HMPL-523, a Novel SYK Inhibitor Showed Anti-Tumor Activities In Vitro and In Vivo

Abstract Introduction: Spleen Tyrosine Kinase (SYK), a non-receptor type of tyrosine kinase, is a member of Syk/ZAP70 tyrosine kinase family. It plays a pivotal role in the regulation of B-cell receptor (BCR) signal pathway, which regulates proliferation, differentiation and survival of B lymphocytes. The abnormal activation of BCR singling is closely related to transformation and development of B cell lymphoma. Targeting BCR downstream molecules, such as Bruton' tyrosine kinase (BTK) and phosphoinositide-3-kinase δ (PI3Kδ) has emerged as new therapeutic approaches and inhibitors of BTK and PI3Kδ were approved recently by FDA for treatment of some subtypes of B-cell malignancies. Currently, a couple small molecular inhibitors against SYK, another BCR downstream molecule, are under the early clinical development and showed initial efficacy in B cell lymphomas. HMPL-523, discovered and currently being developed in Phase I clinical trial by Hutchison MediPharma, is a novel, highly potent and selective SYK inhibitor (IC50: 0.025 μM). The anti-tumor activity of HMPL-523 was evaluated in this study. Methods: Inhibitory effects of HMPL-523 on cell viability were investigated in a panel of B cell lymphoma cell lines with SYK/BCR dysregulation by CellTiter-Glo luminescent or CCK-8 assay. The effect of HMPL-523 on SYK signaling pathway was detected by western blot. Annexin-V- positive and PI-negative population was recognized as apoptotic cells by FACS. Nude mice bearing B cell lymphoma xenograft tumors with SYK/BCR dysregulation were used to determine anti-tumor activity of HMPL-523 in vivo. Result: HMPL-523 blocked phosphorylation of BLNK, downstream protein of Syk, in human mantle cell line REC-1 and human plasma cell line ARH-7777 with IC50 of 0.105 µM and 0.173 μM, respectively. HMPL-523 also inhibited cell viability of Ba/F3 Tel-Syk with IC50 of 0.033 μM. Furthermore, inhibitory effects of HMPL-523 on cell viability were evaluated in a panel of B -cell lymphoma cell lines with SYK/BCR deregulation. Results showed that HMPL- 523 potently inhibited cell survival with IC50s from 0.4 to 2 μM. Consistent with the effect on cell viability, HMPL-523 increased the apoptotic rate of REC-1 cells. Moreover, HMPL-523 showed the synergistic activities on killing human diffused large B cell lymphoma (DLBCL) in combination with other drugs such as BTK inhibitor, PI3Kδ inhibitors and Bcl2 family inhibitor. The detailed mechanisms underlying the synergism are still under investigation. Anti-tumor activity of HMPL-523 was determined in Syk dependent xenograft models. Daily oral administration of 100 mg/kg HMPL-523 showed potent anti-tumor activity in B cell lymphoma REC-1 (TGI: 59%). Conclusion:HMPL-523 is a highly potent SYK inhibitor with good activity against B-cell lymphoma in pre-clinical in vitro and in vivo models, supporting further clinical research for HMPL-523 as either single agent or combination drug with other agents to treat B-cell malignancies e.g. DLBCL Disclosures Yang: Hutchison MediPharma Ltd: Employment, Research Funding. Deng:Hutchison MediPharma Ltd: Employment, Research Funding. Sun:Hutchison MediPharma Ltd: Employment, Research Funding. Liang:Hutchison MediPharma Ltd: Employment, Research Funding. Wang:Hutchison MediPharma Ltd: Employment, Research Funding. Fan:Hutchison MediPharma Ltd: Employment, Research Funding. Tang:Hutchison MediPharma Ltd: Employment, Research Funding. Yu:Hutchison MediPharma Ltd: Employment, Research Funding. Sun:Hutchison MediPharma Ltd: Employment, Equity Ownership. Zhou:Hutchison MediPharma Ltd: Employment, Research Funding. Dai:Hutchison MediPharma Ltd: Employment, Research Funding. Qing:Hutchison MediPharma Ltd: Employment, Research Funding. Su:Hutchison MediPharma Ltd: Employment, Research Funding. Ren:Hutchison MediPharma Ltd: Employment, Research Funding.

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The Synergistic Anti-Tumor Activity of EZH2 Inhibitor SHR2554 and HDAC Inhibitor Chidamide through ORC1 Reduction of DNA Replication Process in Diffuse Large B Cell Lymphoma

Cancers ◽

10.3390/cancers13174249 ◽

2021 ◽

Vol 13 (17) ◽

pp. 4249

Author(s):

Xing Wang ◽

Dedao Wang ◽

Ning Ding ◽

Lan Mi ◽

Hui Yu ◽

...

