Lenalidomide (Revlimid®) Enhances Monoclonal Antibody-Associated Anti-Tumor Activity Against Rituximab-Sensitive and Rituximab-Resistant B-Cell Lymphoma Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2522-2522 ◽  
Author(s):  
Nishitha Reddy ◽  
Raymond Cruz ◽  
Francisco Hernandez-Ilizaliturri ◽  
Joy Knight ◽  
Myron S. Czuczman
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Scid Mouse ◽  
In Vitro Exposure ◽  
Human Lymphoma ◽  
Scid Mouse Model ◽  
Anti Cd20 ◽  
Anti Tumor Activity

Abstract Background: Lenalidomide is a potent thalidomide analogue shown to activate both the innate and adoptive immune system, inhibit angiogenesis, and modify the tumor microenvironment. While lenalidomide has received approval by the U.S. Federal Drug Administration (FDA) for the treatment of various hematological conditions, ongoing clinical trials are addressing its role in the treatment of B-cell lymphomas. There is a dire need to develop novel well-tolerated, therapies which combine various target-specific agents such as lenalidomide and monoclonal antibodies (mAbs). We previously demonstrated that lenalidomide is capable of expanding natural killer (NK) cells in a human-lymphoma-bearing SCID mouse model and improve rituximab anti-tumor activity in vivo. Methods: In our current work we studied the effects of lenalidomide on the biological activity of a panel of mAbs against various B-cell lymphomas, utilizing various rituximab-sensitive (RSCL) and rituximab-resistant cell lines (RRCL) generated in our laboratory from Raji and RL cell lines. Functional assays including antibody-dependant cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CMC) were performed to demonstrate changes in sensitivity to rituximab. RSCL and RRCL (1′105 cells/well) were exposed to either lenalidomide (5 μg/ml) or vehicle with or without mAb at a final concentration of 10μg/ml. The mAb panel consisted of two anti-CD20 mAbs: rituximab (Biogen IDEC, Inc.) and hA20, a humanized anti-CD20 mAb (Immunomedics, Inc.); an anti-CD80 mAb (galixumab, Biogen IDEC Inc.), and an anti-CD52 antibody (Alemtuzumab, Berlex Inc.). Changes in DNA synthesis and cell proliferation were determined at 24 and 48 hrs by [3H]-thymidine uptake. For ADCC/CMC studies, NHL cells were exposed to lenalidomide or vehicle for 24 hrs and then labeled with 51Cr prior to treatment with one of various mAbs (10 mg/ml) and peripheral blood mononuclear cells (Effector: Target ratio, 40:1) or human serum, respectively. 51Cr-release was measured and the percentage of lysis was calculated. Changes in antigen (CD20, CD80, and CD52) expression following in vitro exposure to lenalidomide were studied by multicolor flow cytometric analysis. Results: Concomitant in vitro exposure of various RSCL and RRCL cells to lenalidomide and either galixumab, hA20 or alemtuzumab for 24 hrs resulted in improved anti-tumor activity when compared to controls. In addition, pre-incubation of both RSCL and RRCL with lenalidomide rendered cells more susceptible to alemtuzumab-, hA20- and galixumab-mediated ADCC and CMC. No antigen modulation (i.e., upregulation) was observed following in vitro exposure of lenalidomide to NHL cell lines, suggesting an alternative mechanism involved in the improvement antitumor activity observed. Conclusions: Our data suggest that the augmented antitumor effect of lenalidomide is not limited to its combination with rituximab, but also that it augments the antiproliferative and biological activity of alemtuzumab, hA20 and galixumab. Furthermore, these interactions are observed even in our RRCL. Future studies will be directed towards evaluating whether similar activity will be seen in vivo using a human lymphoma-bearing SCID mouse model. (Supported by USPHS grant PO1-CA103985 from the National Cancer Institute.)

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 ◽  
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.

