The human anti-CD30 antibody 5F11 shows in vitro and in vivo activity against malignant lymphoma

Blood ◽  
2003 ◽  
Vol 102 (10) ◽  
pp. 3737-3742 ◽  
Author(s):  
Peter Borchmann ◽  
John F. Treml ◽  
Hinrich Hansen ◽  
Claudia Gottstein ◽  
Roland Schnell ◽  
...  
Keyword(s):  
Cell Lines ◽  
Specific Binding ◽  
Severe Combined Immunodeficiency ◽  
Scid Mice ◽  
Growth Delay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Complete Regression ◽  
Term Survival

AbstractCD30 is a promising target for antibody-based immunotherapy of Hodgkin lymphoma (HL) and anaplastic large cell lymphoma. To overcome the limitations from currently available murine anti-CD30 monoclonal antibodies (mAbs), a new fully human anti-CD30 antibody was generated. Binding properties were evaluated by recombinant CD30 capture enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell-sorter (FACS) flow cytometry. Activity of this new mAb was assessed in vitro using growth inhibition and antibody-dependent cellular cytotoxicity (ADCC) assays on several cell lines. In vivo activity was determined in a solid as well as in a disseminated xenografted model of HL in severe combined immunodeficiency (SCID) mice. The mAb 5F11 showed specific binding to CD30 (cluster A). The ADCC assays indicated dose-dependent lysis of L540 cells when 5F11 was combined with human effector cells. Upon cross-linking in vitro, 5F11 inhibited the growth of CD30-expressing cell lines. In vivo, treatment with 5F11 induced a marked growth delay or even a complete regression of established xenografted HL in SCID mice. In the disseminated HL model, a high proportion of 5F11-treated mice experienced long-term survival. The new human anti-CD30 monoclonal antibody 5F11 shows promise as a means of CD30-targeted immunotherapy of malignant lymphomas. Based on these results, a clinical phase 1 study in patients with refractory CD30+ lymphoma has been initiated. (Blood. 2003;102:3737-3742)

scholarly journals Identification of Hsp90 inhibitors as potential drugs for the treatment of TSC1/TSC2 deficient cancer

2021 ◽  
Author(s):  
Evelyn M. Mrozek ◽  
Vineeta Bajaj ◽  
Yanan Guo ◽  
Izabela Malinowska ◽  
Jianming Zhang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Xenograft Model ◽  
Growth Delay ◽  
Hsp90 Inhibitors ◽  
Null Cell ◽  
Standard Dosage

Inactivating mutations in either TSC1 or TSC2 cause Tuberous Sclerosis Complex, an autosomal dominant disorder, characterized by multi-system tumor and hamartoma development. Mutation and loss of function of TSC1 and/or TSC2 also occur in a variety of sporadic cancers, and rapamycin and related drugs show highly variable treatment benefit in patients with such cancers. The TSC1 and TSC2 proteins function in a complex that inhibits mTORC1, a key regulator of cell growth, which acts to enhance anabolic biosynthetic pathways. In this study, we identified and validated five cancer cell lines with TSC1 or TSC2 mutations and performed a kinase inhibitor drug screen with 197 compounds. The five cell lines were sensitive to several mTOR inhibitors, and cell cycle kinase and HSP90 kinase inhibitors. The IC50 for Torin1 and INK128, both mTOR kinase inhibitors, was significantly increased in three TSC2 null cell lines in which TSC2 expression was restored.  Rapamycin was significantly more effective than either INK128 or ganetespib (an HSP90 inhibitor) in reducing the growth of TSC2 null SNU-398 cells in a xenograft model. Combination ganetespib-rapamycin showed no significant enhancement of growth suppression over rapamycin. Hence, although HSP90 inhibitors show strong inhibition of TSC1/TSC2 null cell line growth in vitro, ganetespib showed little benefit at standard dosage in vivo. In contrast, rapamycin which showed very modest growth inhibition in vitro was the best agent for in vivo treatment, but did not cause tumor regression, only growth delay.

