Novel 3rd Generation Humanized Type II CD20 Antibody with Glycoengineered Fc and Modified Elbow Hinge for Enhanced ADCC and Superior Apoptosis Induction.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 229-229 ◽  
Author(s):  
Pablo Umana ◽  
Ekkehard Moessner ◽  
Peter Bruenker ◽  
Gabriele Unsin ◽  
Ursula Puentener ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Third Generation ◽  
Type Ii ◽  
Apoptosis Induction ◽  
B Cell Malignancies ◽  
Cd20 Antibody ◽  
Cd20 Antibodies

Abstract Background: Treatment of B-cell non-Hodgkin lymphoma (NHL) with antibodies targeting CD20 in conjunction with combination chemotherapy is standard clinical practice. Two different types of CD20 MAb differing significantly in their mode of CD20 binding and biological activities have been identified (Cragg and Glennie. Blood103: 2738–2743, 2004): type I antibodies, as rituximab, are potent in complement mediated cytotoxicity, whereas type II antibodies, as tositumomab, effectively initiate target cell death via caspase-independent apoptosis with concomitant phosphatidylserine exposure. GA101 is a humanized and optimized, third generation, type II CD20 IgG1 antibody that exhibits enhanced ADCC and superior caspase-independent apotosis induction in comparison with currently available CD20 MAbs. Material and Methods: GA101 was humanized by grafting CDR sequences from the murine monoclonal antibody B-ly1 on framework regions with fully human IgG1-kappa germline sequences. During humanization different elbow hinge sequences in the variable region were studied for their capability to induce apoptosis. Furthermore, the Fc region-carbohydrates were glycoengineered using GlycoMAb™ technology leading to bisected, afucosylated Fc region-carbohydrates. Results: The humanized GA101 antibody bound CD20 as type II antibody with nanomolar affinity. Its glycoengineered Fc region bound with 50-fold higher affinity to human FcgammaRIII receptors compared to a standard, non-glycoengineered antibody. Increased FcgammaRIII binding led to a 10–100-fold increase in ADCC against CD20-expressing NHL cell lines. Modification of elbow hinge sequences within the antibody variable framework regions resulted in a strong apoptosis-inducing activity of GA101 upon CD20 binding on target cells. Direct comparison to other CD20 antibodies GA101 showed enhanced apoptosis induction in both a panel of NHL cell lines and ex vivo in samples from patients with a variety of B-cell malignancies. Furthermore, in B-cell depletion assays with whole blood from healthy donors and B-cell leukemic patients, an assay combining ADCC-, CDC- and apoptosis-mediated mechanisms of action, GA101 was significantly more potent and efficacious than other CD20 antibodies, including rituximab and Fc-variants of rituximab that have increased ADCC. Finally, the in vitro superiority of GA101 also translated into superior efficacy in vivo. In NHL xenograft models of different histological origin, including aggressive DLBCL and MCL, treatment with GA101 results in complete tumor remission and long-term survival (cure) compared to tumor stasis, at best, for rituximab. Conclusion: Compared to existing CD20 antibodies GA101 represents a novel, third generation antibody with significantly enhanced efficacy in a variety of in vitro and in vivo preclinical models. GA101 constitutes the first type II CD20 antibody successfully engineered for increased ADCC. Based on these data, GA101 is a promising therapeutic antibody candidate for the treatment of B-cell malignancies.

Targeting the PI3K/mTOR Pathway in Lymphoma with PQR309 and PQR620: Single Agent Activity and Synergism with the BCL2 Inhibitor Venetoclax

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3017-3017
Author(s):  
Chiara Tarantelli ◽  
Eugenio Gaudio ◽  
Petra Hillmann ◽  
Filippo Spriano ◽  
Ivo Kwee ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Mtor Inhibitor ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Apoptosis Induction ◽  
In Vivo Experiments