Itraconazole, an Oral Antifungal Drug, Is Active in Chemotherapy Resistant B-Cell Non-Hodgkin Lymphoma and Enhances the Anti-Tumor Activity of Chemotherapy Agents

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5138-5138
Author(s):  
Juan J Gu ◽  
Lianjuan Yang ◽  
Cory Mavis ◽  
Matthew J. Barth ◽  
Francisco J. Hernandez-Ilizaliturri
Keyword(s):  
Cell Cycle ◽  
Membrane Potential ◽  
B Cell ◽  
Mitochondrial Membrane Potential ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
In Vitro Exposure ◽  
Chemotherapy Agents ◽  
Anti Tumor Activity

Abstract Background: Relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients previously treated with rituximab-based therapy have poor clinical outcome, according to the results from collaborative trial in relapsed aggressive lymphoma (CORAL) study. It stresses the need to identify and/or optimize novel targeted agents. To better understand the molecular mechanisms underlining the acquired resistance to rituximab, we generated and characterized several rituximab-resistant DLBCL cell lines (RRCLs). Itraconazole, an oral antifungal agent, was reported had novel anticancer activity in basal cell carcinoma, non-small cell lung cancer and prostate cancer. In our current work, we define and characterize the anticancer activity of itraconazole in preclinical rituximab-sensitive or -resistant lymphoma models. Methods: A panel of rituximab-sensitive (RSCL) and rituximab-resistant (RRCL) cell lines were exposed to escalating doses of itraconazole (0-20μM) for 24, 48 and 72h. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 was calculated by Graphpad Prism6 software. Loss of mitochondrial membrane potential (∆ψm) following itraconazole exposure was assessed by DiOC6 and flow cytometry. Subsequently lymphoma cells were exposed to itraconazole or vehicle and various chemotherapy agents such as doxorubicin (1µM), dexamethasone (1µM), cDDP (20μg/ml), bortezomib (20nM), carfilzomib (20nM) or MLN2238 (20nM) for 48 hours. Coefficient of synergy was calculated using the CalcuSyn software. Changes in hexokinase II (HKII), Voltage dependent anion channel protein (VDAC), LC3 and BCL-xL expression levels were determined by western blotting after exposure cells to itraconazole. VDAC-HKII interactions following in vitro exposure to itraconazole were determined by immunoprecipitation of VDAC and probing for HKII in RSCL and RRCLs. Result:Itraconazole consistently showed potent, specific, dose-and time- dependent inhibition of all our sensitive and resistant lymphoma cell lines. In vitro exposure cells to itraconazole resulted in a loss of mitochondrial membrane potential and caused G2 cell cycle arrest. Itraconazole significantly had a synergistic anti-tumor effect combined with various chemotherapeutic agents, including doxorubicin, dexamethasone, cisplatin and different generations of proteasome inhibitors (bortezomib, carfilzomib or ixazomib) in both RSCL and RRCL. Western blot and immunoprecipitation studies demonstrated that following exposure to itraconazole, HKII bound less to mitochondrial specific protein VDAC. Complete silencing of HKII (using HKII siRNA interference) resulted in a rescue of loss in the mitochondrial membrane potential induced by intraconazole. Conclusion: Taking together, our data suggest that itraconazole had a potent anti-tumor activity against rituximab-sensitive or resistant pre-clinical models. The disruption of HKII from mitochondria following itraconazole exposure may contribute to lower the mitochondrial membrane potential and enhance the chemotherapeutic efficacy. Our finding highlights itraconazole as a potential therapeutic agent in the treatment of B-cell malignancies, and strongly supports clinical translation of its use. Disclosures No relevant conflicts of interest to declare.

Comparison of Biodistributions and Therapeutic Efficacies of Pretargeted Radioimmunoconjugates Targeting the CD20, CD22, and DR Molecules on Human B Cell Lymphomas.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 611-611
Author(s):  
Anastasia Pantelias ◽  
John M. Pagel ◽  
Nathan Hedin ◽  
Yukang Lin ◽  
Donald Axworthy ◽  
...  
Keyword(s):  
Cell Lines ◽  
Half Life ◽  
Lymphoma Cell ◽  
In Vivo Studies ◽  
Cell Binding ◽  
Normal Tissues ◽  
Human Lymphoma ◽  
Anti Cd20

