combining venetoclax and CD123-ENG T-cells translated into a significant survival benefit in comparison to single treatment alone (Fig. 2). However, while Ven+CD123-ENG and Ven[2-day-off]+CD123-ENG T-cell treated mice had a survival advantage, they had reduced circulating numbers of human CD3+ T cells on day 8 post T-cells infusion compared to mice that received CD123-ENG T-cells, indicative of potential adverse effect of venetoclax on T-cell survival in vivo. Conclusion: Our data support a concept of combining pro-apoptotic targeted and immune therapy using venetoclax and CD123-ENG T-cells in AML. While it has been reported that venetoclax does not impair T-cell functionality, more in-depth analysis of the effect of Bcl-2 inhibition on T-cell function and survival appears warranted, as it could diminish survival not only of AML blasts but also of immune cells. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents & Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; TESSA Therapeutics: Other: research collaboration. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties. Andreeff:Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; 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.

Target Expression, Preclinical Activity and Mechanism of Action of EM801: A Novel First-in-Class Bcma T-Cell Bispecific Antibody for the Treatment of Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 117-117 ◽  
Author(s):  
Anja Seckinger ◽  
Jose Antonio Delgado ◽  
Laura Moreno ◽  
Brigitte Neuber ◽  
Anna Grab ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
T Cell Activation ◽  
Mechanism Of Action ◽  
Research Funding ◽  
Advisory Committees

Abstract Background. T-cell bispecific antibodies (TCBs) simultaneously binding CD3 on T-cells and individual tumor antigens, activate T-cells and destroy tumor antigen carrying cells. B-cell maturation antigen (BCMA), a surface antigen reported to be expressed on normal and malignant plasma cells (PCs), could represent a potentially promising target for TCBs in multiple myeloma (MM). The Aim of our study was to: i) assess expression of BCMA in normal and malignant PCs as well as cells of the bone marrow (BM) microenvironment by gene expression profiling and flow cytometry to validate it as potential clinical target for TCBs; ii) to evaluate activity of EM801 as member of a novel class of BCMA-TCBs in vitro on primary myeloma cells and in vivo in the H929-xenograft reconstituted NOG mouse model; and iii) to delineate its mechanism of action. Results. Expression. We investigated the expression of BCMA in CD138-purified PCs from BM aspirates obtained from 726 patients including MGUS (n=62), asymptomatic (n=59) and symptomatic MM (605), as well as different BM cellular subsets from healthy donors (n=10 PCs; plasmablasts, memory B-cells, T-cells, CD34+, CD14+, CD15+, n=5 each; n=8 mesenchymal stromal cells) using Affymetrix DNA-microarrays. BCMA expression was observed in malignant PC from 723/726 (99.5%) MGUS and MM patients, 10/10 normal PCs and 5/5 plasmablasts; gene expression of BCMA was undetectable in all other normal BM subsets. Using multiparameter flow cytometry, BCMA surface expression on malignant PCs was confirmed in 40/40 patients while being absent on normal BM cells. BCMA is thus a potential target in virtually all myeloma patients. Activity. In vitro, EM801 induced concentration dependent significant cell death in malignant plasma cells in BM-samples of 21/28 (75%) previously untreated and 8/10 (80%) relapsed/refractory MM patients in concentrations ranging from 10pM to 30nM. No or only minor unspecific toxicity on cells of the BM microenvironment was observed. In vivo efficacy of EM801 was studied in a subcutaneous H929 myeloma cell line xenograft model in NOG (NOD/Shi-scid/IL-2Rγnull) mice reconstituted with human PBMCs. Three doses of EM801, i.e. 0.026, 0.26 and 2.6 nM/kg, the same doses of a BCMAxCD3-(scFv)2 and two control groups were investigated (n=9 mice/group). Three weekly intravenous doses were given, starting on day 19 after tumor cell injection when tumor volumes were 293±135 mm3. On day 47, all mice from control groups had their tumors grown beyond 2000 mm3 and were euthanized for ethical reasons. In contrast, at 2.6 nM/kg (0.5 mg/kg) EM801 tumor regression was already observed after the second i.v. injection in 6/9 animals and the tumor regressed to 16±3 mm3 on day 47. BCMAxCD3-(scFv)2 bispecific antibody without Fc did not show any efficacy at all doses studied. Regarding the mechanism of action, we first demonstrated that EM801 effectively binds myeloma cells and T-cells with a strength of 1622±410 pN (5-10 fold of control) as measured by atomic force microscopy. Secondly, increasing concentrations (0.03-30nM) of EM801 led to progressive T-cell activation in primary BM samples, with significantly increased levels of CD69 (P<0.001), CD25 (P<0.001) and HLADR (P=0.001) expression in both CD4 and CD8 T-cells as compared to an unspecific TCB. Thirdly, EM801 induced significant secretion of interferon-γ (19-3000 pg/ml), granzyme B (68-2986 pg/ml), and perforin (145-3712 pg/ml) as measured by ELISA, together explaining the strong in vitro and in vivo activity of EM801. Conclusions. BCMA is selectively expressed at the RNA (723/726) and protein (40/40) levels on malignant PCs from virtually all MM patients, and thus represents a promising TCB-target. The novel BCMA-TCB EM801 was effective in vitro in 29/38 (76%) primary MM patients' BM samples at picomolar to low nanomolar concentrations, easily achievable in vivo in patients, as well as in the H929-xenograft reconstituted NOG mouse model at 0.5 mg/kg once a week. Neither in vitro (the BM microenvironment) nor in vivo the compound shows significant toxicity or side effects. EM801 confers cytotoxicity by effectively coupling T-cells with malignant PCs, inducing T-cell activation, secretion of interferon-γ, granzyme B and perforin, and thereby effectively killing malignant PCs. EM801 is thus a promising new compound for the treatment of multiple myeloma to be investigated in clinical phase I/II trials. Disclosures Seckinger: EngMab AG: Research Funding; Takeda: Other: Travel grant. Neuber:EngMab AG: Research Funding. Vu:EngMab AG: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Strein:BB Biotech AG: Membership on an entity's Board of Directors or advisory committees; Novimmune SA: Membership on an entity's Board of Directors or advisory committees; EngMab AG: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Hundemer:EngMab AG: Research Funding. San Miguel:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Janssen-Cilag: Honoraria; Millennium: Honoraria; Novartis: Honoraria; Sanofi-Aventis: Honoraria; Onyx: Honoraria. Hose:Takeda: Other: Travel grant; EngMab AG: Research Funding. Paiva:Celgene: Consultancy; Janssen: Consultancy; Binding Site: Consultancy; BD Bioscience: Consultancy; EngMab AG: Research Funding; Onyx: Consultancy; Millenium: Consultancy; Sanofi: Consultancy.