Abstract Background. The PI3K/AKT/mTOR pathway is an important therapeutic target in lymphomas. PQR309 is a dual PI3K/mTOR inhibitor that has shown in vitroanti-lymphoma activity (Tarantelli et al, ASH2015) and is in phase 2 trial (NCT02249429, , NCT02723877, NCT02669511). PQR620 is a novel mTORC1/2 inhibitor that has shown preclinical activity in solid tumor models (Beaufils et al, AACR 2016). Here, we present the in vitro and in vivo anti-lymphoma activity of PQR620 as single agent and also the in vivo results of PQR620 or PQR309 containing combinations with the BCL2 inhibitor venetoclax. Materials and Methods. The drug concentration causing 50% inhibition of cell proliferation (IC50) was obtained in lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), no.=26; mantle cell lymphoma (MCL), no.=8; anaplastic large T-cell lymphoma, no.=5; others, no=5] exposed to increasing doses of PQR620 for 72h using a Tecan D300e Digital Dispenser on 384well plates. For in vivo experiments, NOD-Scid (NOD.CB17-Prkdcscid/J) mice were subcutaneously inoculated with 10 x106 (RIVA) or with 5 x106(SU-DHL-6) cells. Results. PQR620 had a median IC50 of 250 nM (95%CI, 200-269 nM) when tested on 44 lymphoma cell lines. Activity was higher in B cell (no.=36) than in T cell tumors (no.=8) (median IC50s: 250 nM vs 450 nM; P=0.002). At 72h, anti-tumor activityof PQR620 was mostly cytostatic and apoptosis induction was seen only in 6/44 cell lines (13%), Sensitivity to PQR620 or apoptosis induction did not differ between DLBCL and MCL, and they were not affected by the DLBCL cell of origin, by TP53 status or by the presence of MYC or BCL2 translocations. The activity of PQR620 as single agent underwent in vivo evaluation in two DLBCL models, the germinal center B cell type DLBCL (GCB-DLBCL) SU-DHL-6 and the acivated B cell-like DLBCL (ABC-DLBCL) RIVA. Treatments with PQR620 (100mg/kg dose per day, Qdx7/w) started with 100-150 mm3 tumors and were carried for 14 (SU-DHL-6) or 21 days (RIVA). In both models, PQR620 determined a 2-fold decrease of the tumor volumes in comparison with control, with significant differences in both SU-DHL-6 (D7, D9, D11, D14; P < 0.005) and RIVA (D14, D16, D19, D21; P < 0.005). Based on the previously reported synergy between the dual PI3K/mTOR inhibitor PQR309 and venetoclax (Tarantelli et al, ASH 2015), we evaluated the combination of the PQR620 or PQR309 with the BCL2 inhibitor venetoclax (100 mg/kg, Qdx7/w) in the SU-DHL-6 model. Both the venetoclax combination with the dual PI3K/mTOR inhibitor and the venetoclax combination with mTORC1/2 inhibitor were superior to the compounds given as single agents, leading to the eradication of the xenografts. The combination of PQR620 with venetoclax showed highly significant differences either versus control or single agents during all days of the experiment (D4, D7, D9, D11, D14; P < 0.001). Similarly, the combination of PQR309 with venetoclax showed highly significant differences versus venetoclax (D7, D9, D11, D14; P < 0.001) and PQR309 (D7, D9, D11; P < 0.005) alone. Conclusions. The novel mTORC1/2 inhibitor PQR620 had in vitro and in vivo anti-lymphoma activity as single agent. In vivo experiments showed that both PQR620 and the dual PI3K/mTOR inhibitor PQR309 can strongly benefit from the combination with the BCL2 inhibitor venetoclax. Disclosures Hillmann: PIQUR Therapeutics AG: Employment. Fabbro:PIQUR Therapeutics AG: Employment. Cmiljanovic:PIQUR Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees.

A New High Activity Anti-CD22 Chimeric Antigen Receptor (CAR) Targeting B Cell Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2611-2611
Author(s):  
Waleed Haso ◽  
Daniel W. Lee ◽  
Ira Pastan ◽  
Dimiter S. Dimitrov ◽  
Crystal L Mackall ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Cell Lines ◽  
Second Generation ◽  
Third Generation ◽  
Cytokine Release ◽  
Binding Motifs ◽  
Signaling Domains