Keyword(s):

B Cell ◽

Hdac Inhibitor ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Replication Process ◽

Large B Cell Lymphoma ◽

Anti Tumor Activity ◽

Large B Cell

Background: Upregulation of H3K27me3 induced by EZH2 overexpression or somatic heterozygous mutations were implicated in lymphomagenesis. It has been demonstrated that several EZH2-target agents have notable therapeutic effects in EZH2-mutant B-cell lymphoma patients. Here we present a novel highly selective EZH2 inhibitor SHR2554 and possible combination strategy in diffuse large B-cell lymphoma (DLBCL). Methods: Cell proliferation, cell cycle and apoptosis were analyzed by CellTiter-Glo Luminescent Cell Viability Assay and flow cytometry. Western Blot was used to detect the expression of related proteins. The gene expression profiling post combination treatment was analyzed by RNA-Seq. Finally, CDX and PDX models were used to evaluate the synergistic anti-tumor effects of the combination treatment in vivo. Results: The novel EZH2 inhibitor SHR2554 inhibited proliferation and induced G1 phase arrest in EZH2-mutant DLBCL cell lines. The combination of EZH2 inhibitor SHR2554 with histone deacetylase (HDAC) inhibitor chidamide (hereafter referred to as HBI8000) exerted synergistic anti-proliferative activity in vitro and in vivo. Gene expression profile analysis revealed dramatic inhibition of the DNA replication process in combined treatment. Conclusions: SHR2554, a potent, highly selective small molecule inhibitor of EZH2, inhibited EZH2-mutant DLBCL more significantly in vitro and in vivo. The combination of HDAC inhibitor HBI8000 with EZH2 inhibitor SHR2554 exhibited dramatic anti-tumor activity in both mutant and wild-type DLBCL, which may become a potential therapeutic modality for the treatment of DLBCL patients.

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The SMAC Mimetic Lcl-161 Augments the in Vitro and in Vivo anti-Tumor Activity of Rituximab and Chemotherapy in Rituximab Relapse/Refractory Lymphoma Models

Blood ◽

10.1182/blood.v124.21.3110.3110 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3110-3110 ◽

Cited By ~ 2

Author(s):

Kyle L. Runckel ◽

Joseph Skitzki ◽

Francisco Hernandez ◽

Myron S. Czuczman

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

Chemotherapy Resistance ◽

B Cell Lymphoma ◽

Over Expression ◽

Tail Vein Injection ◽

Refractory Lymphoma ◽

Anti Tumor Activity

Abstract The addition of rituximab to front-line therapy regimens in diffuse large B-cell lymphoma (DLBCL) has greatly improved clinical outcomes, but is also associated with a disease that is more resistant to salvage chemotherapy in the second-line setting, reinforcing the need for new therapies targeted at the overlapping resistance pathways between rituximab and chemotherapy. To better understand the mechanisms responsible for rituximab/chemotherapy cross-resistance we developed several rituximab resistance cell lines which exhibited significant concurrent chemotherapy resistance. These multi-therapy resistant cell lines (TRCL) exhibit decreased expression of the pro-apoptotic Bcl-2 family proteins Bak and Bak, along with over-expression of several anti-apoptotic proteins, including the inhibitor of apoptosis proteins (IAP) survivin and livin (determined by Western blot). High IAP expression has been associated with inferior clinical outcomes in a range of hematological malignancies, and solid tumors. To determine the impact of IAP over-expression on TRCL rituximab/chemotherapy resistance we utilized a transient siRNA knockdown of both survivn and livin. TRCLs with livin knockdown had a statistically significant improvement in response to several chemotherapy agents including doxorubicin, vincristine, and the proteasome inhibitor carfilzomib (measured at 48 hours with the Cell Titer-Glo viability assay). These results support livin over-expression as a key lymphoma therapy resistance mechanism, and establish IAPs as potential therapeutic targets. Small molecule IAP inhibitors, like LCL-161 (obtained from Novartis), are chemical mimetics of the endogenous IAP antagonist termed the second mitochondrial inhibitor of caspases (SMAC). Western blot analysis indicated that TRCLs treated in vitro with LCL-161 exhibited a dose dependent decrease in the expression of several IAPs, including livin. In addition, LCL-161 increased rates of TRCL apoptosis, and produced synergistic anti-tumor activity when combined with cytarabine, gemcitabine, and carfilzomib in vitro. LCL-161 also enhanced the ex vivo anti-tumor activity of carfilzomib against primary tumor cells isolated from lymphoma patients with both de novo, and relapse/refractory disease. Cell viability and apoptosis induction were determined at 48 hours with CellTiter-Glo viability assays and flow cytometry respectively. To evaluate the anti-tumor effect of LCL-161 in vivo severe combined immunodeficiency (SCID) mice were inoculated with the TRCL Raji-4RH via tail vein injection (iv), and assigned to observation or treatment arms 7 days after inoculation. Treatments were LCL-161 alone (60mg/kg), the combination of rituximab: 10mg/kg, gemcitabine: 120mg/kg, and vinorelbine: 8mg/kg (RGV), or LCL-161 and RGV together. LCL-161 was administered on day 7 as one dose given p.o. by gavage; RGV was also administered on day 7 as a single i.v. dose given by tail vein injection. Differences in survival (measured as the time to the development of limb paralysis) were evaluated with the Log-rank, Breslow, and Tarone-Ware tests across treatment arms. As a single agent LCL-161 was ineffective in controlling Raji-4RH tumor growth in vivo. However, the combination of LCL-161 with RGV (median survival 133 days) resulted in a statistically significant (P=0.002 with each test) improvement in overall survival when compared to RGV alone (median survival 53 days). In summary, IAPs, especially livin, contribute to rituximab/chemotherapy resistance in relapse/refractory B-cell lymphoma models. However, the IAP inhibitor LCL-161 can disrupt this resistance and augment the effect of chemotherapy in resistant lymphoma cell line models, as well as relapse/refractory lymphoma patient samples. In addition, LCL-161 can improve the anti-tumor activity of the RGV chemotherapy regimen, and increase overall survival in a mouse in vivo model of human rituximab/chemotherapy resistant lymphoma. Our data supports the continued investigation of LCL-161 as a novel and effective targeted agent for the treatment of aggressive rituximab relapse/refractory B-cell lymphomas. (Supported by a NHI SPORE Lymphoma grant: 5 P50 CA130805-04, a NIH grant R01 CA136907-01A1 and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures No relevant conflicts of interest to declare.

