Effects of Oprozomib in Combination with Pomalidomide and/or Dexamethasone on Human Multiple Myeloma Tumors Growing in SCID Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5349-5349 ◽  
Author(s):  
Eric Sanchez ◽  
Mingjie Li ◽  
Abigail Gillespie ◽  
Puja Mehta ◽  
Suzie Vardanyan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Size ◽  
Research Funding ◽  
Single Agent ◽  
Xenograft Model ◽  
Tumor Burden ◽  
Scid Mice ◽  
Human Multiple Myeloma ◽  
Minimal Reduction

Abstract Introduction: Oprozomib (OPZ) is an irreversible, orally administered proteasome inhibitor (PI).It decreases tumor burden and prevents tumor-related bone loss in preclinical multiple myeloma (MM) studies. In recently published clinical trials, the combination of the PI carfilzomib with the immunomodulatory agent (IMiD) lenalidomide and dexamethasone (Dex) has shown high response rates with durable responses for previously treated and untreated MM patients. Carfilzomib with the IMiD pomalidomide (Pom) and Dex for the treatment of RRMM patients shows promising results (Shah et al, 2013). We evaluated OPZ in combination with Pom and Dex using two of our human MM xenograft models in severe combined immunodeficient (SCID) mice. Methods: Each SCID mouse (n=10/group) was surgically implanted with a 20 - 40 mm3 MM tumor piece into the hind limb. Seven days post-implantation mice were randomized into treatment groups based on human immunoglobulin (Ig) G levels. OPZ stock solution (4 mg/ml) was diluted to 40 mg/kg using 1% carboxymethylcellulose (CMC) and administered twice weekly on two consecutive days via oral gavage.Dex stock solution (10 mg/ml) was diluted to 1 mg/kg using NaCl and administered daily via intraperitoneal injection.Pom stock solution (1 mg/ml) was diluted to 10 mg/kg using 1% CMC and administered daily via oral gavage. Tumor size was measured using calipers and IgG levels by ELISA. Results: Using our human MM model LAGk-1A, treatment with single agent OPZ or Pom produced a minimal reduction in tumor volume when compared with vehicle-treated mice, whereas Dex alone or OPZ + Pom produced more anti-MM effects, and no differences were observed between these two groups. Mice treated with OPZ + Dex or Pom + Dex also showed greater anti-MM activity than OPZ + Pom or Dex alone but the differences were not significant. All three agents together resulted in much smaller tumors when compared to OPZ + Pom on days 35, 42, 49 and 56 (P = 0.0006, P = 0.0001, P = 0.0002 and P < 0.0001, respectively). The same triplicate resulted in a smaller tumors when compared to OPZ + Dex on days 35, 42, 49, 56, and 63 (P = 0.0112, P = 0.0030, P = 0.0060, P = 0.0035 and P = 0.0021, respectively). Although Pom + Dex had some anti-MM effects when compared to the three single agents and one of the doublets (OPZ + Pom), mice receiving the triplicate demonstrated markedly smaller tumors when compared with Pom + Dex on days 35, 42, 49, 56, 63, 70, and 77 (P = 0.0250, P = 0.0018, P < 0.0001, P = 0.0014, P = 0.0018, P = 0.0017 and P = 0.0014, respectively). Mice receiving Pom + Dex had to be euthanized on day 77, whereas mice receiving all three drugs had very small tumors at study termination. We obtained similar results in a second MM xenograft model that produces IgG (LAGλ-1). Although day 21 post-tumor implantation mice receiving Pom alone had lower IgG levels compared with vehicle-treated mice (P = 0.0053), mice receiving OPZ + Pom had smaller tumors when compared to Pom alone (P = 0.0387), OPZ alone (P = 0.0004), or vehicle-treated mice (P = 0.0001). Although mice receiving Pom alone had a reduction in tumor size when compared with vehicle-treated mice (P = 0.0021), mice receiving OPZ + Pom had smaller tumors when compared with Pom alone (P = 0.0081), OPZ alone (P = 0.0007), or vehicle-treated mice (P < 0.0001). Mice receiving the triplicate showed smaller tumors on day 28 compared with mice treated with either the doublets of Pom + Dex (P = 0.0120) or OPZ + Pom (P = 0.0043). Conclusions: These in vivo human MM xenograft studies show that the combination of OPZ + Pom + Dex shows greater anti-MM activity than doublets (Pom + Dex, OPZ + Dex or OPZ + Pom) or single agents, and provides further support for this three drug combination to treat MM. Disclosures Tang: Onyx: Research Funding. [email protected]:Onyx: Consultancy, Honoraria, Research Funding, Speakers Bureau.