Abstract Abstract 2611 CD22 is expressed on the surface of B cell hematologic malignancies such as acute lymphoblastic leukemia (ALL). CD22 is a Siglec family lectin present on B cells, starting at the pre-B cell stage of development, but is not expressed on plasma cells. CD22 consists of 7 extracellular Ig domains and is found in 2 isoforms, one of which is missing the second and third N-terminal Ig domains. We generated CAR modified T cells containing anti-CD22 extracellular binding motifs fused to intracellular signaling domains for T cells activation (CD3 zeta) or costimulation (CD28 or 4-1BB). The binding motifs were derived from scFvs that targeted a membrane distal epitope of CD22, Ig domain 3, (BL22 and a higher affinity HA22 motif) or that bound a more membrane proximal, Ig domains 5–7, of CD22 (m971). The CAR constructs we generated were second-generation (CD28 and CD3 zeta; or, 4-1BB and CD3 zeta) or third generation (CD28, 4-1BB and CD3 zeta signaling domains). A CH2CH3 spacer domain from IgG1 was added in some constructs to examine the impact of extending the scFv-derived binding domain away from the transduced T cell membrane. In vitro cellular cytotoxicity and cytokine release experiments with 4 B cell-ALL cell lines (REH, SEM, NALM6, KOPN8) as well as the CD22 (+)ve Daudi and Raji cell lines were performed. Our results demonstrate that addition of the CH2CH3 domain did not improve tumor lysis and that standard affinity BL22 and higher affinity HA22-derived scFv epitopes were equivalent. With regard to signaling domains, second generation constructs were better than third generation constructs both in vitro and in vivo. In comparison between second generation constructs, CD28 containing domains outperformed 4-1BB with regard to lytic activity and cytokine release. Most surprising was the activity of the m971-derived scFv binding epitope. m971-CAR had significantly higher killing activity, a far more robust cytokine release profile, and superior in vivo activity. NSG mice were injected i.v. with 0.5× 106 NALM6-GL cells (pre-B cell ALL line engineered to express luciferase). Three days later, when disease was evident, mice were treated with 1×107 CAR+ T cells, and then followed by bioluminescent imaging to measure disease burden. The m971 CAR was significantly more potent at tumor clearance than our previously developed most active construct expressing the HA22-derived scFv domain (Figure 1). Disease progressed rapidly when non-transduced T cells were used (mock). We are currently examining the activity of different signaling domains on m971 CAR efficacy in vivo and directly comparing the anti-CD22 m971 CAR to the CD19 CAR currently being evaluated in clinical trials. These studies will guide future anti-CD22 CAR-based anti-leukemia immunotherapy trials. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Therapeutic potential of SGN-CD19B, a PBD-based anti-CD19 drug conjugate, for treatment of B-cell malignancies

Blood ◽  
2017 ◽  
Vol 130 (18) ◽  
pp. 2018-2026 ◽  
Author(s):  
Maureen C. Ryan ◽  
Maria Corinna Palanca-Wessels ◽  
Brian Schimpf ◽  
Kristine A. Gordon ◽  
Heather Kostner ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Therapeutic Potential ◽  
Lymphoblastic Leukemia ◽  
Preclinical Models ◽  
B Cell Malignancies ◽  
Key Points

Key Points SGN-CD19B is broadly active in vitro against malignant B-cell lines, including double-hit and triple-hit lymphoma cell lines. SGN-CD19B shows significant antitumor activity in vivo in preclinical models of B-NHL and B-cell–derived acute lymphoblastic leukemia.

Development of REDX05194, a Novel, Potent and Selective Inhibitor of Bruton's Tyrosine Kinase (BTK) As a Potential Treatment for B-Cell Malignancies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4848-4848
Author(s):  
Victoria Walker ◽  
Nicolas E S Guisot ◽  
Stuart Best ◽  
Fatima Talab ◽  
Catherine L Lucas ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
B Cell ◽  
Cell Lines ◽  
Human Pbmcs ◽  
B Cell Malignancies ◽  
In Vivo Efficacy ◽  
Bcr Signaling ◽  
Btk Inhibitor