EB10, an Anti-FLT3 Monoclonal Antibody, Selectively Targets ALL Cell Lines and Primary ALL Blasts without Interfering with Normal Hematopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2744-2744 ◽  
Author(s):  
Obdulio Piloto ◽  
Patrick Brown ◽  
Li Li ◽  
Bao Nguyen ◽  
Kyu-Tae Kim ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Lines ◽  
Scid Mice ◽  
Cytotoxic Agents ◽  
Radioactive Isotopes ◽  
Leukemic Cells ◽  
Class Iii ◽  
Antibody Treatment

Abstract The class III receptor tyrosine kinase, FLT3, is expressed by >90% of B-lineage acute lymphoblastic leukemias (ALL) blasts. In addition, it is expressed at extremely high levels in ALL patients with MLL-rearrangements or hyperdiploidy and sometimes mutated in these same patients. In this report, we investigated the effects of EB10, an anti-human FLT3 monoclonal antibody capable of preventing binding of FLT3 ligand (FL), on ALL cell lines and primary cells. In vitro studies, examining the ability of EB10 to inhibit FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. In some cell lines FLT3 phosphorylation was inhibited and with it, downstream activation of pathways involving MAPK, AKT, and STAT5 phosphorylation. However, several cell lines actually exhibited FLT3 activation upon antibody treatment, possibly because of antibody-mediated receptor dimerization, and subsequent activation of downstream pathways. Nevertheless, through antibody-mediated cellular cytotoxicity (ADCC) such an antibody could still prove efficacious against leukemia cells in vivo. In fact, EB10 treatment significantly prolongs survival and/or reduces engraftment of ALL cell lines and primary ALL blasts in NOD/SCID mice. This effect might be even more pronounced in a host that was less immune compromised than are NOD/SCID mice. The leukemic cells surviving EB10 treatment in the mice were characterized by FACS analysis and found to express low levels or no FLT3. In contrast to the reduction in engraftment of human ALL primary blasts, EB10 treatment of NOD/SCID mice did not reduce engraftment of human hematopoietic CD34+ cells. Taken together, these data demonstrate that EB10 is selectively cytotoxic to ALL blasts while having little effect on normal hematopoiesis. Such an antibody, either naked or conjugated to radioactive isotopes or cytotoxic agents, may prove useful in the therapy of infant ALL as well as childhood and adult ALL patients whose blasts typically express FLT3.

The Multi-Targeted Receptor Tyrosine Kinase Inhibitor, ABT-869, Induces Apoptosis of AML Cells Both In Vitro and In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 616-616 ◽  
Author(s):  
Deepa B. Shankar ◽  
Jenny C. Chang ◽  
Bertrand Parcells ◽  
Salemiz Sandoval ◽  
Junling Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Scid Mice ◽  
Parp Cleavage

