Introduction: TG-1801 is a novel, bispecific anti-CD47 and anti-CD19 fully human IgG1 antibody that targets CD47 selectively on CD19+ B-cells, sparing red blood cells or platelets and blocking the ...CD47-SIRPα macrophage checkpoint on mature B cells. TG-1801 is currently in clinical trial as a single agent or in combination with ublituximab, a glyco-engineered CD20 antibody, in B-cell non-Hodgkin lymphoma (B-NHL). The doublet therapy of ublituximab with the dual PI3Kδ/CK1e inhibitor umbralisib (“U2” regimen), provides a non-chemotherapy backbone regimen on which several novel multidrug combinations are being explored clinically. Here we explored in vitro and in vivo potential synergies between TG-1801 and ublituximab, umbralisib, and the U2 combination, in preclinical models of B-NHL.
Methods: A panel of n=12 human B-cell lymphoma cell lines and primary samples were cultured in vitro in the presence of bone marrow-derived stromal cells (BMSCs), M2-polarized primary macrophages, and primary circulating PBMCs as a source of effector cells. Cell response to TG-1801 +/- U2 treatments was analyzed by proliferation assay, western blot, transcriptomic analysis (qPCR array and RNA sequencing followed by gene set enrichment analysis) and quantification of antibody-dependent cell death (ADCC) and antibody-dependent cell phagocytosis (ADCP). In vivo, drug efficacy was determined in a Raji xenograft model, by dosing tumor-bearing mice for 17 days with ublituximab (5mg/kg, qw) +/- umbralisib (150mg/kg, bid), the combination of both and/or TG-1801 (5mg/kg, qw).
Results: Here we show on a panel of lymphoma cells lines that the number of receptors per cell and the ratio CD47/CD19 do not impact TG-1801-mediated ADCC or phagocytosis (ADCP). In addition, we show that TG-1801 potentiated ublituximab-mediated ADCC and ADCP and exhibited a similar additive effect when added to U2 combination. In vivo, ublituximab alone displayed a tumor growth inhibition (TGI) of 88%, with 3/8 mice harboring a barely palpable tumor, while the umbralisib alone treatment arm showed a TGI of 50%, with 2/8 mice lacking detectable tumors. TG-1801 exhibited a 76% TGI with 1/8 tumor free-mouse. Most importantly, the combination of TG-1801 with umbralisib alone, ublituximab alone, and U2 achieved TGI of 85%, 93% and 93% respectively, with more tumor-free mice 35 days after the last dose in these three groups. Interestingly, this superior anti-tumor effect of the different TG-1801 combinations was associated with a higher infiltration of mouse macrophages within the tumors as assessed by F4/80 IHC labeling. RNA-seq analysis of the Raji xenografts and of n=4 representative in vitro B-NHL co-cultures treated with TG-1801, U2 or the triple combination uncovered the upregulation of the G-protein coupled receptor EBI2/GRP183 as a common event associated with the synergistic antitumor effects of TG-1801 and U2 in vitro and in vivo. A critical role of EBI2 in the regulation of macrophage activity, B cell migration and in the promotion of a pro-inflammatory phenotype was demonstrated upon exposure of the co-cultures with the EBI2 small molecule inhibitor NIBR189, which impaired the U2/TG-1801-evoked ADCP, B-cell cytoskeleton remodeling and inflammatory cytokine production.
Conclusion: The data presented here set the preclinical rationale and support a combination strategy of the novel CD47-CD19 bispecific antibody TG-1801 in B-NHL with other B-cell targeted mechanisms, including umbralisib and ublituximab.
Normant: TG Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Miskin: TG Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Roue: TG Therapeutics, Inc.: Research Funding.
PI3K inhibitors (PI3Ki) are approved for relapsed chronic lymphocytic leukemia (CLL). Although patients may show an initial response to these therapies, development of treatment intolerance or ...resistance remain clinical challenges. To overcome these, prediction of individual treatment responses based on actionable biomarkers is needed. Here, we characterized the activity and cellular effects of 10 PI3Ki and investigated whether functional analyses can identify treatment vulnerabilities in PI3Ki-refractory/intolerant CLL and stratify responders to PI3Ki.