Abstract The B-cell receptor (BCR) signaling pathway is required for the survival, activation, proliferation and differentiation of B-cells. Bruton's Tyrosine Kinase (BTK) is a member of the Tec protein tyrosine kinase family that has emerged as an attractive target for the treatment of B-cell malignancies due to the critical role it plays in BCR signaling. Redx Oncology has developed novel differentiated small molecule inhibitors of BTK, combining current best-in-class potency with distinct selectivity profiles, which are suitable for oral once daily dosing. Here we present REDX05194, the result of a successful lead optimization of our proprietary BTK inhibitor series. REDX05194 is a highly selective, covalent BTK inhibitor displaying subnanomolar binding affinity for BTK (0.39 nM) and nanomolar potency towards BTK in a biochemical assay (3.67 nM). In cell proliferation assays, REDX05194 showed significant in vitro potency against ABC-DLBCL cell lines inhibiting the growth of both TMD-8 (0.89 nM) and OCI-Ly10 (1.36 nM) cells. Analysis of BCR signaling in several lymphoma cell lines, including cell lines of ABC-DLBCL, MCL and FL origin, revealed that treatment with REDX05194 inhibits BTK autophosphorylation and downstream activation of PLCγ2. In human PBMCs, REDX05194 inhibited anti-IgM stimulated upregulation of the CD69 activation marker in CD19 positive B-cells. In addition, using a fluorescent probe that binds to BTK, occupancy of BTK in PBMCs has been demonstrated in response to increasing concentrations of REDX05194. To assess selectivity, 456 kinases were screened at 1 μM, confirming that REDX05194 does not significantly inhibit other kinases involved in BCR signaling (e.g. Syk, Lyn). Furthermore, REDX05194 was shown to have high selectivity versus structurally related cysteine-containing kinases such as ITK in binding assays, and EGFR as demonstrated in both binding and cellular assays. REDX05194 also has a favorable in vitro safety profile and drug-like properties, displaying an improved CYP profile and solubility compared to competitor compounds. REDX05194 demonstrated in vivo efficacy in a mouse collagen-induced arthritis (CIA) model. At 10 mg/kg and 30 mg/kg QD, REDX05194 significantly improved all clinical readouts, including disease severity, compared to the vehicle group. Histological data showed that approximately 1/3 of the mice had no or minimal pannus infiltration and no bone resorption, or had bone resorption restricted to small areas. These findings demonstrate potential clinical efficacy and a dose response. In conclusion, REDX05194 is a highly selective and potent BTK inhibitor with proven efficacy in several lymphoma cell lines and human PBMCs and in vivo efficacy demonstrated in a mouse CIA model. Disclosures No relevant conflicts of interest to declare.

scholarly journals Development of CD19 CAR Engineered Human Placental CD34 +-Derived Natural Killer Cells (CAR19-CYNK) As an Allogeneic Cancer Immunotherapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2779-2779
Author(s):  
Marina Gergues ◽  
Irene Raitman ◽  
Joseph Gleason ◽  
Valentina Rousseva ◽  
Shuyang He ◽  
...  
Keyword(s):  
B Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Publicly Traded ◽  
B Cell Malignancies ◽  
Cd34 Cells ◽  
Anti Tumor Activity