Abstract Children with acute myeloid leukemia (AML) have less than 60% overall survival despite aggressive chemotherapy and bone marrow transplantation. Only one third of the adult patients diagnosed with AML will be cured. AML blast cells from up to 30% of patients express a constitutively active receptor tyrosine kinase, FLT3-ITD, which contains an internal tandem duplication in the juxtamembrane domain. Patients with FLT3-ITD have a worse prognosis. ABT-869 is a novel multi-targeted small molecule inhibitor of receptor tyrosine kinases and is a potent inhibitor of FLT3, c-Kit, and all members of the VEGF and PDGF receptor families. To determine the effects of ABT-896 on AML cells, we treated AML cell lines, primary cells, and tumors in xenograft models with varying concentrations of the drug. In vitro viability assays showed that ABT-869 inhibited the growth of two different cell lines, MV-4-11 (human AML cell line that expresses FLT3-ITD) and BAF3-ITD (murine B-cell line stably transfected with the FLT3-ITD) at an IC50 of 10nM. ABT-869 was also effective against another mutation of FLT3, D835V, but at higher concentrations (IC50 of 100nM). Phosphorylation of FLT3 and activation of downstream signaling molecules, STAT5 and ERK, were inhibited by ABT-869 in a concentration-dependent manner. Cells were also stained with Annexin V-FITC and Propidium Iodide, and analyzed using FACS. ABT-869 induced apoptosis, caspase-3 activation, and PARP cleavage after 48 hours. To examine the in vitro effects of ABT-869 on normal hematopoietic progenitor cells, we performed methylcellulose-based colony assays with human bone marrow. No significant difference was observed in the number and type of colonies formed using BM cells treated with ABT-869 or control, up to a concentration of 1 micromolar. These results suggest that ABT-869 is not toxic to normal bone marrow progenitor cells at concentrations that are effective against AML cells. To examine the effects of ABT-869 in vivo, we treated SCID mice injected with MV-4-11, Baf3-ITD, Baf3-D835V, or Baf3-WT cells, with oral preparations of ABT-869. Complete regression of MV-4-11 tumors was observed in mice treated with ABT-869 at 20 and 40 mg/kg/day. No adverse effects were detected in the peripheral blood counts, bone marrow, spleen or liver. Histology of the tumors from the control-treated group showed a high degree of proliferation by Ki-67 staining, increased mitotic figures, and a well-defined tumor mass. In contrast, the tumors from mice treated with ABT-869 showed a number of apoptotic bodies by TUNEL staining and the presence of reactive, inflammatory cells. Interestingly, we also observed that mice that received ABT-869 the day after injection of AML cells remained tumor-free for over 2 months in contrast to the mice receiving the vehicle alone. Inhibition of FLT3 phosphorylation was demonstrated in the tumors from mice treated with ABT-869. We are evaluating the activity of ABT-869 treatment of SCID mice injected with Baf3-ITD, Baf3-D835V, or Baf3-WT cells. NOD-SCID mouse models are currently being used to analyze the effects of ABT-869 on primary AML cells in vivo. Our preclinical studies demonstrate that ABT-869 is effective and nontoxic, and provide rationale for the treatment and prevention of relapse in AML patients.

FLT3 Immunotherapy Can Eliminate Engraftment of ALL Cells in NOD/SCID Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 869-869
Author(s):  
Obdulio Piloto ◽  
Bao Nguyen ◽  
Patrick Brown ◽  
Kyu-Tae Kim ◽  
David Huso ◽  
...  
Keyword(s):  
Cell Lines ◽  
Scid Mice ◽  
Cytotoxic Agents ◽  
Radioactive Isotopes ◽  
Leukemic Cells ◽  
Pcr Analysis ◽  
Facs Analysis ◽  
Antibody Treatment

Abstract The class III receptor tyrosine kinase, FLT3, is expressed by over 90% of B-lineage acute lymphoblastic leukemias (ALL) blasts. In addition, it is expressed at extremely high levels in ALL patients with MLL-rearrangements or hyperdiploidy and sometimes mutated in these same patients. In this report, we investigated the effects of EB10, an anti-human FLT3 monoclonal antibody capable of preventing binding of FLT3 ligand (FL), on ALL cell lines and primary cells. In vitro studies, examining the ability of EB10 to inhibit FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. In some cell lines FLT3 phosphorylation was inhibited and with it, downstream activation of pathways involving MAPK, AKT, and STAT5 phosphorylation. However, several cell lines actually exhibited FLT3 activation upon antibody treatment, possibly because of antibody-mediated receptor dimerization, and subsequent activation of downstream pathways. Nevertheless, through antibody-mediated cellular cytotoxicity (ADCC) such an antibody could still prove efficacious against leukemia cells in vivo. In fact, EB10 treatment significantly prolongs survival and/or reduces engraftment of several ALL cell lines and some primary ALL samples in NOD/SCID mice, even when EB10 treatment results in FLT3 activation of those cell lines in vitro. Moreover, FACS and PCR analysis of EB10 treated NOD/SCID mice surviving 150 days post leukemic cell injection revealed that FLT3 immunotherapy eliminated leukemic engraftment. The leukemic cells surviving EB10 treatment in the mice were characterized by FACS analysis and found to express lower levels of FLT3. To assess for resistance, cells surviving EB10 treatment were injected into NOD/SCID mice and treated with a single dose of EB10. FACS analysis revealed that these cells remain sensitive to EB10 treatment. Taken together, these data demonstrate that EB10 is cytotoxic to ALL blasts in vivo and EB10 treatment did not select for resistant clones. Such an antibody, either naked or conjugated to radioactive isotopes or cytotoxic agents, may prove useful in the therapy of infant ALL as well as childhood and adult ALL patients whose blasts typically express FLT3.