scholarly journals Predictors of T Cell Expansion and Clinical Responses Following B-Cell Maturation Antigen-Specific Chimeric Antigen Receptor T Cell Therapy (CART-BCMA) for Relapsed/Refractory Multiple Myeloma (MM)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1974-1974 ◽  
Author(s):  
Adam D. Cohen ◽  
J. Joseph Melenhorst ◽  
Alfred L. Garfall ◽  
Simon F Lacey ◽  
Megan Davis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Advisory Committees ◽  
Car T ◽  
Equity Ownership

Abstract Background: Relapsed/refractory (rel/ref) MM is associated with progressive immune dysfunction, including reversal of CD4:CD8 T cell ratio and acquisition of terminally-differentiated T cell phenotypes. BCMA-directed CAR T cells have promising activity in MM, but the factors that predict for robust in vivo expansion and responses are not known. In a phase 1 study of CART-BCMA (autologous T cells expressing a human BCMA-specific CAR with CD3ζ/4-1BB signaling domains) in refractory MM patients (median 7 priors, 96% high-risk cytogenetics), we observed partial response (PR) or better in 12/25 (47%) (Cohen et al, ASH 2017, #505). Recently, we demonstrated in CLL pts receiving CD19-directed CAR T cells that certain T cell phenotypes prior to generation of the CAR T product were associated with improved in vivo expansion and clinical outcomes (Fraietta et al, Nat Med 2018). We thus sought to identify pre-treatment clinical or immunological features associated with CART-BCMA expansion and/or response. Methods: Three cohorts were enrolled: 1) 1-5 x 108 CART cells alone; 2) cyclophosphamide (Cy) 1.5 g/m2 + 1-5 x 107 CART cells; and 3) Cy 1.5 g/m2 + 1-5 x 108 CART cells. Phenotypic analysis of peripheral blood (PB) and bone marrow (BM) mononuclear cells, frozen leukapheresis aliquots, and phenotype and in vitro kinetics of CART-BCMA growth during manufacturing were performed by flow cytometry. CART-BCMA in vivo expansion was assessed by flow cytometry and qPCR. Responses were assessed by IMWG criteria. Results: Responses (≥PR) were seen in 4/9 pts (44%, 1 sCR, 2 VPGR, 1 PR) in cohort 1; 1/5 (20%, 1 PR) in cohort 2; and 7/11 (64%, 1 CR, 3 VGPR, 3 PR) in cohort 3. As of 7/9/18, 3/25 (12%) remain progression-free at 11, 14, and 32 months post-infusions. As previously described, responses were associated with both peak in vivo CART-BCMA expansion (p=0.002) as well as expansion over first month post-infusion (AUC-28, p=0.002). No baseline clinical or MM-related characteristic was significantly associated with expansion or response, including age, isotype, time from diagnosis, # prior therapies, being quad- or penta-refractory, presence of del 17p or TP53 mutation, serum hemoglobin, BM MM cell percentage, MM cell BCMA intensity, or soluble BCMA concentration. Treatment regimen given before leukapheresis or CART-BCMA infusions also had no predictive value. We did find, however, that higher CD4:CD8 T cell ratios within the leukapheresis product were associated with greater in vivo CART-BCMA expansion (Spearman's r=0.56, p=0.005) and clinical response (PR or better; p=0.014, Mann-Whitney). In addition, and similar to our CLL data, we found that a higher frequency of CD8 T cells within the leukapheresis product with an "early-memory" phenotype of CD45RO-CD27+ was also associated with improved expansion (Spearman's r=0.48, p=0.018) and response (p=0.047); Analysis of manufacturing data confirmed that higher CD4:CD8 ratio at culture start was associated with greater expansion (r=0.41, p=0.044) and, to a lesser degree, responses (p=0.074), whereas absolute T cell numbers or CD4:CD8 ratio in final CART-BCMA product was not (p=NS). In vitro expansion during manufacturing did associate with in vivo expansion (r=0.48, p=0.017), but was not directly predictive of response. At the time of CART-BCMA infusion, the frequency of total T cells, CD8+ T cells, NK cells, B cells, and CD3+CD56+ cells within the PB or BM was not associated with subsequent CART-BCMA expansion or clinical response; higher PB and BM CD4:CD8 ratio pre-infusion correlated with expansion (r=0.58, p=0.004 and r=0.64, p=0.003, respectively), but not with response. Conclusions: In this study, we found that CART-BCMA expansion and responses in heavily-pretreated MM patients were not associated with tumor burden or other clinical characteristics, but did correlate with certain immunological features prior to T cell collection and manufacturing, namely preservation of normal CD4:CD8 ratio and increased frequency of CD8 T cells with a CD45RO-CD27+ phenotype. This suggests that patients with less dysregulated immune systems may generate more effective CAR T cell products in MM, and has implications for optimizing patient selection, timing of T cell collection, and manufacturing techniques to try to overcome these limitations in MM patients. Disclosures Cohen: Celgene: Consultancy; Novartis: Research Funding; Oncopeptides: Consultancy; Janssen: Consultancy; Poseida Therapeutics, Inc.: Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; Kite Pharma: Consultancy; GlaxoSmithKline: Consultancy, Research Funding; Seattle Genetics: Consultancy. Melenhorst:Parker Institute for Cancer Immunotherapy: Research Funding; novartis: Patents & Royalties, Research Funding; Casi Pharmaceuticals: Consultancy; Incyte: Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy. Garfall:Amgen: Research Funding; Kite Pharma: Consultancy; Bioinvent: Research Funding; Novartis: Research Funding. Lacey:Novartis Pharmaceuticals Corporation: Patents & Royalties; Parker Foundation: Research Funding; Tmunity: Research Funding; Novartis Pharmaceuticals Corporation: Research Funding. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Vogl:Karyopharm Therapeutics: Consultancy. Pruteanu:Novartis: Employment. Plesa:Novartis: Research Funding. Young:Novartis: Patents & Royalties, Research Funding. Levine:Novartis: Consultancy, Patents & Royalties, Research Funding; CRC Oncology: Consultancy; Incysus: Consultancy; Tmunity Therapeutics: Equity Ownership, Research Funding; Brammer Bio: Consultancy; Cure Genetics: Consultancy. June:Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Stadtmauer:Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AbbVie, Inc: Research Funding; Janssen: Consultancy. Milone:Novartis: Patents & Royalties.

scholarly journals Pharmacologic Inhibition of SUMO-Activating Enzyme Potentiates Interferon Response and T Cell-Mediated Anti-Tumor Immunity in Chronic Lymphocytic Leukemia (CLL) and Lymphoma Models

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3719-3719
Author(s):  
Vi Lam ◽  
Xiaoguang Wang ◽  
Scott R Best ◽  
Nur Bruss ◽  
Tingting Liu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Foxp3 Expression ◽  
Tcr Signaling ◽  
Advisory Committees