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Epratuzumab (Humanized Anti-CD22 MAb) Conjugated with SN-38, a New Antibody-Drug Conjugate (ADC) for the Treatment of Hematologic Tumors: Preclinical Studies Alone and In Combination with Veltuzumab, a Humanized Anti-CD20 MAb

Blood ◽

10.1182/blood.v116.21.3941.3941 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3941-3941

Author(s):

David M Goldenberg ◽

Serengulam Govindan ◽

Tom M Cardillo ◽

Robert M Sharkey

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

In Vivo Studies ◽

Anti Cd20 ◽

Anti Tumor Activity

Abstract Abstract 3941 Background: Monoclonal antibody (MAb) therapy has had a significant impact on the management of B-cell malignancies, but is most often used in combination with chemotherapy. We developed an ADC that combines SN-38, the active component of irinotecan, a topoisomerase I inhibitor, with the internalizing, humanized, anti-CD22 IgG, epratuzumab, and determined its activity alone and in combination with an anti-CD20 antibody therapy (veltuzumab). Methods: Epratuzumab was conjugated with SN-38 (E-SN-38) at a mole ratio of ∼6:1. The conjugate is designed specifically to be released slowly in the presence of serum (50% released over ∼1.5 days), allowing liberation of the drug when internalized, but also being released locally after being bound to the tumor. In vitro and in vivo studies were performed to assess the activity of the conjugate against several subcutaneously- or intravenously-inoculated B-cell lymphoma cell lines. In vivo studies also examined combination therapy using E-SN-38 and the veltuzumab (V). Results: In vitro studies in 4 B-cell lymphoma cells lines (Daudi, Raji, Ramos, WSU-FSCCL) and 4 acute lymphoblastic lymphoma cell lines (697, REH, MN-60, and RS4;11) expressing varying amounts of CD22 showed an IC50 for E-SN-38 in the nanomolar range, confirming potent activity. Nude mice bearing SC Ramos human lymphoma had significant selective anti-tumor activity compared to a control, non-targeting, IgG-SN-38 conjugate, at a dosing regimen of 75 to 250 μg of the conjugates given twice-weekly for 4 weeks. Significant anti-tumor activity was also found in several other cell lines. When combined with veltuzumab, significant improvement in therapeutic activity was observed. For example, median survival in a WSU-FSCCL human follicular B-cell lymphoma IV model with treatment initiated 5 days after implantation was 42 d (0/10 surviving at 160 d) and 91 d (2/10 surviving) for untreated and veltuzumab-treated animals, respectively; 63d (0/10 surviving after 160 d) and >160 d (with 6/10 surviving) for E-SN-38 and E-SN-38 + V, respectively; and 63 d (0/10) and 91 d (2/10) for non-targeting IgG-SN-38 conjugate alone and combined with V). The E-SN-38 conjugate combined with V was significantly better than all treatment or control groups (P ≤ 0.05). Conclusion: E-SN-38 ADC is a potent therapeutic, even at non-toxic dose levels, and shows significantly enhanced efficacy when combined with anti-CD20 immunotherapy, representing an important new ADC treatment regimen. Disclosures: Goldenberg: Immunomedics, Inc.: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Govindan:Immunomedics, Inc.: Employment. Cardillo:Immunomedics, Inc.: Employment.

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Comprehensive in Vitro and in Vivo Analysis of Phosphatidylinositol-3-Kinase Delta (PI3Kδ) Inhibition Using GS-1101 (CAL-101) in Combination with Mammalian Target of Rapamycin (mTOR) Inhibition Using Temsirolimus or Everolimus in Mantle Cell Lymphoma (MCL)

Blood ◽

10.1182/blood.v120.21.3719.3719 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3719-3719

Author(s):

Paul M. Barr ◽

Francisco J. Hernandez-Ilizaliturri ◽

Thomas Murante ◽

Shannon P. Hilchey ◽

Derick R Peterson ◽

...