Improved Therapy of Multiple Myeloma by Combining Milatuzumab (Anti-CD74 Humanized mAb) and Bortezomib: In Vitro and In Vivo Results.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1176-1176
Author(s):  
Rhona Stein ◽  
David M. Goldenberg
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Leukemic Cells ◽  
Tumor Type ◽  
Term Survival ◽  
Significant Difference ◽  
Highly Effective ◽  
Ic50 Values

Abstract Background: The humanized anti-CD74 monoclonal antibody, milatuzumab (hLL1, or IMMU-115; Immunomedics, Inc, Morris Plains, NJ), is in clinical evaluation for therapy of multiple myeloma (MM) after preclinical evidence of activity in this tumor type (Stein et al, Blood2004;104:3705). Here we examine the ability of milatuzumab to increase the efficacy of drugs in MM cell lines. Methods: MTT cytotoxicity assays were performed on a panel of MM cell lines, including CAG, KMS11, KMS12-PE, and MC/CAR, to examine the effects of bortezomib, doxorubicin (dox), and dexamethasone (dex) alone and combined with milatuzumab or milatuzumab + crosslinking 2nd Ab (goat anti-human IgG, GAH). In vivo studies used a CAG-SCID mouse model of disseminated disease. Results: Without drugs, crosslinked milatuzumab, but not milatuzumab alone, yielded significant anti-proliferative effects on the four MM cell lines. In combination studies, crosslinked milatuzumab produced significant reductions in the IC50 values of the anti-MM drugs. For example, in CAG, milatuzumab+GAH decreased the IC50 values 58%, 78%, and 98% for bortezomib, dox, and dex, respectively (P=0.0034, 0.0073, and 0.078, respectively). In vivo, milatuzumab at 100 μg/injection, 2x weekly for 4 weeks, starting 1 day after injection of CAG cells, more than doubled the median survival time (MST) from 42 days in untreated CAG-bearing SCID mice to 103 days. Combination therapy with milatuzumab and bortezomib or dox was compared to milatuzumab alone, with treatments initiated 5 days after injection of CAG cells. Bortezomib alone (1.0 mg/kg) increased MST from 33 to 44 days (P=0.0021 vs. untreated). Treatment with milatuzumab alone (100 μg/mouse) increased the MST to 73 days (P<0.0001 vs. untreated). When bortezomib and milatuzumab treatments were combined, the MST increased to 93 days (P=0.0441 vs. milatuzumab and P=0.0065 vs. bortezomib). Thus, the combination of milatuzumab and bortezomib increased survival significantly compared to either single treatment. Given alone, dox yielded little or no effect on survival compared with untreated animals, and there was no significant difference between milatuzumab monotherapy and milatuzumab plus doxorubicin in this model. In contrast, a milatuzumabdox immunoconjugate was found to be a highly effective therapeutic agent, with all mice achieving long-term survival. The inhibition of the NF-κB survival pathway of B-leukemic cells by milatuzumab supports its complementary effects when combined with drugs having different mechanisms of action, such as bortezomib. Conclusions: The therapeutic efficacies of bortezomib, dox, and dex are enhanced in vitro in MM cell lines when given in combination with milatuzumab. In vivo, milatuzumab alone or especially in combination with bortezomib is highly effective in MM. (Supported in part by USPHS grant P01CA103985 from the NCI, and grants from the Thomas and Agnes Carvel Foundation and the Walter and Louise Sutcliffe Foundation.)