Abstract Radioimmunotherapy (RIT) using anti-CD20 monoclonal antibodies (Ab) produces response rates of 60–95% in relapsed non-Hodgkin’s lymphoma (NHL) patients; however, tumor-to-normal organ ratios of absorbed radiation are low and many patients relapse. The efficacy of RIT is limited by non-specific delivery of radiation to normal tissues due to the long circulating half-life of radiolabeled antibodies. Pretargeted RIT (PRIT) using streptavidin (SA)-Ab conjugates followed by a clearing agent and radiolabeled biotin can augment the efficacy of RIT and decrease toxicity compared with conventional RIT. Although PRIT using anti-CD20-SA Abs have been studied with promising results, targeting multiple antigens may increase efficacy. Since successful clinical trials have been conducted with directly radiolabeled anti-DR and anti-CD22 Abs, we initiated in vitro and in vivo studies comparing pretargeted anti-CD20 Ab-SA conjugate (1F5/SA) with pretargeted anti-CD22-SA (HD39/SA) and anti-HLA-DR-SA (Lym-1/SA) conjugates in three different human B-lymphoma cell lines, RAMOS (Burkitt), RAJI (Burkitt) and FL-18 (transformed follicular). Using standard flow cytometry techniques all three Ab-SA conjugates bound to ≥ 97% of FL18 cells. Cell binding for 1F5/SA, Lym-1/SA, and HD39/SA was 99%, 99%, and 83% to RAJI cells, respectively, and 99%, 22%, and 85% to RAMOS cells. The blood half-life of each conjugate in vivo was measured by injecting groups of 4 mice i.v. with 0.7nmol (150μg) of 125I labeled Ab-SA conjugate and drawing 10μl of blood at various time points to determine the percent injected dose per gram (% ID/g). The half-lives of 1F5/SA, Lym-1/SA and HD39/SA were 18.38, 14.92 and 16.23 hours, respectively. When 5.8nmol (50μg) of a clearing agent (synthetic biotin-N-acetyl-galactosamine) was given 24 hours post 125I-Ab-SA injection, the % ID/g in blood fell by more than 80% of the initial dose within a half-hour. Blood, tumor and non-specific organ uptake was determined by biodistribution experiments in mice (Balb/c nu/nu) bearing human lymphoma xenografts. Athymic mice with s.c. RAMOS, RAJI, or FL-18 xenografts received 1.4nmol (300μg) of either 1F5/SA, HD39/SA, or Lym-1/SA i.v. followed 24 hours later by 5.8nmol (50μg) clearing agent to remove non-localized conjugate from circulation, and 3 hours later by an 111In labeled DOTA-biotin ligand (1μg). The biodistributions of each conjugate were evaluated by sacrificing mice at 24 and 48 hours after 111In-DOTA-biotin. At 24 hours, the ID/g was 18.2±13.6% in FL18 xenografts for pretargeted Lym-1/SA, 18.2±17.4% ID/g for 1F5/SA and 3.3±0.7% ID/g for HD39/SA. Conversely, at 24 hours pretargeted Lym-1/SA uptake in RAJI tumors was 10.8±2.1% ID/g, and 1F5/SA and HD39/SA RAJI tumor localization was 5.2±1.9% ID/g and 2.2±0.5% ID/g. respectively. 1F5/SA had superior uptake (7.1±3.3% ID/g) in RAMOS xenografts compared with Lym-1/SA (3.5±1.5% ID/g) and HD39/SA (2.7±1.0% ID/g). These data suggest a strong correlation between in vitro cell binding results and in vivo biodistributions for all three Ab-SA conjugates in all three human lymphoma cell lines. Using these agents in combination may result in a synergistic effect that has the potential to increase the efficacy of PRIT over using any one of the agents alone. Biodistribution and therapy studies using the Ab-SA conjugates in combination in tumored mice are on-going.

scholarly journals Properties and structure-function relationships of veltuzumab (hA20), a humanized anti-CD20 monoclonal antibody

Blood ◽  
2009 ◽  
Vol 113 (5) ◽  
pp. 1062-1070 ◽  
Author(s):  
David M. Goldenberg ◽  
Edmund A. Rossi ◽  
Rhona Stein ◽  
Thomas M. Cardillo ◽  
Myron S. Czuczman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Lines ◽  
Single Amino Acid ◽  
High Dose ◽  
Non Hodgkin Lymphoma ◽  
Single Amino Acid Change ◽  
Human Lymphoma ◽  
Anti Cd20

Abstract Veltuzumab is a humanized anti-CD20 monoclonal antibody with complementarity-determining regions (CDRs) identical to rituximab, except for one residue at the 101st position (Kabat numbering) in CDR3 of the variable heavy chain (VH), having aspartic acid (Asp) instead of asparagine (Asn), with framework regions of epratuzumab, a humanized anti-CD22 antibody. When compared with rituximab, veltuzumab has significantly reduced off-rates in 3 human lymphoma cell lines tested, aswell as increased complement-dependent cytotoxicity in 1 of 3 cell lines, but no other in vitro differences. Mutation studies confirmed that the differentiation of the off-rate between veltuzumab and rituximab is related to the single amino acid change in CDR3-VH. Studies of intraperitoneal and subcutaneous doses in mouse models of human lymphoma and in normal cynomolgus monkeys disclosed that low doses of veltuzumab control tumor growth or deplete circulating or sessile B cells. Low- and high-dose veltuzumab were significantly more effective in vivo than rituximab in 3 lymphoma models. These findings are consistent with activity in patients with non-Hodgkin lymphoma given low intravenous or subcutaneous doses of veltuzumab. Thus, changing Asn101 to Asp101 in CDR3-VH of rituximab is responsible for veltuzumab's lower off-rate and apparent improved potency in preclinical models that could translate into advantages in patients.