Keyword(s):

Cell Viability ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Mtor Inhibitors ◽

B Cell Lymphoma ◽

Mtor Inhibition ◽

Sequential Administration

Abstract Abstract 3719 The clinical efficacy of mTOR inhibition in MCL is limited by known resistance pathways mediated through IRS-1 and mTORC2. Simultaneous inhibition of other molecules downstream of the B cell receptor, such as PI3Kδ, may abrogate such negative feedback mechanisms. PI3Kδ inhibition using GS-1101 has demonstrated early efficacy in MCL. Taken together, the combination of mTORC1 and PI3Kδ inhibition may represent a rationale combination to test in MCL. To this end, we utilized a panel of B cell lymphoma lines including established MCL cell lines (Granta, Jeko, Mino, Rec-1, HBL-2, Z-138), cytarabine resistant MCL lines (MinoAraCR, JekoAraCR, Rec-1AraCR, HBL-2AraCR) and primary MCL cells isolated from patients. In all cell lines, dose-finding experiments using GS-1101 and the mTOR inhibitors temsirolimus and everolimus were performed in triplicates. Cell viability was determined using an Alamar Blue reduction assay. Proteins downstream of PI3K – mTOR signaling were evaluated by western blot analysis. Synergy between the agents was evaluated using Laska et al's model–free test. For in vivo studies, severe combined immunodeficiency mice were injected with 10×106 Z-138 cells on day 0. GS-9820, a PI3Kδ inhibitor optimized for murine studies, was used in lieu of GS-1101. Upon detection of tumor engraftment, animals were divided into 6 groups, each containing 5 mice; Control, GS-9820 at 10 and 20mg/kg/dose, temsirolimus at 10 and 20mg/kg/dose, and GS-9820 plus temsirolimus at 10mg/kg/dose each. GS-9820 was administered by gastric lavage twice daily on days +15 to +19 and +22 to +26. Temsirolimus was administered via tail vein injection on days +15, +17, +19, +22, +24, and +26. Tumor measurements were used to determine therapeutic activity. The initial screen of lymphoma histologic subtypes demonstrated that cell viability was reduced across Burkitt, diffuse large B cell and MCL lines exposed to GS-1101. In MCL lines, the cell viabilities observed after 48 h treatments with GS-1101 (5uM) were 80% ± 6.9, 66% ± 2.2 and 68% ± 4.7 in Granta, Jeko and Rec-1 cells respectively. No difference was observed in cytarabine resistant cells suggesting non-cross resistance with cytarabine. The activity in primary MCL cells was similar using GS-1101 (5uM) [viability range 55%-65%] while peripheral blood mononuclear cells (PBMCs) appeared less sensitive to GS-1101 [78% ± 2.4]. Both mTOR inhibitors provided moderate reductions in viability after 48 h exposures. Compared to untreated controls, the viabilities of Granta, Jeko and Rec-1 cell lines after 48 h exposures to temsirolimus (5nM) were 73% ± 1.3, 53% % ± 6.9 and 54% ± 2.0 respectively as well as 68% ± 2.9, 50% ± 7.4 and 55% ± 2.0 respectively after everolimus (5nM). Similar results were observed in primary MCL cells using temsirolimus (5nM) [range 80%-85%] while PBMCs were largely unaffected [90% ± 2.2]. The combination of GS-1101 and either mTOR inhibitor produced largely additive reductions in cell viability. Synergistic interactions were observed in Rec-1 cells for 8 dose combinations of GS-1101 (0.1–5.0uM) and either temsirolimus (1–5nM) or everolimus (1–5nM) (unadjusted p < 0.05 for all 8 combinations). Evidence of synergy was insufficient at any combination after adjustment for multiple comparisons over the 3 cell lines. Sequential administration using 24 h pretreatment with each agent was evaluated; no benefit over simultaneous administration was demonstrated. Consistent with known mechanisms of action, immunoblotting revealed decreased 4EBP1 and S6K phosphorylation with mTOR inhibition while PI3K inhibition consistently decreased Akt phosphorylation. In vivo, GS-9820 appears active in the Z-138 xenografts at early time points. Tumor size was reduced to 60% ± 5.5 of control at day 18 and 23 using either 10 or 20 mg/kg of GS-9820. Testing of GS-9820 in combination with temsirolimus in this model is ongoing. Our findings indicate that PI3Kδ inhibition using GS-1101 and GS-9820 is active in vitro and also in a MCL murine xenograft. GS-1101 in combination with mTORC1 inhibition largely produced additive in vitro anti-lymphoma effects in MCL. Ongoing work is aimed at understanding the differences in molecular events downstream of PI3K and mTOR inhibition comparing Rec-1 cells, where synergy was demonstrated, with other cell lines to provide insight into optimal therapeutic combinations and to determine in which molecularly defined subsets of MCL they may be most active. Disclosures: Johnson: Gilead Sciences: Employment. Lannutti:Gilead Sciences Inc: Employment.

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FF-10102-01: A Novel, Highly Selective Spleen Tyrosine Kinase Inhibitor, to Be in Clinical Application for Treatment of Autoimmune Diseases and B-Cell Malignancies

Blood ◽

10.1182/blood.v128.22.3745.3745 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3745-3745

Author(s):

Chieko Kinouchi ◽

Kazuya Taguchi ◽

Susumu Shimoyama ◽

Tadaaki Ioroi ◽

Hayato Ogura ◽

...