Abstract Introduction: CLL is characterized by deficient immunity which clinically manifests as increased predisposition towards malignancies and infectious complications. T-cells from patients with CLL exhibit a skewed repertoire with predominance of Tregs as well as impaired immune synapse formation and cytotoxic function. Small ubiquitin-like modifier (SUMO) family proteins regulate a variety of cellular processes, including nuclear trafficking, gene transcription and cell cycle progression, via post-translational modification of target proteins. Sumoylation regulates NFκB signaling, IFN response and NFAT activation, processes indispensable in immune cell activation. Despite this, the role of sumoylation in T cell biology in context of cancer is not known. TAK-981 is a small molecule inhibitor of the SUMO-activating enzyme (SAE) that forms a covalent adduct with an activated SUMO protein, thereby preventing its transfer to the SUMO-conjugating enzyme (Ubc9). Here, we investigated the immunomodulatory effects of TAK-981 in CLL. Methods: T cells from patients with CLL were purified using Dynabeads. For polarization assays, FACS-sorted naïve CD4+ T cells were cultured for 7 days in control or differentiation media. For gene expression profiling (GEP; Clariom S), RNA was harvested after 3 and 24 hours of TCR engagement from FACS-sorted naïve CD4+ T cells. For in vivo immunization experiments, CD4+KJ1-26+ cells were inoculated IV into BALB/cJ mice. Mice received 100 µg IV ovalbumin ± R848 followed by TAK-981 7.5 mg/kg or vehicle control IV twice weekly for 10 days prior to spleen collection. Both recipient and transplanted splenocytes were analyzed. For analysis of tumor-infiltrating lymphocytes (TILs), BALB/c mice were injected with 1x10 6 A20 lymphoma cells and treated as above. TAK-981 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA). Results: T cells from patients with CLL demonstrated high baseline protein sumoylation that slightly increased following TCR engagement (αCD3/CD28). Treatment with TAK-981 significantly downregulated SUMO1 and SUMO2/3-modified protein levels yet did not disrupt early TCR signaling as evidenced by sustained ZAP70, p65/NFκB and NFAT activation detected by immunoblotting, immunocytochemistry and GEP. Treatment with TAK-981 resulted in dose-dependent upregulation of the early activation marker CD69 in CD4 + T cells following 72 and 96 hours of TCR stimulation vs. control. Meanwhile, expression of CD25, HLA-DR and CD40L was delayed in the presence of TAK-981. Interestingly, CD38, an IFN response target, was induced two-fold in TAK-981-treated cells after 24 hours and persisted at high levels at subsequent timepoints. T cell proliferation was reduced in the presence of high (1 μM) but not low/intermediate concentrations of TAK-981, accompanied by reduced S phase entry and decreased synthesis of IL-2. However, T cells did not undergo apoptosis under those conditions. Targeting SAE in either control or Th1/Treg polarizing conditions facilitated an increase in IFNγ and loss of FoxP3 expression (accompanied by decreased IL-2/STAT5), suggesting a shift towards Th1 and away from Treg phenotype, respectively. GEP (Reactome, GSEA) confirmed a dramatically upregulated IFN response in TAK-981-treated CD4 + naïve T cells. Furthermore, targeting SAE enhanced degranulation (CD107a), IFNγ and perforin secretion in cytotoxic CD8+ T cells and potentiated T cell cytotoxicity in allogeneic assays with lymphoma cells (OCI-LY3, U2932) as targets. Consistent with our in vitro data, OVA-stimulated transplanted transgenic KJ1-26+ splenocytes, as well as total CD4+ T cells from recipient mice treated with TAK-981 in vivo exhibited a significant reduction in expression of FoxP3 and an increased production of IFNγ (Figure 1). In the A20 syngeneic model, treatment with TAK-981 similarly downregulated FoxP3 expression in CD4+ TILs and induced IFNγ secretion in CD8+ TILs. Conclusion. Using a combination of in vitro and in vivo experiments, we demonstrate that pharmacologic targeting of sumoylation with TAK-981 does not impair proximal TCR signaling in T cells obtained from patients with CLL, but leads to rebalancing toward healthy immune T cell subsets via induction of IFN response and downmodulation of Tregs. These data provide a strong rationale for continued investigation of TAK-981 in CLL and lymphoid malignancies. Figure 1 Figure 1. Disclosures Siddiqi: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; TG Therapeutics: Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncternal: Research Funding; Janssen: Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Danilov: SecuraBio: Research Funding; Bayer Oncology: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy, Research Funding; Rigel Pharm: Honoraria; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding.

scholarly journals Dual Inhibition of PI3K-δ and PI3K-γ By Duvelisib Eliminates CLL B Cells, Impairs CLL-Supporting Cells, and Overcomes Ibrutinib Resistance in a Patient-Derived Xenograft Model

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4420-4420 ◽  
Author(s):  
Shih-Shih Chen ◽  
Jeffery Lorne Kutok ◽  
Gerardo Ferrer ◽  
Priyadarshini Ravichandran ◽  
Michael Ibrahim ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Supporting Cells ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Ibrutinib Resistance