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<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<0.05 for both Nalm-6 and Daudi), IFN-g (p<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<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.

scholarly journals Evaluation of Otlertuzumab (TRU-016), an Anti-CD37 ADAPTIRTM Therapeutic in Preclinical Combination Studies with Kinase Inhibitors and a Next Generation Anti-CD20 Mab in Vitro and in Animal Models of Non-Hodgkin’s Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3333-3333
Author(s):  
Jane Gross
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Combination Index ◽  
Pi3k Inhibitor ◽  
Next Generation ◽  
Pi3k Inhibitor Ly294002 ◽  
Anti Cd20

Abstract Background: CD37 is a 50-55 kDa heavily glycosylated member of the tetraspanin superfamily of molecules. This cell surface protein is expressed on normal and transformed B-cells, and has been implicated in diverse processes including cellular activation and proliferation, cell motility, and cell-cell adhesion. Otlertuzumab is a novel humanized anti-CD37 therapeutic, built on the ADAPTIRTM (modular protein technology) platform that has been shown to mediate caspase-independent direct killing of normal and malignant B-cells, a mechanism of action that appears to be distinct from CD20 therapies. In addition, otlertuzumab results in killing through antibody directed cellular cytotoxicity (ADCC), mediated in part by NK cells. The therapeutic potential of otlertuzumab in the treatment of chronic lymphocytic leukemia (CLL) is currently being investigated in Phase 2 clinical studies in combination with bendamustine or rituximab. Preclinical vitro and in vivocombination studies for NHL to evaluate otlertuzumab in combination with other emerging drugs including kinase inhibitors (PI3K and BTK) and the next generation anti-CD20 mAb obinituzumab are reported here. Methods: The ability of otlertuzumab to interact and increase malignant B cell killing with kinase inhibitors was investigated, including a pan PI3K inhibitor (LY294002), a PI3K delta inhibitor (CAL101(GS-1101, idelalisib)), a PI3K delta/gamma inhibitor (IPI-145, (INK1197)) and an inhibitor of BTK (PCI-32765). Combination studies were assayed in vitro using the Minos (mantle cell lymphoma), DoHH-2 (follicular lymphoma) and Ramos (Burkitt’s B cell lymphoma) cell lines. In addition, studies were performed in vitro to test the combination of otlertuzumab and obinituzumab. Individual drugs were tested alone or in combination with otlertuzumab. Combination index analyses were performed for drug combinations over the 20-90% effect levels. To determine whether in vitro synergy could be repeated in vivo, the tumor line with the best in vitro combination characteristics was utilized in xenograft tumor models and treated with otlertuzumab ± LY294002 or otlertuzumab ± PCI-32765 or otlertuzumab ± obinituzumab. Results: Combination index analyses determined that the killing effects of otlertuzumab were synergistic with the pan PI3K inhibitor LY294002, demonstrating comparable results in all three cell lines tested in vitro. The combination of otlertuzumab and the BTK inhibitor PCI-32765 demonstrated synergy in vitro with the Ramos and Minos cell lines. The PI3K delta inhibitor (idelalisib) also demonstrated synergistic activity with all three cell lines when tested in combination with otlertuzumab in vitro. Finally, combination index analyses determined that the killing effects of otlertuzumab were synergistic with the obinituzumab, demonstrating comparable results in all three cell lines tested in vitro. In vivo, the combination of otlertuzumab plus the pan PI3K inhibitor (LY294002) or otlertuzumab plus obinituzumab resulted in greater efficacy relative to each agent alone in the DoHH-2 xenograft tumor models. The combination of otlertuzumab with PCI-32765 resulted in significant delay of tumor outgrowth compared to PCI-32765 alone in the MINO xenograft model of NHL. In vivo results indicated that the in vitro synergy results were applicable to a more complex in vivodisease model. Conclusions: Otlertuzumab tested in combination with multiple kinase inhibitors or next generation anti-CD20 had increased cell killing of NHL cell lines in vitro over that observed for each agent alone. Furthermore, the combinations of otlertuzumab with either obinituzumab, LY294002 or PCI-32765 displayed greater anti-tumor activity in vivo than each of the agents alone. These results provide preclinical rationale for the potential combinations of otlertuzumab with several emerging therapeutics for the treatment of NHL and related B-cell malignancies, including CLL. Disclosures Gross: Emergent BioSolutions Inc: Employment.