Keyword(s):

B Cells ◽

Tyrosine Kinase ◽

Autoimmune Diseases ◽

B Cell ◽

Research Funding ◽

B Cell Malignancies ◽

Ic50 Value ◽

Syk Inhibitor

Abstract Spleen tyrosine kinase (Syk) is essential for downstream pathways in signaling from B-cell receptor (BCR) in B cells and Fc-gamma receptors in macrophages. Because of the essential roles it plays in signaling, Syk has been targeted in drug development for autoimmune and allergic diseases, in which B cells and macrophages have pivotal roles in the pathophysiology. In addition, accumulated data suggest that aberrant BCR signaling is deeply involved in the pathogenesis of B-cell malignancies. Here we report the pharmacological profile of a novel and highly selective Syk inhibitor, FF-10102-01 (FF-10102 hydrochloride). FF-10102-01 was evaluated in the following in vitro assays: Kinase inhibitory activities were performed with ProfilerPro Kit (PerkinElmer, USA). The effects on cell signaling and functions were studied using THP-1, a human monocyte-derived cell line, after differentiation with human recombinant interferon gamma (IFNγ). The effects on CD69, an activated B-cell marker, expression on B cells were evaluated using whole blood samples from healthy volunteers and mice, and on lymphoma cell growth using SU-DHL-6, a human diffuse large B-cell lymphoma-derived cell line. FF-10102-01 was also evaluated in vivo in animal models of antigen-induced antibody production, anti-platelet antibody-induced thrombocytopenia, and collagen-induced arthritis (CIA). FF-10102-01 showed high potency against recombinant human Syk enzyme activity, with a 50% inhibitory concentration (IC50) value of 1.2 nmol/L. Kinase profiling assay in a panel of 216 kinases revealed that FF-10102-01 is highly selective for Syk, with an IC50 value more than 25 times lower than those of the other kinases. FF-10102-01 exhibited suppressive effects on phosphorylation of downstream molecules of Syk, SLP76, and ERK1/2 in THP-1 cells. FF-10102-01 inhibited tumor necrosis factor α secretion under stimulation by immunoglobulin (Ig) G and phagocytosis of IgG-opsonized beads in IFNγ-induced differentiated THP-1 cells. CD69 expression under stimulation with anti-BCR antibodies in human and mouse blood was also inhibited by FF-10102-01, with IC50 values of 314 nmol/L and 307 nmol/L, respectively. FF-10102-01 was the most potent inhibitor of the growth of SU-DHL-6 cells among other known Syk, JAK, and Bruton's tyrosine kinase inhibitors compared, with an IC50 value of 318 nmol/L. FF-10102-01 is orally available with a good PK profile, and showed inhibitory effect at daily doses 50 mg/kg (as free base) on specific antibody production in ovalbumin-immunized mouse model. Orally administered FF-10102-01 showed significant effects in a mouse model of thrombocytopenia and a rat model of rheumatoid arthritis (CIA) at 25 mg/kg and 10 mg/kg (as free base), respectively. In the thrombocytopenia model, the prevention of platelet decrease by FF-10102-01 was well correlated with suppressions of platelet-phagocytotic macrophages in spleen and CD69-positive B cells in blood. These data indicate that FF-10102-01 is a potent and highly selective orally available Syk inhibitor, with pharmacological effects through inhibition of both B-cell and macrophage activities represented in inhibitions of cell signaling and functions in vitro, and in animal models of autoimmune diseases in vivo. The results suggest that FF-10102-01 is a promising agent for treatment of autoimmune diseases such as immune thrombocytopenia, rheumatoid arthritis, and B-cell malignancies. A clinical trial of FF-10102-01 is planned to commence in 2017. Disclosures Kinouchi: FUJIFILM Corporation: Employment. Taguchi:FUJIFILM Corporation: Employment. Shimoyama:FUJIFILM Corporation: Employment. Ioroi:FUJIFILM Corporation: Employment. Ogura:FUJIFILM Corporation: Employment. Yamamoto:Toyama Chemical Co., Ltd.: Employment. Iino:Toyama Chemical Co., Ltd.: Employment. Maeda:Toyama Chemical Co., Ltd.: Employment. Kato:Toyama Chemical Co., Ltd.: Employment. Fujiwara:FUJIFILM Corporation: Employment. Hagiwara:FUJIFILM Corporation: Employment. Iwamura:FUJIFILM Corporation: Employment. Kuter:Rigel: Consultancy, Research Funding; Genzyme: Consultancy; Bristol-Myers Squibb: Research Funding; GlaxoSmithKline: Consultancy; ONO: Consultancy; Amgen: Consultancy, Paid expert testimony; Protalex: Research Funding; MedImmune: Consultancy; Pfizer: Consultancy; Syntimmune: Consultancy; Shire: Consultancy; Eisai: Consultancy; 3SBios: Consultancy; CRICO: Other: Paid expert testimony; Shionogi: Consultancy. Nakamura:Toyama Chemical Co., Ltd.: Employment. Shimada:FUJIFILM Corporation: Employment.

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In Vivo Efficacy of Anti-CD20-Interferon-Gamma Fusion Protein Against Syngeneic B Cell Lymphoma Is Mediated By Natural Killer Cells

Blood ◽

10.1182/blood-2019-131890 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1575-1575

Author(s):

Alex Vasuthasawat ◽

Reiko E Yamada ◽

Kham R Trinh ◽

Neiki Rokni ◽

Sherie L Morrison ◽

...