Abstract Background: Natural killer (NK) cells exhibit anti-tumor activity in a non-antigen-specific manner without causing graft-versus-host disease. T cell and cord blood NK cells expressing chimeric antigen receptor (CAR) targeting CD19 have demonstrated remarkable clinical efficacies against B cell lymphomas (Maude et al, N Engl J Med 2018; Neelapu et al, N Engl J Med 2017; Liu et al, N Engl J Med 2020). Celularity has developed a platform for the expansion and differentiation of human placental CD34 + stem cells towards NK cells. The introduction of CD19 CAR enables generation of CAR19-CYNK cells that can be used as an off-the-shelf, cryopreserved, allogeneic cell therapy for CD19 + B cell malignancies. Reported here are the in vitro and in vivo results evaluating anti-tumor activity of CAR19-CYNK against CD19 + B cell malignancies. Methods: CAR19-CYNK cells were generated by retroviral transduction of human placental CD34 + cells with an anti-CD19 CAR (CD19scFv-CD28CD3ζ, Sorrento Therapeutics), followed by culture expansion in the presence of cytokines. CD19 CAR expression and phenotype of CAR19-CYNK cells were characterized by flow cytometry using the following surface markers: CD56, CD3, CD226, CD16, CD11a, CD94, NKG2D, NKp30, NKp44, NKp46. The in vitro anti-tumor activity of CAR19-CYNK against the B cell lymphoma cell lines, Daudi and Nalm-6, was assessed at various effector to target (E:T) ratios using a flow cytometry-based cytotoxicity assay and multiplex Luminex analysis for cytokine profiling. Non-transduced (NT) NK cells were used as control. In vivo efficacy of CAR19-CYNK was assessed using a disseminated B-cell lymphoma xenograft model in B-NDG-hIL15 mice. B-NDG-hIL15 mice lack T, B, and NK cells and are transgenic for human IL-15 to support CAR19-CYNK persistence and maturation. Luciferase expressing Daudi cells (3×10 6) were intravenously (IV) injected on Day 0 three days after the mice were preconditioned with a myeloablative dose of busulfan to allow for better tumor cell engraftment. CAR19-CYNK cells (1x10 7) were IV injected on Day 7. Tumor burden was assessed weekly by bioluminescence imaging (BLI) and the mice were followed for assessment of their survival (n=5 mice per group). Results: Placental CD34 + cells were genetically modified using a retroviral vector and achieved an average of 29.2% ± 12.4% (range 17.5% to 50.1%; n=5 donor lots) CD19 CAR expression on CAR19-CYNK cells at the end of 35-day culture. The average fold expansion of CAR19-CYNK was 6186 ± 2847 with the range of 2692 to 10626 (n=5 donor lots). Post-thaw evaluation of CAR19-CYNK (n=5 donor lots) revealed 93.8 ± 3.9% of CD56 +CD3 - NK cells, and transduction of CD19 CAR on CYNK did not significantly alter NK cell phenotype based on various activation and lineage markers (CD226, CD16, CD11a, CD94, NKG2D, NKp30, NKp44, NKp46). CAR19-CYNK displayed enhanced in vitro cytotoxicity against lymphoma cell lines, Daudi and Nalm-6, compared to that of NT NK cells. At the E:T ratio of 10:1, CAR19-CYNK (n=5 donor lots) elicited significant increased cytotoxicity against Nalm-6 compared to that of NT NK cells, with 75.9 ± 14.8% vs. 0.00 ± 0.00% at 24h (p&lt;0.005). Under the same condition, CAR19-CYNK (n=4 donor lots) showed higher cytotoxicity against Daudi compared to that of NT NK cells with 23.6 ± 18.9% vs. 4.9 ± 4.0%. When cocultured with tumor cell lines, CAR19-CYNK showed increased secretion of the proinflammatory cytokines GM-CSF (p&lt;0.05 for both Nalm-6 and Daudi), IFN-g (p&lt;0.05 for Nalm-6), and TNF-a compared to that of NT NK cells at an E:T ratio of 1:1 for 24h. To evaluate the in vivo efficacy of CAR19-CYNK, a disseminated Daudi xenograft B-NDG-hIL15 model was used. CAR19-CYNK treated mice demonstrated a significant survival benefit with a median survival of 39 days versus a median survival of 28 days for the vehicle treated group (p&lt;0.05). Conclusions: In summary, we have successfully established a process for generating CAR19-CYNK cells from human placental CD34 + cells. CAR19-CYNK demonstrated enhanced in vitro cytotoxicity against CD19 + B cell malignancies and in vivo survival benefit in a disseminated lymphoma xenograft B-NDG-hIL15 model. Further development of CAR19-CYNK for CD19 + B cell malignancies is warranted. Disclosures Gergues: Celularity Inc: Current Employment, Current equity holder in publicly-traded company. Raitman: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Gleason: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Rousseva: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. He: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Van Der Touw: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Ye: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Kang: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang: Sorrento Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Pai: Sorrento Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Guo: Sorrento Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Ji: Sorrento Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Hariri: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang: Celularity Inc.: Current equity holder in publicly-traded company, Ended employment in the past 24 months.