Peripheral blood mononuclear cell samples (n = 51 in total) from treatment-naïve and PI3Ki-treated patients with CLL were studied. Cells were profiled against 10 PI3Ki and the Bcl-2 antagonist venetoclax. Cell signaling and immune phenotypes were analyzed by flow cytometry. Cell viability was monitored by detection of cleaved caspase-3 and the CellTiter-Glo assay.
pan-PI3Kis were most effective at inhibiting PI3K signaling and cell viability, and showed activity in CLL cells from both treatment-naïve and idelalisib-refractory/intolerant patients. CLL cells from idelalisib-refractory/intolerant patients showed overall reduced protein phosphorylation levels. The pan-PI3Ki copanlisib, but not the p110δ inhibitor idelalisib, inhibited PI3K signaling in CD4+ and CD8+ T cells in addition to CD19+ B cells, but did not significantly affect T-cell numbers. Combination treatment with a PI3Ki and venetoclax resulted in synergistic induction of apoptosis. Analysis of drug sensitivities to 73 drug combinations and profiling of 31 proteins stratified responders to idelalisib and umbralisib, respectively.
Our findings suggest novel treatment vulnerabilities in idelalisib-refractory/intolerant CLL, and indicate that ex vivo functional profiling may stratify PI3Ki responders.
Heat shock protein 90 (HSP90) is required for the proper folding, function, and stability of various client proteins, two of which (KIT and PDGFRα) are critical in the pathogenesis and progression of ...gastrointestinal stromal tumors (GIST). This phase I study investigated the safety and maximum tolerated dose (MTD) of retaspimycin hydrochloride (IPI-504), a novel potent and selective HSP90 inhibitor, in patients with metastatic and/or unresectable GIST or other soft-tissue sarcomas (STS).
IPI-504 was administered intravenously at doses ranging from 90 to 500 mg/m(2) twice weekly for 2 weeks on/1 week off. Safety, pharmacokinetic, and pharmacodynamic profiles were determined. Response was assessed by Response Evaluation Criteria for Solid Tumors (RECIST) 1.0 and optionally via 18-fluorodeoxyglucose positron emission tomography (18-FDG-PET) imaging.
Fifty-four patients received IPI-504; 37 with GIST and 17 with other STS. The MTD was 400 mg/m(2) twice weekly for 2 weeks on/1 week off. Common related adverse events were fatigue (59%), headache (44%), and nausea (43%). Exposure to IPI-504, 17-AAG, and 17-AG increased with IPI-504 dose. Stable disease (SD) was observed in 70% (26 of 37) of patients with GIST and 59% (10 of 17) of patients with STS. There was one confirmed partial response (PR) in a patient with GIST and one PR in a patient with liposarcoma. Metabolic partial responses occurred in 11 of 29 (38%) patients with GIST.
In this study of advanced GIST or other STS, IPI-504 was generally well-tolerated with some evidence of antitumor activity, serving as a clinical proof-of-concept that HSP90 inhibition remains a promising strategy.
Despite the remarkable activity of BTK inhibitors (BTKi) in relapsed B-cell non-Hodgkin lymphoma (B-NHL), no clinically-relevant biomarker has been associated to these agents so far. The relevance of ...phosphoproteomic profiling for the early identification of BTKi responders remains underexplored.
A set of six clinical samples from an ongoing phase I trial dosing patients with chronic lymphocytic leukemia (CLL) with TG-1701, a novel irreversible and highly specific BTKi, were characterized by phosphoproteomic and RNA sequencing (RNA-seq) analysis. The activity of TG-1701 was evaluated in a panel of 11 B-NHL cell lines and mouse xenografts, including two NF-κB- and BTK
-driven BTKi-resistant models. Biomarker validation and signal transduction analysis were conducted through real-time PCR, Western blot analysis, immunostaining, and gene knockout (KO) experiments.