Keyword(s):

T Cells ◽

Fusion Protein ◽

B Cell ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

In Vivo Efficacy ◽

Anti Cd20

Background: The interferons, including IFNα/IFNβ (type I) and IFNγ (type II) are essential mediators of anti-cancer immunity. To achieve efficient targeting of IFNs to tumor sites, we have developed antibody (Ab)-IFN fusion protein technology. We previously reported the antigen-specific targeting of IFNα to CD20+ target cells with efficient inhibition of proliferation, induction of apoptosis, and in vivo tumor eradiation dependent upon IFNα receptors on the tumor cell surface (Xuan et al, Blood, 2010). A fusion protein targeting human CD20 (anti-CD20-IFNα) exhibited stronger direct anti-proliferative effects, complement-dependent cytotoxicity (CDC), Ab-dependent cell-mediated cytotoxicity (ADCC), and in vivo potency against B-cell lymphoma xenograft models compared to the parent Ab rituximab (Timmerman et al, Blood 2015). Based on these results, a phase I, first-in-human, dose-escalation trial of anti-CD20-IFNα for B cell non-Hodgkin lymphoma is now underway (NCT02519270). Given the distinct properties of IFNγ from type I IFNs, including upregulation of antigen presentation, control of immune cell trafficking, and activation of T cells, NK cells, and macrophages, we hypothesized that Ab-targeted IFNγ may have anti-tumor effects mechanistically-distinct from those of Ab-IFNα fusions. We now report on the construction and characterization of anti-CD20 fusions containing IFNγ. Methods: The VH and VL regions from antibody 2B8 recognizing human CD20 were engineered in recombinant form with mouse IgG2a constant regions, and fused at the C-terminus with mIFNγ, yielding anti-hCD20-mIFNγ. Tumor cell proliferation in vitro was measured by [3H]-thymidine incorporation, ADCC by LDH release using mouse splenocyte effectors, CDC by propidium iodide (PI) exclusion, and in vivo tumor growth assessed using the huCD20-expressing syngeneic mouse B cell lymphoma 38C13-huCD20. Tumor-infiltrating lymphocytes were measured by flow cytometry. Results: Anti-hCD20-mIFNγ displayed potent IFNγ bioactivity comparable to free recombinant mIFNγ, and suppressed the in vitro proliferation of 38C13-huCD20 lymphoma cells by up to 70% (at 1 nM), though not as potently as anti-hCD20-mIFNα, which inhibited proliferation by 98% (Figure 1). Anti-hCD20-mIFNγ showed enhanced ADCC against lymphoma cells compared with the unfused, parent antibody (16-20% at E:T ratio of 20:1, versus 9-12%, respectively, p=0.0024)(Figure 2), while CDC was identical to unfused antibody. In vivo efficacy was demonstrated in mice bearing established subcutaneous 38C13-huCD20 tumors, with systemic (i.v.) injection of 100 μg anti-hCD20-mIFNγ fusion protein on days 5, 6, 7, or 5, 6, 7, 9 after tumor inoculation resulting in cure of approximately 70-80% of mice in repeated experiments. In contrast, therapy with equimolar doses of unfused, native anti-hCD20 Ab resulted in no cures. Mechanistic studies in anti-hCD20-mIFNγ fusion protein-treated mice showed that depletion of natural killer (NK) cells (using anti-asialo-GM1) significantly abrogated tumor clearance (p=0.01), while depletion of macrophages (clodronate liposomes) had lesser, borderline effects (p= 0.05)(Figure 2), and depletion of complement (cobra venom factor) or T cells (CD4+ or CD8+) had no significant effects on tumor eradication. Subcutaneous mouse B cell lymphomas treated with intratumoral injections of anti-hCD20-mIFNγ displayed increased tumor-infiltrating CD8+ T cells (mean 20.6% versus 5% in PBS-treated controls, p=0.008), and CD4+ T cells (mean 15.3% versus 6.6%). Conclusions: Anti-hCD20-mIFNγ fusion protein has in vitro and in vivo efficacy in a syngeneic, immunocompetent model of B cell lymphoma, with NK cells and possibly macrophages implicated in the mechanism(s) of tumor eradication. Ab-targeted mIFNγ can also promote infiltration of immune cells into the tumor microenvironment. These findings may suggest a novel approach for the immunotherapy of B cell lymphomas and other cancers. Disclosures Vasuthasawat: Qwixel therapeutics LLC: Other: stake;which receives some funding through UCLA. Trinh:Qwixel therapeutics LLC: Other: stake;which receives some funding through UCLA. Morrison:Qwixel therapeutics LLC: Other: stake;which receives some funding through UCLA. Timmerman:ImmunGene: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Other: travel support, Research Funding; Merck: Research Funding; Kite, A Gilead Company: Consultancy, Honoraria, Other: travel support, Research Funding; Spectrum Pharmaceuticals: Research Funding.

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Antibody-Targeted Chemotherapy with Immunoconjugates of Calicheamicin: Differential Anti-Tumor Activity of Conjugated Calicheamicin Targeted to B-Cell Lymphoma Via B-Cell Lineage Specific Molecules CD19, CD20 and CD22.

Blood ◽

10.1182/blood.v104.11.2490.2490 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2490-2490 ◽

Cited By ~ 1

Author(s):

John F. DiJoseph ◽

Douglas C. Armellino ◽

Maureen M. Dougher ◽

Arthur Kunz ◽

Erwin R. Boghaert ◽

...