Targeting B-Cell Malignancies With Anti-CD20-Interleukin-21 Fusokine

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 377-377 ◽  
Author(s):  
Shruti Bhatt ◽  
Daxing Zhu ◽  
Xiaoyu Jiang ◽  
Seung-uon Shin ◽  
John M Timmerman ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cd20 Antibody ◽  
Anti Cd20

Abstract The anti-CD20 antibody rituximab has revolutionized the treatment for B cell non-Hodgkin lymphomas (NHLs). However, rituximab has limited effectiveness as a single agent in some NHL subtypes and its clinical efficacy is compromised by acquired drug resistance. As a result, many patients still succumb to NHLs. Hence, strategies that enhance the activity of anti-CD20 antibody may improve patient outcome. Interleukin-21 (IL21), a member of the IL2 cytokine family, exerts diverse regulatory effects on natural killer (NK), T and B cells. IL21 has been reported to possess potent anti-tumor activity against a variety of cancers not expressing IL21 receptor (IL21R) through activation of the immune system and is in clinical trials for renal cell carcinoma and metastatic melanoma. We have recently reported that apart from immuno-stimulatory effects, IL21 exerts direct cytotoxicity on IL21R expressing diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cell lines and primary tumors both in vitro as well in vivo (Sarosiek et al Blood 2010; Bhatt et al AACR 2013). Herein we designed a fusion protein comprising IL21 linked to the N-terminus of anti-CD20 antibody (αCD20-IL21 fusokine) to improve efficacy of its individual components and prolong IL21 half-life. We have verified the expression of full length fusion protein and demonstrated that αCD20-IL21 fusokine retained binding ability to its individual components; CD20 and IL21R, as analyzed by immunofluorescence and flow-cytometry analyses. Similar to our previous study of IL21 in DLBCL, treatment of B cell lymphoma cell lines with fusokine lead to phosphorylation of STAT1 and STAT3, upregulation of cMYC and BAX and downregulation of BCL-2 and BCL-XL, implying the activation of IL21R dependent signaling to trigger cytotoxic effects. In vitro, direct cell death induced by αCD20-IL21 fusokine in DLBCL (RCK8, WSU and Farage) and MCL (Mino, HBL2 and SP53) cell lines was markedly increased compared to its individual components (IL21 and parent αCD20-IgG1 antibody). More importantly, fusokine treatment resulted in cell death of MCL cell lines (L128, G519 and UPN1) that were found to be resistant to IL21 alone treatment. Furthermore, treatment of freshly isolated primary NHL cells with the αCD20-IL21 fusokine also exhibited a 40-50% increase in direct cell death compared to its individual components. Previous studies reported that IL21 enhances antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies by activation of NK cells. ADCC assays using chromium release with purified human NK cells demonstrated that ADCC induced by the parent antibody was enhanced in the presence of IL21 while IL21 alone had minimal effect on the lysis of Raji, Daudi, and Jeko1 target cells. Notably, αCD20-IL21 fusokine demonstrated increased ADCC activity in comparison to parent antibody plus IL21 in Raji, Daudi and Jeko-1 cells (p<0.001, p<0.005 and p<0.001, respectively). Similar results were obtained in primary MCL tumor cells. Consistent with this finding, fusokine treatment resulted in enhanced activation of the NK cells as assessed by CD69 upregulation and CD16 downregulation using flow-cytometry. Complement dependent cytotoxicity (CDC) of the fusokine was similar to the parent antibody and rituximab in Raji cells. Studies analyzing in vivo effects of the fusokine are in progress and will be presented at the meeting. These data strongly suggest that together with direct apoptotic potential, an anti-CD20 IL21 fusokine retains the ability to trigger indirect cell killing mediated via activation of immune effector cells. These dual effects may give remarkable advantage to the fusokine over existing anti-CD20 antibodies for the treatment of NHL tumors. Collectively, our study demonstrates that anti-tumor effects of IL21 and anti-CD20 antibodies can be enhanced by conjugation of IL21 with anti-CD20 antibody that may serve as a novel anti-lymphoma therapy. Disclosures: Rosenblatt: Seattle Genetics, Inc.: Research Funding.

Phosphatidylinositol-3 Kinase Delta (PI3Kδ) Inhibitor AMG 319 Is a Potent, Selective and Orally Bioavailable Small Molecule Inhibitor That Suppresses PI3K-Mediated Signaling and Viability in Neoplastic B Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4964-4964 ◽  
Author(s):  
Angus Sinclair ◽  
Daniela Metz ◽  
Tim Cushing ◽  
Liqin Liu ◽  
Rachael Brake ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Small Molecule ◽  
Single Agent ◽  
Chemotherapeutic Agents ◽  
B Cell Malignancies ◽  
Molecule Inhibitor ◽  
Human B Cell