A nonsupervised, phosphoproteomic-based clustering did match the early clinical outcomes of patients with CLL and separated a group of "early-responders" from a group of "late-responders." This clustering was based on a selected list of 96 phosphosites with Ikaros-pSer442/445 as a potential biomarker for TG-1701 efficacy. TG-1701 treatment was further shown to blunt Ikaros gene signature, including
and
, in early-responder patients as well as in BTKi-sensitive B-NHL cell lines and xenografts. In contrast, Ikaros nuclear activity and signaling remained unaffected by the drug
and
in late-responder patients and in BTK
, BTK
, and noncanonical NF-κB models.
These data validate phosphoproteomic as a valuable tool for the early detection of response to BTK inhibition in the clinic, and for the determination of drug mechanism of action.
Abstract
Enhancer of Zeste Homolog 2 (EZH2) is the histone lysine methyltransferase (HKMT) component of the Polycomb Repressive Complex 2 (PRC2). In conjunction with other members of the complex, ...EZH2 represses gene expression through methylation of histone H3 on lysine 27 (H3K27). EZH2 overexpression is implicated in tumor progression and correlates with poor prognosis in several tumor types and enzymatic hyperactivity of EZH2 has been linked to aberrant repression of tumor suppressor genes in diverse cancers. Recently, direct inhibition of EZH2 HKMT activity by small molecules has been shown to be effective in inhibiting the proliferation of EZH2 mutant diffuse large B-cell lymphoma (DLBCL) cell lines and the growth of tumors in EZH2 mutant DLBCL xenografts.
We have identified and optimized a series of small molecule EZH2 inhibitors that is structurally distinct from previously published chemotypes. CPI-169, a representative compound from that effort, inhibits the catalytic activity of PRC2 with an IC50 of < 1nM, decreases cellular levels of H3K27me3 with an EC50 of 70 nM, and triggers cell cycle arrest and apoptosis in a variety of cell lines. Importantly, compound treatment triggers a sequence of downstream functional consequences of EZH2 inhibition whereby apoptosis is not induced before ten days of continuous target engagement.
Administered subcutaneously at 200 mpk twice daily (BID), CPI-169 is well tolerated in mice with no observed toxic effect or body weight loss. In the present study we show that CPI-169 treatment led to tumor growth inhibition (TGI) of an EZH2 mutant KARPAS-422 DLBCL xenograft. The TGI is proportional to the dose administered and to the reduction of the pharmacodynamic marker H3K27me3. The highest dose, 200 mpk, BID led to complete tumor regression.
Since CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) is the standard treatment of advanced DLBCL, we were interested in combining a suboptimal dose of CPI-169 (100 mpk, BID) with a single dose of CHOP in the KARPAS-422 model. After a week of combinatorial treatment the tumors rapidly regressed and became unpalpable. Four weeks after the last dose only a single mouse presented a palpable tumor.
The immunohistochemical analysis of tumor samples revealed a strong correlation between the global decrease of H3K27me3, the decrease in the proliferation marker Ki-67 and the increase in cleaved-caspase 3 positive cells.
In conclusion, we identified a strong synergistic anti-tumor activity between the standard of care CHOP and CPI-169, a distinct EZH2 inhibitor in an in vivo model of DLBCL.