A Humanized Anti-Ganglioside GM2 Antibody, BIW-8962, Exhibits ADCC/CDC Activity against Multiple Myeloma Cells and Potent Anti- Tumor Activity in Mouse Xenograft Models.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1718-1718 ◽  
Author(s):  
Toshihiko Ishii ◽  
Asher Alban Chanan-Khan ◽  
Jazur Jafferjee ◽  
Noreen Ersing ◽  
Takeshi Takahashi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Phase 1 ◽  
Xenograft Model ◽  
Surface Expression ◽  
Scid Mice ◽  
Subcutaneous Xenograft ◽  
Anti Tumor Activity

Abstract BIW-8962 is a humanized anti-ganglioside GM2 (GM2) monoclonal antibody, produced by Poteligent technology to enhance ADCC activity. GM2 is expressed on many cancer cells including multiple myeloma (MM), small cell lung cancer and glioma cells. In this study, we evaluated the anti-myeloma activity of BIW-8962 in preclinical myeloma models both in vitro and in vivo. Expression of GM2 was analyzed in 15 human MM cell lines by FCM. Eleven out of 15 MM cell lines had positive surface expression of GM2. GM2 as a potential target was then verified in primary MM samples obtained from patients. Eleven out of 15 samples were positive for GM2. We then used two GM2 positive MM cell lines (U266B1 and KMS-11) and evaluated ADCC and CDC activity of BIW-8962 in vitro. BIW-8962 exhibited a potent ADCC and less potent CDC activity. In vivo anti-tumor activity of BIW-8962 was then examined using the standard subcutaneous xenograft model; KMS-11 was inoculated in the flank of SCID mice. BIW-8962 (intravenously administered biweekly for 3 weeks) exhibited a potent anti-tumor activity from as low a dose level as 0.1 mg/kg. Furthermore, in a more clinically relevant model, in which OPM-2/GFP (GM2 positive MM cell line) cells were intravenously inoculated into SCID mice with preferentially tumor growth within the bone marrow microenvironment, BIW-8962 (intravenously administered biweekly for 4 weeks, 10 mg/kg) suppressed OPM-2/GFP cell growth and serum M protein elevation, demonstrating in vivo anti-myeloma effect of BIW-8962. Our preclinical investigations rationalize clinical evaluation of BIW-8962 in patients with MM. Currently BIW-8962 is being investigated in a Phase 1 study in patients with multiple myeloma.

The Monoclonal Antibody nBT062 Conjugated to Cytotoxic Maytansinoids Has Potent and Selective Cytotoxicity against CD138 Positive Multiple Myeloma Cells in Vitro and in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1716-1716 ◽  
Author(s):  
Hiroshi Ikeda ◽  
Teru Hideshima ◽  
Robert J. Lutz ◽  
Sonia Vallet ◽  
Samantha Pozzi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Tumor Cells ◽  
Cell Lines ◽  
Xenograft Model ◽  
Growth Delay ◽  
Selective Cytotoxicity ◽  
Bystander Killing