Abstract Abstract 4964 Immune receptors such as the B cell receptor (BCR) require key signaling intermediate phosphatidylinositol-3 kinase delta (PI3Kδ) for normal immune cell survival, development and function. PI3Kδ is a class IA lipid kinase, is expressed primarily within the hematopoietic system and is composed of a catalytic subunit p110δ and a regulatory subunit p85. Recently, deregulated BCR-PI3Kδ signaling has been reported to play a role in B-cell malignancies such as chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL) by mediating abnormal B-cell growth and survival. Indeed, the constitutive phosphorylation of downstream signaling intermediate AKT is associated with poor prognosis in several B cell malignancies. Here, we have investigated the potential of a novel small molecule inhibitor of PI3Kδ, AMG 319, to suppress PI3K signaling in human B cell lines and assessed the subsequent effects on viability as a single agent and in combination with chemotherapeutic drugs in preclinical models. Small molecule AMG 319 is a potent and selective inhibitor of PI3Kδ with excellent preclinical pharmacokinetic (PK) properties. AMG 319 was found to potently inhibit PI3Kδ in enzyme assays (IC50 <10 nM). AMG 319 also potently suppressed the phosphorylation of AKT (pAKTS473) in primary murine splenocytes (IC50<5 nM) after BCR cross linking and demonstrated a less than 10 fold shift in human whole blood B cells using a similar BCR pAKT assay in vitro. In a cell based selectivity screen, AMG 319 was selective for PI3Kδ against other PI3K class I isoforms (200 to >5000 fold). Furthermore, AMG 319 was considered inactive at 10 μM on non-PI3K class I kinases in a broader kinome screen of 402 kinases. In preclinical PK studies, AMG 319 had low systemic clearance, T1/2 range of 2–4 hr, oral bioavailability of >45% and unbound fractions in plasma of 5–19%. Here, we have investigated the potential for AMG 319 to inhibit constitutive PI3K mediated signaling and effects on human B cell line viability. In a broad screen of >20 cell lines derived from B cell malignancies, the majority of lines were found to express PI3Kδ protein, all cells lines expressed the PI3Kα and β isoforms and variable levels of constitutive pAKTS473 were detected. AMG 319 was found to potently suppress constitutive pAKTS473 in the cell lines with IC50 in the low single to double digit nM range. Cellular viability was inhibited by AMG 319 though lines were variably sensitive to drug (range low double digit nM to μM IC50). As cell lines were variably sensitive to AMG 319 as a single agent, we examined if AMG 319 could enhance the efficacy of chemotherapeutic agents in vitro and in vivo. These studies focused on a DLBCL cell line HT which was relatively insensitive to AMG 319 as a single agent (IC50 ∼10 μM) in viability assays even though pAKTS473 was potently suppressed (IC50 ∼ 0.030 μM). Treatment with AMG 319 was found to synergize with the effects of vincristine to reduce cell viability in vitro using a 72 hr viability assay. Next we examined whether the enhanced cytotoxicity using these drugs in combination could be observed in vivo. Using the human B-cell lymphoma HT xenograft model, we found that AMG 319 in combination with vincristine enhanced tumor growth inhibition above that observed with either agent alone. Taken together, these findings suggest that the inhibition of PI3Kδ with AMG 319 may enhance the effects of chemotherapeutic agents in B cell malignancies. In conclusion, AMG 319 is a potent and selective inhibitor of PI3Kδ with excellent PK properties. AMG 319 inhibited constitutive pAKTS473, reduced the viability of B cell lines and synergized with vincristine in vitro and in vivo. The safety, PK and preliminary efficacy of AMG 319 are currently being investigated in a Phase I trial in patients with relapsed or refractory lymphoid malignancies. Disclosures: Sinclair: Amgen: Employment, Stock and Options. Metz:Amgen, Inc: Employment, Stock and Options. Cushing:Amgen, Inc: Employment, Stock and Options. Liu:Amgen, Inc: Employment, Stock and Options. Brake:Amgen, Inc: Employment, Stock and Options. Starnes:Amgen, Inc: Employment, Stock and Options. Means:Amgen, Inc: Employment, Stock and Options. Henne:Amgen, Inc: Employment, Stock and Options. Archibeque:Amgen: Employment, Stock and Options. Mattson:Amgen, Inc: Employment, Stock and Options. Drew:Amgen, Inc: Employment, Stock and Options. Busse:Amgen, Inc: Employment, Stock and Options. Wang:Amgen, Inc: Employment, Stock and Options. Al-Assaad:Amgen, Inc: Employment, Stock and Options. Molineux:Amgen: Employment, Stock and Options.