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 ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2490-2490 ◽  
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.

First Bispecific Antibody Immunocytokine (Anti-CD20/HLA-DR-Interferon-á2b) Is Highly Toxic for Human Lymphoma Cells in Vitro.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1695-1695
Author(s):  
Diane L Nordstrom ◽  
Edmund A Rossi ◽  
David M. Goldenberg ◽  
Chien-Hsing Chang
Keyword(s):  
Bispecific Antibody ◽  
Size Exclusion ◽  
Biological Entity ◽  
Lymphoma Cells ◽  
Hla Dr ◽  
Human Lymphoma ◽  
Anti Cd20 ◽  
Anti Tumor Activity

Abstract Abstract 1695 Poster Board I-721 Background IFN-á2 is indicated for the therapy of a variety of hematopoietic tumors. As with most cytokines, the short serum half-life and severe side effects of IFN-á2 are major factors affecting its dosing schedule and efficacy. Fusion or conjugation of IFN-á2 to a tumor-targeting IgG has the potential to enhance in vivo potency due to increased tumor localization and more favorable pharmacokinetics. We have recently demonstrated that 20-2b, a monospecific immunocytokine generated by the dock-and-lock (DNL) method to comprise tetrameric IFN-á2b covalently linked to veltuzumab, a humanized anti-CD20 mAb, exhibited very potent anti-tumor activity in vitro and in human lymphoma xenografts (Rossi et al., Blood, in press). However, lymphomas and leukemias that express little or no CD20 are expected to be resistant to therapy with 20-2b. HLA-DR is expressed on many hematopoietic tumors and some solid cancers. A bispecific immunocytokine that could target IFN-á to both CD20 and HLA-DR might be a more effective therapeutic against a wide variety of hematopoietic malignancies, including those that express CD20, HLA-DR, or both. Since each component of the multifunctional complex (veltuzumab, anti-HLA-DR F(ab)2, and IFN-á2b) has anti-tumor activity independently, we evaluated if the bispecific immunocytokine can potentially be even more potent than the monospecific immunocytokine, 20-2b. Methods One strategy of the modular DNL method is to fuse either the dimerization-and-docking domain (DDD) derived from protein kinase A, or the anchoring domain (AD) of a cognate A-kinase anchoring protein, to a biological entity, resulting in respective DDD- and AD-modules that are readily combined to quantitatively generate stably-tethered structures of defined composition with retained bioactivity. We have selectively combined recombinant DDD-modules of both IFN-á2b and anti-HLA-DR Fab (derived from humanized L243) together with a recombinant AD-module of anti-CD20 IgG (veltuzumab) to generate the first bispecific antibody-based immunocytokine, designated 20-C2-2b, which comprises two copies of IFN-á2b and a stabilized F(ab)2 of hL243 site-specifically linked to veltuzumab. Results Each of the three modules, veltuzumab-AD, hL243-Fab-DDD, and IFN-á2b-DDD, was produced recombinantly in separate myeloma cell cultures. Combining equimolar amounts of the three modules under mild redox conditions resulted in the formation of 20-C2-2b, which was purified by sequential chromatographic processes involving Protein A, IMAC and anion exchange chromatography to remove potential side-products such as 20-2b and 20-C2 (the hexavalent bispecific antibody comprising veltuzumab and four Fabs of hL243). Size-exclusion HPLC analysis indicated a major peak of a retention time consistent with a ∼310 kDa protein. Reducing SDS-PAGE of 20-C2-2b revealed the presence of all three constituents. The complex was immunoreactive with an anti-IFN-á2b, an anti-idiotype to hL243, as well as an anti-idiotype to veltuzumab, and showed increased binding to Raji lymphoma cells compared to either veltuzumab or hL243. More importantly, 20-C2-2b was found to be extremely cytotoxic to Daudi, an IFNá-sensitive Burkitt lymphoma cell line, having an IC50 = 0.035 pM, which was 100,000-fold more potent than hL243 IgG, 100-fold more potent than a combination of veltuzumab, hL243 IgG and a structural analog comprising an irrelevant IgG and IFN-á2b, and 5-fold more potent than 20-2b. In the same assay, we have also determined that 20-C2-2b was about 2-fold more potent than C2-2b, which comprises hL243 IgG linked to four molecules of IFN-á2b. Conclusions The DNL method provides a modular approach to enable the creation of novel multifunctional complexes. Based on our experience with 20-2b, the bispecific immunocytokine 20-C2-2b is expected to have greater in vivo potency than IFN-á due to improved pharmacokinetics and endowed targeting specificity, and may potentially be useful for therapy of a variety of hematopoietic tumors that express either CD20 or HLA-DR. Disclosures Nordstrom: Immunomedics, Inc.: Employment. Rossi:Immunomedics, Inc: Employment. Goldenberg:Immunomedics Inc.: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Chang:Immunomedics Inc.: Employment.