Citation Format: Vidya Balasubramanian, Priya Iyer, Shilpi Arora, Patrick Troyer, Emmanuel Normant. CPI-169, a novel and potent EZH2 inhibitor, synergizes with CHOP in vivo and achieves complete regression in lymphoma xenograft models. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1697. doi:10.1158/1538-7445.AM2014-1697
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Background: TG-1701 is a selective, covalent BTK inhibitor administered once daily (QD). Both the “U2” combination (anti-CD20 mAb ublituximab + the PI3Kδ-CK1ε inhibitor umbralisib) ...and BTK inhibition are highly active in treatment-naïve (TN) and relapsed/refractory (R/R) CLL, each having previously demonstrated superiority over standard chemoimmunotherapy. Here we report the results of the dose escalation of TG-1701 monotherapy and TG-1701+U2. Methods: Pts with R/R CLL and lymphoma were enrolled in a Ph 1 study initially evaluating dose escalation (DE) of oral TG-1701 QD continuously administered in 28-day cycles (100, 200, 300, and 400 mg). After characterizing the safety profile of TG-1701 monotherapy, we implemented a parallel DE arm of TG-1701+U2. Select dose levels of TG-1701 monotherapy were expanded in CLL, MCL and Waldenström's (WM). All pts were treated until disease progression. The primary objectives are to characterize the safety profile and define the recommended Ph 2 doses for the drugs alone and in combination. Results: As of 03 February 2021, 123 pts were treated with TG-1701: 25 in the monotherapy DE arm, 61 in the 200 mg disease-specific cohorts (20 CLL 5 TN, 21 MCL 4 TN, 20 WM 8 TN), 20 in the 300 mg CLL cohort (4 TN), and 17 in the 1701+U2 DE arm. The median # of prior therapies was 1 (range, 1 - 10). All pts were BTKi-naïve. All 123 pts were evaluable for safety. TG-1701 was well tolerated and the maximum tolerated dose (MTD) for monotherapy was not reached at 400 mg (demonstrating near 100% saturation of the BTK at all dose levels studied). Treatment emergent adverse events (TEAE) of clinical interest included atrial fibrillation (AF 4.0% of pts, G ≥3 in 1 case), G ≥3 hypertension (2.4%), and bleeding events (18.7%, all G1-2). No cases of ventricular tachyarrhythmia were reported. TEAEs leading to TG-1701 dose reduction occurred in 6.5% of pts. TEAEs leading to treatment discontinuation occurred in 1.6% of pts (AF, COVID-19). At the data cut-off, 119 pts were evaluable for response, including 40 in DE (Table). The median duration of response has not been reached among responders overall. The median follow-up (mFU range) was 15.9 mos (1.3 - 28.6+) in DE and 8.5 mos (1.4 -15.6+) in disease-specific cohorts. Conclusions: TG-1701 exhibits an encouraging safety and efficacy profile. The combination of 1701+U2 has been well tolerated and dose escalation continues. The combination shows enhanced depth of response over TG-1701 monotherapy. Recruitment to this study continues. Response per investigator review by treatment group. Clinical trial information: NCT03671590. Table: see text
Introduction
Agents targeting BTK have demonstrated activity in a variety of B-cell malignancies, however not all patients respond to therapy, and amongst those that do respond, complete remissions ...are rare. BTK-based combination regimens have the potential to increase depth of response and permit time-limited therapy, however innovative approaches to accelerate the development of combination regimens are needed. TG-1701 is a novel, orally available and covalently-bound BTK inhibitor that exhibits superior selectivity for BTK compared with ibrutinib in in vitro whole kinome screening (Abstr 3973, EHA 2018). Herein we report the first results of a unique Phase 1 parallel dose-escalation study of TG-1701 monotherapy and TG-1701 in combination with umbralisib, a novel PI3K-δ and casein kinase-1ε inhibitor, and ublituximab, a glycoengineered anti-CD20 mAb (1701 + U2).
Methods
The primary objectives of the study are to characterize the safety profile and to determine the recommended Phase 2 dose of TG-1701 as a single agent and in combination with U2. Other objectives include assessment of pharmacokinetics (PK), preliminary antitumor activity, and pharmacodynamics (PD BTK occupancy). Eligible patients must have B-cell malignancy relapsed or refractory (R/R) to one or more prior standard therapy. Treatment consists of escalating doses of oral TG-1701 once daily (QD), continuously administered in 28-day (D) cycles (C). Patients in the 1701 + U2 combination arm receive escalating TG-1701 oral QD + umbralisib 800 mg oral QD + ublituximab 900 mg IV on D1, 8, 15 of C1, and D1 of C2-6. All patients are treated until disease progression, unacceptable toxicity, or investigator/patient decision to withdraw study consent.