Abstract Novel agents targeting the B-cell receptor signaling pathway, such as ibrutinib and idelalisib, are effective in patients with chronic lymphocytic leukemia (CLL), but complete responses are infrequent and residual disease often remains. Patients may discontinue ibrutinib because of unacceptable adverse events or if they develop ibrutinib resistance due to Bruton tyrosine kinase (BTK) or phospholipase C gamma 2 (PLCG2) mutations or other less-well-defined mechanisms. Therefore, alternative therapies that can overcome ibrutinib intolerance and resistance are needed. Duvelisib (DUV) is an oral dual phosphatidylinositol 3-kinase (PI3K)-δ and -γ inhibitor with activity in patients with relapsed/refractory CLL and a manageable safety profile. Preclinical studies point to distinct roles of PI3K-δ and -γ in CLL biology, suggesting that dual isoform inhibition may enhance efficacy by targeting both CLL and CLL-supporting cells. Importantly, DUV potently inhibits PI3K-δ and -γ in human peripheral blood mononuclear cells and CLL cells. Here, we further characterize the distinct functions of PI3K-δ and -γ in CLL and examine the mechanisms of action of DUV in vitro and in a patient-derived xenograft (PDX) model, highlighting the ability of DUV to overcome ibrutinib resistance. We first assessed the in vivo efficacy of PI3K-δ and/or PI3K-γ inhibition on CLL B cells using a PDX mouse model in which activated patient-derived T cells and CLL B cells were injected into alymphoid NOD-scid IL-2Rγnull (NSG) mice. Cells from 4 patients were transferred and allowed to expand for 2 weeks, after which mice were treated with DUV, a PI3K-δ inhibitor, or a PI3K-γ inhibitor for 3 weeks. DUV and the PI3K-δ inhibitor, but not the PI3K-γ inhibitor, significantly decreased the number of CLL B cells in the spleens of mice. In another set of experiments, combination treatment with the PI3K-δ and -γ inhibitors more potently impaired CLL B-cell survival than the PI3K-δ inhibitor alone. Next, the effects of PI3K-δ or -γ inhibition on CLL-supporting cells, ie, autologous T cells and murine macrophages, were examined in the PDX model using the same 4 patient samples. Significant decreases in both patient-derived T cells and murine macrophages were observed in the spleens of mice treated with DUV or the PI3K-γ inhibitor but not with the PI3K-δ inhibitor. Therefore, the function of PI3K-δ and -γ in macrophages and macrophage-supported CLL cell survival were examined in vitro. Murine bone marrow-derived macrophages were polarized via interleukin 4 and macrophage colony stimulating factor in the presence of DUV, a PI3K-δ inhibitor, or a PI3K-γ inhibitor. Both DUV and the PI3K-γ inhibitor impaired M2 polarization, assessed by arginase 1 (ARG1) mRNA expression. Culture with M2 macrophages increased CLL B-cell viability, and the addition of DUV inhibited this survival-promoting activity more than the PI3K-δ or -γ inhibitors alone. To assess whether DUV could inhibit CLL B cells from ibrutinib-unresponsive patients, activated T cells and CLL B cells from 2 patients who progressed on ibrutinib (1 with a BTK C481S mutation and 1 without a BTK mutation) were transferred into NSG mice and allowed to expand for 2 weeks, and then mice were treated with DUV or ibrutinib for 3 weeks. For both patient samples, there was a > 10-fold reduction in the number of CLL B cells recovered from the spleens of mice treated with DUV, as well as a significant reduction in the percentage of proliferating CLL B cells. In contrast, ibrutinib did not have significant impact on CLL B-cell numbers or proliferation in the spleen. In conclusion, DUV inhibits the in vivo survival and proliferation of leukemic B cells from CLL patients, including those who have progressed on ibrutinib. Dual PI3K-δ and -γ inhibition is more effective at inhibiting CLL B cells in vivo than PI3K-δ inhibition alone. Moreover, PI3K-γ inhibition shifts macrophage polarization away from a CLL-supportive M2 phenotype. Thus, DUV exerts inhibitory effects on CLL B cells and on CLL-supporting T and myeloid cells. Overall, these findings elucidate the non-redundant roles of PI3K-δ and -γ in CLL and demonstrate the potent antitumor activity of dual PI3K isoform inhibition by DUV in ibrutinib-resistant patient CLL cells in vivo. Further investigation of DUV as a therapeutic option for patients who are refractory to or intolerant of ibrutinib or other BTK inhibitors is ongoing in a phase 2 clinical trial (BRIO; NCT03370185). Disclosures Chen: Janssen: Research Funding; ArgenX: Research Funding; Beigene: Research Funding; Pharmacyclics: Research Funding; Verastem: Research Funding. Kutok:Infinity Pharmaceuticals: Employment, Equity Ownership. Barrientos:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics/AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Weaver:Verastem Oncology: Employment, Other: Stockholder; Agios Pharmaceuticals: Employment; Femto Dx: Equity Ownership. Pachter:Verastem: Employment, Other: Stockholder. Rai:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees. Chiorazzi:AR Pharma: Equity Ownership; Janssen, Inc: Consultancy.

scholarly journals Nedd8-Activating Enzyme Inhibition Enhances Anti-Tumor Immunity and PD1 Blockade in In Vivo lymphoma Models

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2414-2414
Author(s):  
Xiaoguang Wang ◽  
Vi Lam ◽  
Dan Vuong ◽  
Tingting Liu ◽  
Olga Danilova ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Tumor Immunity ◽  
Research Funding ◽  
Checkpoint Inhibitors ◽  
Dependent Manner ◽  
Advisory Committees