Target-Directed Development of a Proposed Biosimilar Rituximab (GP2013): Comparability of Antibody-Dependent Cellular Cytotoxicity Activity and Pre-Clinical Pharmacokinetics and Pharmacodynamics with Originator Rituximab

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4867-4867
Author(s):  
Antonio da Silva ◽  
Ulrich Kronthaler ◽  
Ines Meyer ◽  
Anastassia Papandrikopoulou ◽  
Thomas Stangler ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
B Cell Depletion ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Originator Product ◽  
Acceptance Range ◽  
Anti Tumor Activity

Abstract Abstract 4867 Background: Biosimilars are biologics approved by highly-regulated markets to be similar to existing agents that aim to offer more affordable treatment, thereby increasing patient access. Development of a biosimilar involves extensive characterization of the originator product over several years and a target-directed iterative development process to ensure a product that is highly comparable to the originator with similar clinical efficacy, safety and quality. Using antibody-dependent cellular cytotoxicity (ADCC), a main mode of action of rituximab, we illustrate how functional/structural relationship can be engineered into a biosimilar to ensure comparability at the in vitro level. Here we also present pre-clinical data confirming in vivo comparability for the proposed biosimilar rituximab GP2013, in terms of pharmacokinetics (PK), pharmacodynamics (PD) and efficacy. Methods: By employing a highly sensitive glycan quantitation method, relevant post-translational glycosylation patterns were assessed for their impact on in vitro ADCC relative potency data using the Raji and NK3.3 cell lines as target- and effector cells, respectively. Subsequently, bioactivity of GP2013 and originator rituximab were evaluated in a dose-response manner across a wide concentration range against SU-DHL-4 (diffuse large B-cell lymphoma) and Daudi (Burkitt's lymphoma) cell lines using freshly purified human NK cells. In vivo anti-tumor activity was assessed in two xenograft SCID mouse models of non-Hodgkin's lymphoma (SU-DHL-4 and Jeko-1 cell lines). Comparative PK and PD were assessed in single (5 mg/kg, n=14) and multiple (20 or 100 mg/kg, n=8) dose studies in cynomolgus monkeys, the pharmacologically most relevant species. Results: GP2013 and originator rituximab showed similar ADCC potency against both SU-DHL-4 and Daudi cells, with ADCC being reflective of engineered glycosylation patterns and structure-function relationships. In both xenograft mouse models, GP2013 and originator rituximab inhibited tumor growth to a similar extent, including at the more sensitive sub-optimal dose levels that are most likely to identify any potential differences. In primates, PK analysis confirmed bioequivalence between GP2013 and originator rituximab with nearly identical AUC values and 90% CIs entirely within the standard acceptance range of 0.8–1.25. Bioequivalence of PD response (B-cell depletion) was also shown, with 95% CIs of areas under the effect-time curves (AUEC) ratios for relative change from baseline in B-cell populations within the 0.8–1.25 acceptance range. The use of different doses indicated that comparable exposure and PD response can be expected for GP2013 and originator rituximab using indication-specific dosing regimens. Conclusions: This pre-clinical comparability exercise confirms that GP2013 and originator rituximab are pharmacologically similar with regard to ADCC potency, anti-tumor activity, PK exposure (AUC) and B-cell depletion. As such, it is hypothesized that GP2013 will show similar efficacy and safety as the originator product in ongoing clinical trials across different clinical indications. Disclosures: da Silva: Sandoz Biopharmaceuticals/HEXAL AG: Employment. Kronthaler:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Meyer:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Papandrikopoulou:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Stangler:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Visser:Sandoz Biopharmaceuticals/HEXAL AG: Employment.

Sign in / Sign up

Export Citation Format

Share Document