MLN9708 Elicits Pharmacodynamic Response in the Bone Marrow Compartment and Has Strong Antitumor Activity in a Preclinical Intraosseous Model of Plasma Cell Malignancy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1834-1834 ◽  
Author(s):  
Edmund Lee ◽  
Bret Bannerman ◽  
Michael Fitzgerald ◽  
Jennifer Terkelsen ◽  
Daniel Bradley ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Antitumor Activity ◽  
Plasma Cell ◽  
Research Funding ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Tumor Burden ◽  
Human Multiple Myeloma ◽  
Cell Malignancy

Abstract Abstract 1834 Poster Board I-860 Introduction The clinical success of VELCADE® (bortezomib) for Injection has validated the proteasome as a therapeutic target for the treatment of human cancer. The novel proteasome inhibitor MLN9708 is a potent, reversible, and specific inhibitor of the b5 site of the 20S proteasome identified in preclinical studies. MLN9708 is currently in human clinical development for both hematological and non-hematological malignancies. Here we describe the pharmacodynamic (PD) response of MLN9708 in the murine bone marrow compartment and its strong antitumor activity in an intraosseous xenograft model of plasma cell malignancy. Materials MLN9708 immediately hydrolyzes to MLN2238, the biologically active form, upon exposure to aqueous solutions or plasma. MLN2238 was used for all preclinical studies described below. Methods It has been previously shown that double transgenic iMycCa/Bcl-XL mice develop de novo plasma cell malignancies (J. Clin. Invest. 113:1763-1773, 2004) in which neoplastic plasma cell development is driven by the targeted expression of the transgene Myc (c-myc; myelocytomatosis oncogene) and Bcl-x (Bcl2l1; encodes the oncoprotein Bcl-XL). DP54 is a plasma cell tumor cell line derived from the bone marrow of a syngeneic mouse previously inoculated with an iMycCa/Bcl-XL tumor (Cancer Res. 67:4069-4078, 2007). In vitro, DP54 cells express both the Myc and Bcl-XL transgenes, various plasma cell and B-cell markers including CD38, CD138 and B220, and has gene expression profile very similar to human multiple myeloma. To establish a preclinical intraosseous model of plasma cell malignancy for efficacy studies, freshly dissociated DP54-Luc cells (constitutively expressing firefly luciferase under a mouse Ig-k promoter) were aseptically injected into the bone marrow space of the upper shaft of the right tibia of NOD-SCID mice. Once tumor growth has been established, mice were randomized into treatment groups and then treated intravenously (IV) with vehicle, bortezomib (at 0.8 mg/kg twice weekly [BIW]) or MLN2238 (at 11 mg/kg BIW) for 3 consecutive weeks. Tumor burden was measured by bioluminescent imaging. Results MLN2238 strongly inhibited proteasome activity in the blood and bone marrow compartments of mice (maximum b5 inhibition of 84% and 83%, respectively). In vivo, when DP54 cells were aseptically injected into the bone marrow space of the mouse tibia, signs of bone erosion in the tibia, femur and cranial sagittal sultures (as determined by ex-vivo mCT imaging) were observed which resembled osteolytic lesions frequently seen in human multiple myeloma. Dissemination of DP54-Luc cells after intratibia inoculations were detected by in vivo bioluminescent and confirmed by ex vivo imaging where luminescent tumor nodules were detected in the spleen, kidneys, intestine, lymph nodes and bones including right tibia, spine and cranium. To assess the antitumor activity of MLN2238 in the bone marrow compartment, an efficacy study was performed using the DP54-Luc intraosseous xenograft model of plasma cell malignancy. Tumor burden (bioluminescence), osteolytic lesions (mCT) and overall survival after treatment with bortezomib and MLN2238 will be presented. Conclusion The novel proteasome inhibitor MLN9708 demonstrates strong activity in the bone marrow compartment in vivo. MLN9708 is currently in human clinical development for both hematological and solid tumor indications. Disclosures Lee: Milllennium: Employment, Equity Ownership. Bannerman:Milllennium: Employment. Terkelsen:Milllennium: Employment. Bradley:Milllennium: Employment, Equity Ownership, Research Funding. Li:Milllennium: Employment. Li:Milllennium: Employment. Janz:Milllennium: Research Funding. Van Ness:Milllennium: Research Funding. Manfredi:Milllennium: Employment. Kupperman:Milllennium: Employment.