Abstract CD138 is expressed on differentiated plasma cells and is involved in the development and/or proliferation of multiple myeloma (MM), for which it is a primary diagnostic marker. In this study, we report that immunoconjugates comprised of the murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives (nBT062-SMCC-DM1, nBT062-SPDB-DM4 and nBT062-SPP-DM1) showed cytotoxic activity against CD138-positive MM cells both in vitro and in vivo. These agents demonstrated cytotoxicity against OPM1 and RPMI8226 (CD138-positive MM cell lines) in a dose and time-dependent fashion and were also cytotoxic against primary tumor cells from MM patients. Minimal cytotoxicity was noted in CD138-negative cell lines and no activity was observed against peripheral blood mononuclear cells from healthy volunteers, suggesting that CD138-targeting is important for immunoconjugate-mediated cytotoxicity. Examination of the mechanism of action whereby these immunoconjugates induced cytotoxicity in MM cells demonstrated that treatment triggered G2/M cell cycle arrest, followed by apoptosis associated with cleavage of PARP and caspase-3, -8 and -9. Neither interleukin-6 nor insulin-like growth factor-I could overcome the apoptotic effect of these agents. The level of soluble (s)CD138 in the BM plasma from 15 MM patients was evaluated to determine the potential impact of sCD138 on immunoconjugate function. The sCD138 level in BM plasma was found to be significantly lower than that present in MM cell culture supernatants where potent in vitro cytotoxicity was observed, suggesting that sCD138 levels in MM patient BM plasma would not interfere with immunoconjugate activity. Because adhesion to bone marrow stromal cells (BMSCs) triggers cell adhesion mediated drug resistance to conventional therapies, we next examined the effects of the conjugates on MM cell growth in the context of BMSC. Co-culture of MM cells with BMSCs, which protects against dexamethasoneinduced death, had no impact on the cytotoxicity of the immunoconjugates. The in vivo efficacy of these immunoconjugates was also evaluated in SCID mice bearing established CD138-positive MM xenografts and in a SCID-human bone xenograft model of myeloma. Significant tumor growth delay or regressions were observed at immunoconjugate concentrations that were well tolerated in all models tested. The ability of these agents to mediate bystander killing of proximal CD138-negative cells was also evaluated. While nBT062-SPDB-DM4 was inactive against CD138-negative Namalwa cells cultured alone, significant killing of these CD138-negative cells by nBT062-SPDB-DM4 was observed when mixed with CD138-positive OPM2 cells. This bystander killing may contribute to the eradication of MM tumors by disrupting the tumor microenvironment and/or killing CD138-negative MM tumor cells, such as the putative CD138 negative myeloma stem cells. These studies demonstrate strong evidence of in vitro and in vivo selective cytotoxicity of these immunoconjugates and provide the preclinical framework supporting evaluation of nBT062-based immunoconjugates in clinical trials to improve patient outcome in MM.

A gp130 Interleukin-6 Transducer-Dependent SCID Model of Human Multiple Myeloma

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4727-4737 ◽  
Author(s):  
Cosette Rebouissou ◽  
John Wijdenes ◽  
Patrick Autissier ◽  
Karin Tarte ◽  
Valerie Costes ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
Scid Mice ◽  
Term Survival ◽  
Myeloma Cells ◽  
Tumoral Cells ◽  
Human Myeloma Cells

Agonist antihuman gp130 transducer monoclonal antibodies (MoAbs) were used in SCID mice to grow myeloma cells whose survival and proliferation is dependent on gp130 transducer activation. The agonist anti-gp130 MoAbs neither bound to murine gp130 nor activated murine cells and, as a consequence, did not induce interleukin-6 (IL-6)–related toxicities in mice. They have a 2-week half-life in vivo when injected in the peritoneum. The agonist antibodies made possible the in vivo growth of exogenous IL-6–dependent human myeloma cells as well as that of freshly explanted myeloma cells from 1 patient with secondary plasma cell leukemia. Tumors occurred 4 to 10 weeks after myeloma cell graft and weighed 3 to 5 g. They grew as solid tumors in the peritoneal cavity and metastasized to the different peritoneal organs: liver, pancreas, spleen, and intestine. Tumoral cells were detected in blood and bone marrow of mice grafted with the XG-2 myeloma cells. Tumoral cells grown in SCID mice had kept the phenotypic characteristics of the original tumoral cells and their in vitro growth required the presence of IL-6 or agonist anti-gp130 MoAbs. Myeloma cells from 4 patients with medullary involvement persisted for more than 1 year as judged by detectable circulating human Ig. However, no tumors were detected, suggesting a long-term survival of human myeloma cells without major proliferation. These observations paralleled those made in in vitro cultures as well as the tumor growth pattern in these patients. This gp130 transducer-dependent SCID model of multiple myeloma should be useful to study various therapeutical approaches in multiple myeloma in vivo.