Results
As of July 15, 2019, 19 patients (WM = 6, CLL = 4, FL = 4, MZL = 2, DLBCL = 2, MCL = 1) have been treated with TG-1701: 3 patients at 100 mg QD, 9 patients at 200 mg QD (expansion before opening combination), 3 patients at 300 mg QD single agent arm, and 4 patients at 100 mg QD combination arm. Patients had a median of 2 prior systemic therapies (range, 1 - 5), and all of them received previous anti-CD20 therapy. Four patients were refractory to their last prior therapy, 8 had extranodal disease, and 8 had bulky disease. To date, patients have received a median of 4 (range, 2 - 10) cycles of TG-1701.
No dose-limiting toxicity (DLT) have been observed to date. The most common treatment-related adverse events (TRAE) are: neutropenia (21%), diarrhea (16%), nausea (16%), bruising (16%), infection (16%), ALT/AST elevation (11%), rash (11%), abdominal pain (11%), and fatigue (11%). Grade 3 TRAE are: asymptomatic and isolated lipase elevation (N=1), nausea (N=1), rash (N=1), and acute respiratory tract infection (N=1). There have been no grade 4 TRAE nor treatment discontinuations due to adverse events.
Linear kinetics are apparent, evidenced by approximately dose proportional increase in AUC on C1D1 and C1D8. High systemic clearance (CL) has been observed with a mean CL/F of 55.4 L/hr and half-life of 2.24 hours. Tmax is observed between 1 to 4 hours post dose. PK-PD (BTK occupancy) relationship at 200 mg on C1D1 is presented in Figure 1. Decrease from baseline in tumor burden is presented in Figure 2. Two patients at the lowest dose (100 mg QD) have achieved a partial response (PR): MCL = ↓78% and WM = ↓88%. All 3 evaluable patients treated with 100 mg 1701 + U2 have achieved a response at the first response assessment: 1 CR (FL) and 2 PR (FL ↓88%, and MZL ↓65%). All patients remain on study treatment, except for both patients with DLBCL that discontinued due to disease progression. All patients with CLL have shown lymphocytosis during the first 2 cycles of therapy. Near complete BTK occupancy has been achieved in all patients at all dose levels (Figure 1).
Conclusions
TG-1701, a once daily BTK inhibitor has an encouraging preliminary safety profile, with clinical and pharmacodynamic activity at all dose levels evaluated. This study (NCT03671590) continues enrollment in TG-1701 single-agent and 1701 + U2 combination arms.
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Cheah:Roche, Janssen, MSD, Gilead, Loxo Oncology, Acerta, BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene, Roche, Abbvie: Research Funding; Roche: Other: Travel expenses. Wickham:Roche: Honoraria; Celgene: Other: Travel/Meeting Expenses . Turpuseema:TG Therapeutics Inc.: Employment, Equity Ownership. Miskin:TG therapeutics Inc.: Employment, Equity Ownership. Tang:TG Therapeutics Inc., Roche, Alexion: Equity Ownership; TG Therapeutics Inc.: Employment. Normant:TG Therapeutics Inc.: Employment, Equity Ownership. Ricart:TG Therapeutics, Pfizer, Merck: Equity Ownership; TG therapeutics Inc.: Employment. Tam:Abbvie, Janssen, Beigene, Roche, Novartis: Honoraria; Abbvie, Janssen: Research Funding.