Abstract Introduction: Immune checkpoint inhibitors have limited single agent activity in B-cell non-Hodgkin lymphoma (NHL). Hence, it is important to develop strategies which will thwart immune evasion in this disease. Neddylation is a sequential enzyme-based process which ultimately regulates protein turnover. In the initial step, NAE activates NEDD8 in an ATP-dependent reaction in which a high-energy thioester bond is formed between NEDD8 and the catalytic cysteine of NAE. Active NEDD8 is then transferred to the NEDD8-specific E2 conjugating enzyme (UBE2M) and is conjugated to cullin proteins which are part of the Cullin-RING E3 ubiquitin ligases (CRL). Pevonedistat (pevo) forms a covalent adduct with NEDD8, thereby inhibiting NAE and thus reduces CRL activity and diminishes ubiquitination and proteasomal degradation of CRL substrates (IκB, HIF-1α, etc). We have recently reported that neddylation regulates T cell activation and polarization (Best et al, Leukemia 2021). Here we investigate how pharmacologic targeting of neddylation modulates anti-tumor immunity using NHL models. Methods: Peripheral blood mononuclear cells were isolated from patients with NHL and T cells were purified using Dynabeads. A20 cells were transplanted into flanks of syngeneic BALB/c mice. When tumors reached 100 mm 3, mice were randomized into groups and treated with pevo 60 mg/kg subcutaneously daily for 10 days or vehicle control. Once moribund, mice were sacrificed, tumors were processed into single-cell suspension and analyzed by flow cytometry. Pevo was provided by Takeda Development Center Americas Inc. (Cambridge, MA). Results: Primary patient-derived CD3/28-stimulated CD3 + T cells exhibited upregulation of TNFα and IFNγ in vitro in the presence of pevo. Concurrently, we observed increased expression of PD-1 and CTLA-4. Pre-treatment of T cells with pevo enhanced killing of NHL cell lines (JeKo-1, Mino, Maver-1 and VAL) in allogeneic cytotoxicity assays. Expectedly, treatment with pevo resulted in increased expression of HIF-1α in TCR-stimulated T cells. shRNA-mediated knockdown of HIF-1α abrogated the pevo effect, suggesting that NAE inhibition modulates T cell function in HIF-1α-dependent manner. While A20 cells showed resistance in vitro, treatment with pevo delayed lymphoma progression in A20 mice in vivo (Fig 1A). This was accompanied by an increase of tumor-infiltrating lymphocytes (TILs; Fig 1B). CD8 + TILs from pevo-treated mice exhibited activated phenotype as manifested by increased secretion of IFNγ (Fig 1C). Meanwhile, expression of the exhaustion molecules CTLA-4 and PD-1 by CD4/CD8 + TILs remained unchanged. To further investigate the role of T-cell immunity in this setting, we employed 1) CD8 depletion by pre-treatment with 12.5 mg/kg anti-CD8 antibody (IV); or 2) CRISPR/Cas9-mediated knockout of β2-microglobulin (MHC class I protein) in A20 cells. Either approach led to a partial decrease of pevo efficacy in vivo compared with respective controls. To exclude tumor-intrinsic effect of NAE inhibition, we knocked down UBE2M in A20 cells. Loss of UBE2M had no effect on growth of control tumors, or pevo therapeutic effect, implying that the anti-tumor efficacy of NAEi was T cell-mediated in this model. Since pevo modulates PD-1 on human T cells, we explored its effect on PD-L1 expression. Treatment with pevo upregulated PD-L1 expression in A20 cells in a MYC-dependent manner. Hence, we explored a combination of pevo and αPD-1 blockade in A20 model. Combination treatment significantly increased the CD4 + and CD8 + TILs. A decrease in tumor growth was significantly more pronounced than with either drug alone (Fig 1A). The combination benefit was fully reversed by loss of B2M, again highlighting the importance of immune mechanism . We observed expansion of IL-2, IL-4 and IL-17-secreting CD4+ TILs following the combined treatment, compared with either drug alone. In addition, CD4+ and CD8+ TILs derived from these mice secreted high levels of IFNγ (Fig. 1C). Conclusions: NAE inhibition enhanced T cell-mediated cytotoxicity in vitro. Treatment with pevo promoted activation of TILs and restricted tumor growth in an A20 mouse lymphoma model. Pevo-treated tumors were sensitized to αPD-1 . Thus, targeting NAE enhances anti-tumor immunity. Our data provide a strong rationale for future studies of pevo in combination with immune checkpoint inhibitors in lymphoma and other tumors. Figure 1 Figure 1. Disclosures Siddiqi: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; TG Therapeutics: Research Funding; Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncternal: Research Funding. Berger: Takeda Development Center Americas, Inc.: Current Employment. Danilov: Bayer Oncology: Consultancy, Honoraria, Research Funding; SecuraBio: Research Funding; Genentech: Consultancy, Honoraria, Research Funding; TG Therapeutics: Consultancy, Research Funding; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Research Funding; Takeda Oncology: Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Rigel Pharm: Honoraria.

Role Of B Cell Tolerance In PF4/Heparin Antibody Production

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2396-2396
Author(s):  
Yongwei Zheng ◽  
Alexander W Wang ◽  
Mei Yu ◽  
Anand Padmanabhan ◽  
Benjamin E Tourdot ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Cell Tolerance ◽  
Inflammatory Stimulus ◽  
Wild Type ◽  
B Cell Tolerance ◽  
Advisory Committees