Effect of inducing HR deficiency using AVB500, a receptor tyrosine kinase AXL inhibitor, on response to olaparib in uterine serous cancer.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e18108-e18108
Author(s):  
Michael Driscoll Toboni ◽  
Barbara Blachut ◽  
Mary M Mullen ◽  
Jo'an Tankou ◽  
Hollie M Noia ◽  
...  
Keyword(s):  
Treatment Period ◽  
Cell Lines ◽  
Day Treatment ◽  
Parp Inhibitor ◽  
Tumor Burden ◽  
Scid Mice ◽  
In Vivo Studies ◽  
Treatment Groups

e18108 Background: Evidence suggests DNA repair is a therapeutic target in endometrial cancer (EC). Given this, we determined whether combination therapy with AVB500, an AXL inhibitor, could improve response in a uterine serous cancer (USC) model. Methods: Two USC cell lines (ARK1 & ARK4) were treated with AVB500 (Aravive Biologics, Houston, TX) in combination with the poly ADP ribose polymerase (PARP) inhibitor, olaparib. Colony forming assays were assessed after 4 days of treatment with either AVB500 alone, olaparib alone or combination treatment (olaparib + AVB500); colonies were stained and absorbance was obtained to calculate relative cell viability using Graph Pad Prism. Baseline homologous recombination (HR) status was determined after radiating cells with 10Gy and identifying RAD51 foci by immunofluorescence (IF). Cell lines were considered to be HR proficient if over 30% of the cells expressed RAD51 ( > 5 foci per cell). IF was conducted using a Leica confocal microscope and foci were quantified using FociCounter. In vivo studies were performed using NOD-SCID mice injected with 1 x 107 ARK1 cells intraperitoneally followed by treatment q3 days for a 14 and 21 day treatment period. Treatment groups were vehicle control, AVB500 alone, olaparib alone and olaparib with AVB500. Results: The absorbance for olaparib + AVB500 was significantly less than the olaparib only group in two assays involving ARK1s (0.417nm vs 0.756nm, p = 0.001; 0.320nm vs 0.620nm, p = 0.008) as well as in ARK4s (0.186nm vs 0.641nm, p = 0.003). The HR assay indicated both cell lines were HR proficient. After baseline HR proficiency was established, the cell lines were pretreated with AVB500 prior to radiation. When compared to cells without treatment with AVB500, IF showed a decrease in RAD51 foci per cell in ARK1 (2.7 vs 7.3, p = 0.0003) and ARK4 (6.3 vs 13.0, p = 0.0054). The proportion of ARK1 cells expressing RAD51 decreased to 21%, indicating HR deficiency. Lastly, NOD-SCID mice receiving olaparib + AVB500 had less tumor weight than those treated with olaparib alone (0.008g vs 0.138g, p = 0.002) and AVB500 alone (0.008g vs 0.145g, p = 0.0006) in a 14 day and a 21 day treatment period (0.212g vs 0.586g, p = 0.027 and 0.212 vs 0.494g, p = 0.005, respectively). Conclusions: HR proficient USC cell lines treated in vitro and in vivo with the combination of AVB500 and olaparib demonstrate an improved response to olaparib or AVB500 alone with a greater decrease in tumor burden. AVB500 appears to induce HR deficiency. Additional therapeutic and mechanistic experiments are ongoing.