Abstract
BET (bromodomain and extra-terminal) proteins bind to acetylated lysine residues on chromatin and participate in the regulation of gene transcription. Inhibition of BET protein binding to ...chromatin with small molecules selectively suppresses the transcription of a set of oncogenes, including MYC and BCL-2. TG-1601 is a novel, selective and potent small molecule inhibitor of BET bromodomains. TG-1601 binds to the first and second bromodomains (BD1, BD2) of the BET protein family, BRD2, BRD3, BRD4, and BRDT, with Kd values ranging from 0.5 nM to 9.1 nM. MYC protein expression is strongly inhibited in the MV4-11 cancer cell line with an EC50 of 5 nM, with GI50 comprised between 15 nM and 85 nM in a variety of leukemia and myeloma cancer cell lines, indicating potent inhibition of cell proliferation. Time course and dose-response studies conducted in mice bearing subcutaneous MV4-11 xenografts showed that MYC protein was undetectable 3 hours following a single 25 mg/kg oral dose, with a TG-1601 tumor concentration of 6uM achieved. Interestingly, at 24h post-dose, while TG-1601 is cleared from the tumor, MYC protein level remains below 40% of its initial level, indicating a long-lasting effect pharmacodynamic of TG-1601, potentially attributable to enhanced binding affinity compared to earlier generation molecules. In agreement with this long-lasting effect, efficacy studies in MV4-11 tumor-bearing mice, dosed with a 20 mg/kg/day PO regimen interrupted by increasing drug holiday periods, showed that drug holidays of 2, 3 and 4 days per week only modestly affected efficacy (3%, 15% and 12% TGI respectively), suggesting discontinuous dosing of TG-1601 in clinic may not significantly impact efficacy. Anti-cancer agents have been shown - in vitro or in vivo - to synergize with BET inhibitors. Here we show that TG-1601 and anti PD-1 antibody demonstrated synergistic anti-tumor activity when combined in the B16F10 model, an aggressive syngeneic model of melanoma. TG-1601 inhibition of MYC, CCR-2 and IL1RN gene expression was tested in a whole blood ex-vivo experiment, and the genes were validated as pharmacodynamic markers to monitor TG-1601 activity in clinic. In conclusion, TG-1601 is a novel BET inhibitor with remarkably strong affinity and a potent ability to inhibit MYC expression and cell growth, with favorable pharmacokinetic properties supporting clinical development. Its properties in vivo provide an opportunity for the rational development of TG-1601 as an anti-cancer agent, taken alone or in combination with other small molecules or antibodies. IND enabling studies are underway, with clinical evaluation expected to commence in the first half of 2018.
Citation Format: Emmanuel Normant, Leonid Gorelik, Rama Shmeis, Henry Le, Robert Nisch, Karen TenHuisen, Teja Turpuseema, James Oliviero, Hari P. Miskin, Peter Sportelli, Michael S. Weiss. TG-1601 is a novel BET inhibitor with strong binding affinity and long-lasting effect in pre-clinical models abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5790.
Despite the promising introduction of the proteasome inhibitor bortezomib in the treatment of mantle cell lymphoma (MCL), not all patients respond, and resistance often appears after initial ...treatment. By analyzing a set of 18 MCL samples, including cell lines with constitutive or induced resistance to bortezomib, we found a high correlation between loss of sensitivity to the proteasome inhibitor and up-regulation of the prosurvival chaperone BiP/Grp78. BiP/Grp78 stabilization was ensured at a posttranscriptional level by an increase in the chaperoning activity of heat shock protein of 90 kDa (Hsp90). In bortezomib-resistant cells, both BiP/Grp78 knockdown and cell pretreatment with the Hsp90 inhibitor of the ansamycin class, IPI-504, led to synergistic induction of apoptotic cell death when combined with bortezomib. Cell exposure to the IPI-504–bortezomib combination provoked the dissociation of Hsp90/BiP complexes, leading to BiP/Grp78 depletion, inhibition of unfolded protein response, and promotion of NOXA-mediated mitochondrial depolarization. The IPI-504–bortezomib combination also prevented BiP/Grp78 accumulation, thereby promoting apoptosis and inhibiting the growth of bortezomib-resistant tumors in a mouse model of MCL xenotransplantation. These results suggest that targeting unfolded protein response activation by the inhibition of Hsp90 may be an attractive model for the design of a new bortezomib-based combination therapy for MCL.
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The discovery and optimization of a series of small molecule EZH2 inhibitors is described. Starting from dimethylpyridone HTS hit (2), a series of indole-based EZH2 inhibitors were ...identified. Biochemical potency and microsomal stability were optimized during these studies and afforded compound 22. This compound demonstrates nanomolar levels of biochemical potency (IC50=0.002μM), cellular potency (EC50=0.080μM), and afforded tumor regression when dosed (200 mpk SC BID) in an EZH2 dependent tumor xenograft model.
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