Abstract Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of heparin, platelet factor 4 (PF4) and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. However, heparin, a glycosoaminoglycan, and PF4 are normal body constituents and it is as yet unclear what triggers the initial induction of pathogenic antibodies. Here we described detection of B cells among peripheral blood mononuclear cells (PBMCs) from each of 9 healthy adults that produced PF4/heparin-specific IgM antibodies following in vitro stimulation with ubiquitous pro-inflammatory molecules containing unmethylated CpG dinucleotides derived from bacterial and viral DNA. PF4/heparin-specific IgM-generating B cells were present at a frequency of at least 0.03 to 1 per thousand B cells present in the PBMC population. Similarly, splenic B cells isolated from unmanipulated wild-type mice consistently produced PF4/heparin-reactive antibodies following in vitro stimulation with CpG. In addition, wild-type mice produced PF4/heparin-reactive antibodies upon in vivo challenge with CpG whereas unchallenged wild-type mice did not. These findings demonstrate that both humans and mice possess pre-existing, inactive and tolerant PF4/heparin-specific B cells. We suggest that tolerance can be broken by a strong inflammatory stimulus, leading to activation of these B cells and production of antibodies that recognize PF4/heparin in vitro and in vivo. Consistent with this concept, mice lacking protein kinase Cd (PKCd), a signaling molecule of the B-cell survival factor BAFF (B-cell activation factor), that are known to have breakdown of B-cell tolerance to self-antigens, spontaneously produced anti-PF4/heparin antibodies in the absence of an inflammatory stimulus. Taken together, these findings demonstrate that breakdown of tolerance can lead to PF4/heparin-specific antibody production and that B-cell tolerance plays an important role in HIT pathogenesis. Disclosures: White II: Bayer: Membership on an entity’s Board of Directors or advisory committees; CSL-Behring: Membership on an entity’s Board of Directors or advisory committees; NIH: Membership on an entity’s Board of Directors or advisory committees; Asklepios: Membership on an entity’s Board of Directors or advisory committees; Wyeth: Membership on an entity’s Board of Directors or advisory committees; Entegrion: Membership on an entity’s Board of Directors or advisory committees; Biogen: Membership on an entity’s Board of Directors or advisory committees; Baxter: Membership on an entity’s Board of Directors or advisory committees.

Phase I/Ib Study of the Novel Immunotoxin MT-3724 in Relapsed/Refractory Non-Hodgkin's B-Cell Lymphoma (NHL)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5112-5112
Author(s):  
Paul A Hamlin ◽  
Catherine S. Diefenbach ◽  
David J. Valacer ◽  
Jack Higgins ◽  
Michelle A. Fanale
Keyword(s):  
B Cells ◽  
Phase I ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
B Cell Lymphoma ◽  
Advisory Committees ◽  
Dose Cohort ◽  
Mixed Response

Abstract Background CD20 is selectively expressed on the surface of early pre-B-cells, remains throughout B-cell development, and is then lost from plasma cells. Because CD20 is present on the majority of B-cell lymphomas, anti-CD20 monoclonal antibody (MAb) therapy is widely employed in the treatment of NHL. However a majority of NHL patients eventually become refractory to CD20 MAb(s). Resistance mechanisms may include increased MAb catabolism, initial or post treatment selection of low CD20 expressing tumor cells, trogocytosis of surface CD20, failure of MAb effector mechanisms and/or impaired patient immune cell function. MT-3724 is a recombinant fusion protein consisting of a CD20 