Preclinical Rationale for the Use of the Multikinase Inhibitor Sorafenib in the Treatment of Human Lymphomas

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2605-2605
Author(s):  
Carmelo Carlo-Stella ◽  
Cristiana Lavazza ◽  
Arianna Giacomini ◽  
Loredana Cleris ◽  
Daniela Sia ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tumor Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Scid Mice ◽  
Multikinase Inhibitor ◽  
Preclinical Data

Abstract Introduction: The multikinase inhibitor Sorafenib (Nexavar, Bayer) exerts a remarkable activity against a variety of nonhematological tumors by blocking tumor cell proliferation and angiogenesis through the inhibition of the RAF/MEK/ERK pathway, as well as the receptor tyrosine kinases vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptor (PDGFR), c-KIT, Flt3, and RET. Several lines of evidence suggest that sorafenib might have a relevant clinical impact in the therapy of malignant lymphomas by overcoming the cytoprotective effects of ERK, Mcl-1, and Bcl-XL. However, preclinical data establishing a rationale for the clinical use of sorafenib in lymphomas are still lacking. The present studies aimed to investigate the activity and the mechanism(s) of action of sorafenib in human lymphomas. Methods: The effects of sorafenib were evaluated in vitro using a panel of six human cell lines of different phenotypes, including JVM-2 (B-Chronic Lymphocytic Leukemia), Granta-519 (Mantle Cell Lymphoma), DOHH2 (Follicular Lymphoma), SU-DHL-4V (Diffuse Large B-Cell Lymphoma), HD-MY-Z (Hodgkin Lymphoma), and KMS-11 (Multiple Myeloma) cell lines. Additionally, the antitumor efficacy and mechanism of action of sorafenib were investigated in vivo by means of five lymphoma xenograft models in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Results: In vitro, the response of cell lines to sorafenib (1–10 μM for 24–48 hours) was evaluated by detecting apoptotic cell death with the annexin-V/propidium iodide double staining assay, and viable cell countings with the Trypan blue dye exclusion test. All 6 cell lines responded to sorafenib with values of 50% inhibitory concentrations ranging from 1 to 7.5 μM. In contrast, normal CD34+ cells remain insensitive to the drug up to 15 μM. Despite significant rates of sorafenib-induced apoptosis were seen in all cell lines, activation of caspase-3 analyzed by fluorescent-activated cell sorter was only detected in DOHH-2 and JVM-2 cell lines. The phosphorylation status of mitogen-activated protein kinase (MAPK) was investigated using the human phospho-MAPK Array kit (R&D systems), analyzed with the open source imaging software ImageJ (http://rsb.info.nih.gov/ij/), and then validated by Western blotting. Sorafenib induced a significant reduction of pAkt1, pAkt2, and pAkt3 in SU-DHL-4V, Granta-519, and JVM-2 cell lines, whereas p38 phosphorylation levels were significantly reduced in all but one cell line (KMS-11). Reduced levels of pMEK, pERK1 and pERK2 were detected in SU-DHL-4V, KMS-11, Granta-519, and HD-MY-Z cell lines. Down-regulation of MCL-1 was seen in HD-MY-Z, JVM-2, and DOHH-2 cell lines. In vivo, the activity of sorafenib was evaluated in NOD/SCID mice bearing subcutaneous tumor nodules. Animals with tumors averaging from 140 to 160 mg were randomly grouped to receive sorafenib (90 mg/kg body weight, IP, once daily for 15 days) or control vehicle. Sorafenib significantly (P ≤0.001) reduced the growth of subcutaneous HD-MY-Z, KMS-11, Granta-519, SU-DHL-4V, and JVM-2 nodules, with values of tumor growth inhibition of 70%, 52%, 40%, 37%, and 24%, respectively. In control mice, TUNEL staining of tumor sections showed large areas of viable cells without significant necrosis, whereas a 2- to 5-fold increase of necrotic areas was detected in sorafenib-treated mice bearing the different lymphoma xenografts. Analysis of tumor vasculature by means of in vivo biotinylation of endothelial cells with sulfo-NHS-LC-biotin showed a 30% to 60% reduction of vessel density in sorafenib-treated mice bearing the different lymphoma xenografts. Conclusions: Sorafenib efficiently targets a variety of human lymphomas representative of different phenotypes by inhibiting tumor angiogenesis and directly affecting tumor cell survival. Our preclinical data establish a rationale for exploring the clinical activity of sorafenib in human lymphomas.

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