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Targeted Chemotherapy ◽

Phase I Clinical Trials ◽

Anti Cd20 ◽

Anti Tumor Activity ◽

Acid Labile

Abstract Antibody-targeted chemotherapy using tumor-targeted immunoconjugates of the cytotoxic agent, calicheamicin, is a clinically validated strategy for the treatment of acute myeloid leukemia. Calicheamicin is a potent cytotoxic natural product that binds DNA in the minor groove and causes double strand DNA breaks. B lymphoid lineage-specific antigens CD19, CD20, and CD22 have been studied extensively as potential targets for therapeutic applications of immunotoxins. In order to determine which one of these three antigens is most suitable for antibody-targeted calicheamicin therapy, we conjugated monoclonal antibodies, BU12 (murine anti-CD19 mAb), rituximab (chimeric anti-CD20 mAb), and m5/44 (murine anti-CD22 mAb) to a hindered disulfide derivative of N-acetyl gamma calicheamicin and evaluated the anti-tumor activity of these conjugates against three human B-cell lymphoma lines (BCL), Ramos, Raji and RL. Each of these three mAb bound to their respective antigens on the surface of BCL and was modulated, indicative of their potential internalization. Immunoconjugates of these mAbs, prepared by covalently linking calicheamicin via either acid-labile or acid-resistant linkers, caused a potent inhibition of BCL growth in vitro (IC50s ranged from 7 pM for the acid-labile linked m544 up to 6.8 nM for the acid-resistant linked anti-CD20 conjugates of calicheamicin). Immunoconjugates with acid-labile linkers were more potent than their counterparts with the acid-stable linker and conjugates targeted to either CD19 or CD22 were more potent than those targeted to CD20 in inhibiting BCL growth in vitro. In contrast, unconjugated mAb to CD19 or CD22 had no effect on BCL growth in vitro whereas anti-CD20 mAb, at concentrations >1 μg/ml, had an inhibitory effect of 30% on in vitro BCL growth. When examined for their effects on the growth of established subcutaneous BCL xenografts in nude mice, calicheamicin conjugated to anti-CD22 was by far the most efficacious conjugate against each of the three BCL xenografts studied. Calicheamicin conjugated to rituximab caused significant inhibition of BCL growth but was less effective than the conjugates of anti-CD22 or anti-CD19 mAb. Interestingly, anti-CD19 conjugates of calicheamicin, while effective in vitro against both Raji and Ramos BCL and effective against Raji BCL xenografts, had no effect on the growth of Ramos BCL xenografts in vivo. The reasons underlying the lack of anti-tumor activity of CD19-targeted calicheamicin conjugate against Ramos xenografts in vivo remain unknown. Based on a number of factors including the potent and consistent anti-tumor activity of the anti-CD22-conjugated calicheamicin, CD22 was selected as the molecular target for further development. A calicheamicin conjugate containing an acid-labile linker of humanized anti-CD22 mAb, CMC-544, is currently being evaluated in phase I clinical trials in non-Hodgkin’s B-cell lymphoma.

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Prolonged Inhibition of BCR Signaling and Suppression of B Cell Lymphoma through Irreversible Inhibition of Bruton’s Tyrosine Kinase

Blood ◽

10.1182/blood.v112.11.2608.2608 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2608-2608

Author(s):

Erica Evans ◽

Michael Sheets ◽

Hugues Bernard ◽

Thia St. Martin ◽

Mariana Nacht ◽

...

Keyword(s):

Tyrosine Kinase ◽

B Cell ◽

Signaling Pathway ◽

Kinase Activity ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Irreversible Inhibition ◽

Bcr Signaling

Abstract B-cell receptor (BCR) signaling is essential for normal B cell development and proliferation. In addition, functional BCR signaling contributes to the proliferation and survival of many B cell lymphomas through constitutive low level or tonic signaling. Disruption of this tonic signaling pathway results in cell death or decreased viability in many B cell lymphoma cells. Bruton’s tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases and plays a crucial role in the development and activation of B cells through association with the BCR signalosome. We have developed a novel, potent, irreversible inhibitor of Btk that selectively disrupts the BCR signaling pathway as measured by inhibition of both Btk Y223 autophosphorylation and phosphorylation of the Btk substrate PLCg2. AVL101 inhibits Btk but leaves upstream BCR kinase activity intact; both Syk phosphorylation at Y525/526 and transphosphorylation of Btk on residue Y551 are unaltered. By mass spectrometry, we have shown that AVL101 covalently modifies Btk specifically at Cys-481, and in cell-based assays, this irreversible binding leads to prolonged inhibition of Btk kinase activity. After removal of the compound, inhibition of Btk activity is maintained in cultured B cell lymphoma cells for > 8 hours. This prolonged inhibition correlates with the half-life of the Btk protein, which we have found to be greater than 8 hours, irrespective of BCR activation state or modification by the inhibitor. We have demonstrated that AVL101 functionally inhibits BCR signaling in vitro and in vivo. In vitro, AVL101 inhibits BCR ligand-induced calcium flux as well as proliferation of the B cell lymphoma cell lines DOHH2 and WSU-DLCL2. In vivo, we have shown AVL101 is orally bioavailable and can inhibit Btk-dependent B cell function. Our data demonstrate that Btk is a valid therapeutic target for B cell lymphomas and other disorders dependent on BCR signaling. Furthermore, irreversible inhibition of Btk enables complete and prolonged inactivation of the target kinase, and suggests that clinically, a rapid and sustained therapeutic response may be possible using an orally available, irreversible Btk inhibitor.