binding variable fragment (scFv) fused to the enzymatically active Shiga-like toxin-I A1 subunit (SLT-I A1). SLT-I A1 is an N-glycosidase that catalytically inactivates 60S ribosomal subunits causing inhibition of protein synthesis. Upon its scFv binding to cell surface CD20 in vitro, SLT-I A1 forces MT-3724 internalization which then routes in a predictable fashion to the cytosol and irreversibly inactivates the cell ribosomes triggering cell death. MT-3724 has been shown to specifically bind and kill CD20+ malignant human B-cells in vitro and non-human primate (NHP) B-cells in vivo. MT-3724 was tested for safety in healthy NHPs: 6 intravenous (IV) doses of MT-3724 were given over 12 days at doses of 50, 150, and 450 mcg/kg. There were no deaths or effects on serum chemistries in the NHP studies. The major observed toxicity (inappetence) resolved within 48 hours of last dose. There was a significant, dose-dependent NHP B-cell depletion by Day 3 at all doses. Given the preclinical activity and mechanism of action, a Phase I/Ib study of MT-3724 was initiated in NHL. Methods MT-3724 is being tested for safety and tolerability in a first-in-human, open label, ascending dose study (3 + 3 design) in sequential cohorts of 5, 10, 20 and 50 mcg/kg/dose. Eligible subjects who previously responded to a CD20 MAb containing therapy followed by relapse/recurrence of NHL receive 6 doses by 2 hour IV infusions over the first 12 days of a 28 day cycle (first cycle). With continued safety, tolerability and lack of tumor progression, subjects may receive up to 4 additional 6-dose cycles (21 days) with tumor assessments after cycles 2, 4 and 5. Dose escalation is based on < 33% dose limiting toxicities (DLTs) observed during the first 28 day cycle. Results Three NHL subjects (2 transformed DLBCL, 1 FL) have completed at least one cycle in the 5 mcg/kg/dose cohort with no protocol DLTs or infusion related reactions and are evaluable for safety. Non-DLTs included grade (Gr) 2-3 transient hyperglycemic episodes related to pre-infusion corticosteroid therapy (n=1); transient Gr 4 neutropenia, possibly related to MT-3724 during cycle 1, week 4 (n=1); Gr 4 hypercalcemia and acute kidney injury with Gr 3 hypophosphatemia during cycle 1, week 4 due to leukemic disease progression (n=1). Subject 1 completed 5 cycles of therapy, with a partial response achieved post cycle 2 sustained through cycle 5; Subject 3 had a mixed response (both subjects had transformed DLBCL). Three subjects have now initiated treatment in the 10 mcg/kg/dose cohort with updated data to be presented at the meeting. Conclusions MT-3724 at 5 mcg/kg/dose has been safely administered for up to 5 cycles in this first-in-human study in relapsed/refractory NHL subjects. Treatment with the 10 mcg/kg cohort has commenced with continuing dose ascension planned. There is early evidence of clinical activity. Disclosures Diefenbach: Gilead: Equity Ownership, Research Funding, Speakers Bureau; Jannsen Oncology: Consultancy; Idera: Consultancy; Immunogen: Consultancy; Incyte: Research Funding; Genentech: Research Funding; Celgene: Consultancy; Molecular Templates: Research Funding; Seattle Genetics: Consultancy, Honoraria, Research Funding. Valacer:Molecular Templates: Employment. Higgins:Molecular Templates: Employment. Fanale:Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Research Funding; Infinity: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Honoraria, Research Funding; Genentech: Research Funding; Medimmune: Research Funding; Novartis: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Molecular Templates: Research Funding; ADC Therapeutics: Research Funding; Onyx: Research Funding; Gilead: Research Funding.