Pre-Clinical Development of Adct-402, a Novel Pyrrolobenzodiazepine (PBD)-Based Antibody Drug Conjugate (ADC) Targeting CD19-Expressing B-Cell Malignancies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1564-1564 ◽  
Author(s):  
Francesca Zammarchi ◽  
David G. Williams ◽  
Lauren Adams ◽  
Karin Havenith ◽  
Simon Chivers ◽  
...  
Keyword(s):  
Single Dose ◽  
B Cell ◽  
Cell Lines ◽  
Xenograft Model ◽  
B Cell Malignancies ◽  
Employment Equity ◽  
Equity Ownership ◽  
Anti Tumor Activity

Abstract Human CD19 antigen is a 95 kilodalton type I transmembrane glycoprotein belonging to the immunoglobulin superfamily (Wang, Wei, & Liu, 2012). The role of CD19, both in health and disease, is well studied, and the therapeutic efficacy and safety of CD19 modulation have been well defined over several decades (Scheuermann & Racila, 1995). In normal human tissue, expression of CD19 is limited to the various stages of B-cell development and differentiation (except plasma cells) and its expression is maintained on the majority of B-cell malignancies, including B-cell leukemia and non-Hodgkin lymphomas of B-cell origin. CD19 has rapid internalization kinetics and it is not shed into the circulation (Blanc et al., 2011; Gerber et al., 2009). All these features make CD19 an attractive target for the development of an ADC to treat B-cell malignancies. ADCT-402 is an ADC composed of a humanized antibody directed against human CD19, stochastically conjugated via a valine-alanine cleavable, maleimide linker to a PBD dimer cytotoxin. PBD dimers are highly efficient anticancer drugs that covalently bind in the minor groove of DNA and form cytotoxic DNA interstrand cross-links. The average drug to antibody ratio of ADCT-402 is 2.3 ± 0.3, as shown by hydrophobic interaction chromatography and reverse-phase HPLC. In vitro, ADCT-402 demonstrated potent cytotoxicity in a panel of human-derived cell lines of differing levels of CD19, while its potency was strongly reduced in CD19-negative cell lines. In vivo, ADCT-402 demonstrated dose-dependent anti-tumor activity in a subcutaneously implanted human Burkitt's lymphoma-derived Ramos xenograft model, where a single dose at 0.33 mg/kg induced significantly delayed tumor growth compared to the vehicle-treated mice and at 0.66 mg/kg and 1 mg/kg gave 4/10 and 10/10 tumor-free survivors, respectively. In the same model, ADCT-402 showed remarkably superior anti-tumor activity compared to both maytansinoid- and auristatin-based CD19-targeting ADCs, when they were tested at the same dose and schedule (1 mg/kg, single dose). Moreover, ADCT-402 mediated an impressive increase in survival compared to both vehicle-treated and isotype control ADC-treated mice in the disseminated Ramos xenograft model when tested as a single dose at 0.33 mg/kg or 1 mg/kg. For example, a single dose of ADCT-402 at 1 mg/kg resulted in 10/10 survivors at day 91, while there were 0/10 survivors at day 19 in the group of animals treated with either the vehicle control or with a single dose of the non-binding, control ADC at 1 mg/kg. In rat, a single dose of ADCT-402 at 2 mg/kg was well tolerated with no adverse signs or hematologic effects. Altogether, these data show the potent and specific anti-tumor activity of ADCT-402 against CD19-expressing B-cell malignancies, both in vitro and in vivo, and warrant further development of this ADC into the clinic. Disclosures Zammarchi: ADC Therapeutics: Employment. Williams:Spirogen/Medimmune: Employment. Adams:Spirogen/Medimmune: Employment, Equity Ownership. Havenith:ADC Therapeutics: Employment. Chivers:ADC Therapeutics: Employment. D'Hooge:Spirogen/Medimmune: Employment, Equity Ownership. Howard:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Patents & Royalties. Hartley:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. van Berkel:ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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