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The Addition of the BTK Inhibitor Ibrutinib to Anti-CD19 Chimeric Antigen Receptor T Cells (CART19) Improves Engraftment and Antitumor Responses Against Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v126.23.704.704 ◽

2015 ◽

Vol 126 (23) ◽

pp. 704-704

Author(s):

Marco Ruella ◽

Saad S Kenderian ◽

Olga Shestova ◽

Joseph A. Fraietta ◽

Sohail Qayyum ◽

...

Keyword(s):

B Cell ◽

Cell Line ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

University Of Pennsylvania ◽

Tumor Control

Abstract Introduction: The bruton tyrosine kinase (BTK) inhibitor ibrutinib demonstrates considerable activity in mantle cell lymphoma (MCL). However, approximately 30% of patients do not respond to this treatment and the therapy invariably leads to drug resistance with a median response of 17.5 months. Infusion of autologous T cells transduced with chimeric antigen receptors (CAR) against the B-cell specific CD19 antigen (CART19) leads to dramatic clinical responses in the majority of patients with acute lymphoblastic leukemia and the activity in B cell lymphoma is currently being evaluated in clinical trials. Bulky disease, as sometimes seen in MCL, may impair T cell infiltration. The features of ibrutinib that make it an interesting addition to CART19 include its efficacy in reducing tumor masses and its ability to mobilize neoplastic B cells into the peripheral blood, thereby potentially exposing them to the killing activity of CART19. Therefore, we sought to investigate the combination of the two novel targeted therapies, ibrutinib and CART19 in MCL. Results: In vitro studies with established MCL cell lines and with a novel cell line (MCL-RL) showed a range of responses to ibrutinib with an IC50 ranging from 10 nM to 10 µM; MCL-RL was the most sensitive cell line evaluated with an IC50 of 10nM, similar to primary MCL. Both ibrutinib-sensitive and ibrutinib-resistant cell lines strongly activated CART19 in an antigen-specific manner as detected by CD107a degranulation, cytokine production and CFSE proliferation assays. Importantly, in vitro assays with MCL cell lines co-cultured with increasing doses of CART19 (E:T= 2:1, 1:1, 0.5:1, 0.25:1) combined with increasing concentrations of ibrutinib (0, 10, 100, 1000 nM) demonstrated strong additive tumor killing (Figure 1). Notably, supra-therapeutic doses of Ibrutinib (>/=1 uM) impaired cytokine production and T cell proliferation in vitro. In order to test this combination in vivo we established a novel MCL model, injecting i.v. luciferase-positive MCL-RL cells into NSG mice. This resulted in 100% MCL engraftment in liver and spleen, with eventual dissemination into lymph nodes and bone marrow. Treatment with three different doses of CART19 (0.5, 1 and 2 million cells/mouse) led to a dose dependent anti-tumor effect. A similar dose response to CART19 was also observed in the ibrutinib-resistant Jeko-1 cell line. We also treated MCL-RL xenografts with different doses (0, 25 and 125 mg/Kg/day) of ibrutinib, with a median overall survival respectively of 70, 81 and 100 days (p<0.001). Importantly, a direct in vivo comparison of the highest ibrutinib dose (125 mg/kg) and CART19 showed a significantly improved tumor control for mice treated with CART19. However, treatment with either CART19 or ibrutinib as single agents invariably led to late relapse. Therefore we sought to treat MCL-RL xenografts with the combination of CART19 and ibrutinib and compare it to the single agent activity. The combination resulted in significant improvement in tumor control compared to mice treated with the single agents with 80% of mice achieving long-term disease-free survival ( p=0.007 at day 110, representative mice shown in Figure 2A). Intriguingly, we found that mice treated with ibrutinib had higher numbers of circulating CART19 cells (Figure 2B). Conclusions: Combining CART19 with ibrutinib represents a rational way to incorporate two of the most recent therapies in MCL. Our findings pave the way to a two-pronged therapeutic strategy in patients with MCL and other types of B-cell lymphoma. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Ruella: Novartis: Patents & Royalties, Research Funding. Kenderian:Novartis: Patents & Royalties, Research Funding. Maus:Novartis: Consultancy, Patents & Royalties, Research Funding. Milone:Novartis: Patents & Royalties, Research Funding. Lacey:Novartis: Patents & Royalties, Research Funding. Mato:Genentech: Consultancy; Pronai Pharmaceuticals: Research Funding; Celgene Corporation: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; TG Therapeutics: Research Funding; AbbVie: Consultancy, Research Funding; Janssen: Consultancy. Schuster:Genentech: Consultancy; Pharmacyclics: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Hoffman-LaRoche: Research Funding; Janssen: Research Funding; Gilead: Research Funding; Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding. Kalos:Novartis: Patents & Royalties, Research Funding. June:Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. Gill:Novartis: Patents & Royalties, Research Funding. Wasik:Janseen and Novartis: Research Funding.

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