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 ADG152, a Novel CD20xCD3 T Cell Engager Prodrug with Enhanced Therapeutic Index, Demonstrates Strong Anti-Tumor Activity with Improved Safety

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1204-1204
Author(s):  
Bin Cai ◽  
Aaron N Nguyen ◽  
Songmao Zheng ◽  
Jianfeng Shi ◽  
Guizhong Liu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cytokine Release ◽  
Publicly Traded ◽  
Advisory Committees ◽  
The Past

Abstract Recent clinical data illustrate the effectiveness of CD20xCD3 T cell engagers (TCEs) that redirect the patient's endogenous T cells to eliminate CD20-positive tumor cells. While several of these products have demonstrated promising clinical activities in B-cell malignancies, their potential therapeutic utility is limited by cytokine release syndrome (CRS), even after strategies such as step-up dosing are implemented. ADG152 is a novel CD20xCD3 TCE prodrug engineered using Adagene's SAFEbody technology to minimize or eliminate CRS and on-target/off-tumor toxicities. The anti-CD20 arm of ADG152 has been engineered for enhanced binding to CD20 compared to other clinical stage or approved antibodies, while its anti-CD3 arm has modulated affinity for CD3 and is also masked by a conditionally activable peptide. In normal tissues and in circulation, the masking moiety on the anti-CD3 arm can function to block the binding of ADG152 to T cells; however, in an activable condition such as the tumor microenvironment where protease activity has been reported to be elevated, the masked antibody can be activated, enabling the activated ADG152 to simultaneously engage T cells and neighboring CD20-expressing tumor cells. In vitro studies showed that ADG152 has enhanced binding to human B cells and CD20-positive Raji tumor cells compared with the benchmark CD20xCD3 TCE plamotamab. On the other hand, ADG152 has significantly reduced binding to the human CD3 δ/ε protein dimer and no binding to human CD3+, CD4+, and CD8+ T cells isolated from PBMCs of normal human donors. Consistent with these results, ADG152 shows significantly decreased ability (more than 100-fold) compared with the benchmark and the unmasked parental molecule to activate CD8+ T cells and to induce T cell-mediated killing in the presence of tumor cells in vitro. ADG152 demonstrated strong anti-tumor effects in vivo. In a human PBMC-engrafted Raji xenograft mouse tumor model, dosing with ADG152 resulted in almost complete tumor growth inhibition at 1.5 mg/kg. In exploratory toxicology studies in cynomolgus monkeys, ADG152 resulted in significantly less cytokine release in monkey blood compared with benchmark, giving ~100-fold safety margin for ADG152 for cytokine induction (Figure). In addition, ADG152 was as effective as the benchmark at inducing B cell depletion from peripheral blood of cynomolgus monkeys. In summary, the preclinical characterization of ADG152 demonstrates that our SAFEbody platform can be used to engineer safe and potent bispecific T cell engagers with increased therapeutic index by allowing for strong anti-tumor activities in mice at doses with minimal cytokine release in monkeys, thereby supporting its advancement to clinical development either as a single agent or in combination with other therapies for the treatment of CD20-expressing B cell malignancies. Figure 1 Figure 1. Disclosures Cai: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Nguyen: Sparcbio, LLC: Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Zheng: Janssen Pharmaceuticals: Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Shi: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Liu: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Li: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Du: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Frankel: Cytovia Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company; Adagene Inc.: Consultancy, Current equity holder in publicly-traded company; Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; IMV: Consultancy; Precision Biosciences: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Sutro: Membership on an entity's Board of Directors or advisory committees; Immunai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Minerva Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Myeloid Therapeutics: Consultancy; RAPT Therapeutics: Consultancy; Syros: Consultancy. Luo: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Xu: Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company.

Sign in / Sign up

Export Citation Format

Share Document