Carfilzomib Platform Study Using The LAGκ-1A Multiple Myeloma Xenograft Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4452-4452
Author(s):  
Eric Sanchez ◽  
Mingjie Li ◽  
Suzie Vardanyan ◽  
Jillian Gottlieb ◽  
Kevin Delijani ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Disease Progression ◽  
Tumor Size ◽  
Single Agent ◽  
Xenograft Model ◽  
In Vivo Studies ◽  
Human Igg ◽  
Similar Treatment ◽  
Agent Treatment

Introduction We previously demonstrated that severe combined immunodeficient (SCID) mice bearing the human multiple myeloma (MM) xenograft LAGκ-1A treated with single agent carfilzomib or the alkylating agent (AA) cyclophosphamide (CY) did not show a reduction in tumor growth compared to vehicle-treated mice. In contrast, carfilzomib with CY resulted in a significant decrease in tumor size and IgG levels when compared to mice treated with single agent carfilzomib or CY or vehicle alone. We have also shown that the combination of carfilzomib and another AA, bendamustine, decreased tumor size and IgG levels, when compared to mice treated with single agents or vehicle alone. However, no data is available regarding sequencing of the proteasome inhibitors (PI) carfilzomib or bortezomib with the AA melphalan (MEL). Thus, we used our SCID-hu MM models to evaluate the sequencing of these drugs with MEL. These studies are critical as both PIs are now being used to treat MM. Thus, we evaluated the response, toxicity and survival of animals treated sequentially with these drugs. Methods Each naïve SCID mouse was surgically implanted with a 20 – 40 mm3 MM tumor piece into the left hind limb superficial gluteal muscle. Seven days post–implantation mice were randomized into treatment groups based on human immunoglobulin (Ig) G levels. Carfilzomib stock solution (2 mg/ml) was diluted to 3 mg/kg using 5% dextrose and administered twice weekly on two consecutive days via intravenous (i.v.) injection. Bortezomib stock solution (1 mg/ml) was diluted to 0.25 mg/kg using NaCl and administered twice weekly (Thursdays and Saturdays) via i.v. injection. MEL stock solution (3 mg/ml) was diluted to 1 mg/kg using PBS and administered once weekly via intraperitoneal injection. Mice (n = 10/group) were initially treated with carfilzomib or MEL alone until tumor progression. Progression was defined as an increase in paraprotein equal to or above 25% confirmed on one consecutive assessment. Mice initially treated with carfilzomib were randomized to continue to receive single agent carfilzomib, add in MEL alone or combine it with ongoing carfilzomib, substitute single agent bortezomib, or discontinue treatment altogether. A similar treatment strategy was evaluated with mice treated initially with MEL. At progression, these animals were continued on single agent MEL, carfilzomib added alone or with continuation of MEL, or discontinued treatment. Tumor size was measured using standard calipers and human IgG levels with an ELISA (Bethyl Laboratories, Montgomery, TX). This study was conducted according to protocols approved by the Institutional Animal Care and Use Committee. Results When carfilzomib was administered first, followed by the addition of MEL, a modest nonsignificant reduction in tumor size was observed compared to either drug alone. In addition, substitution of single agent bortezomib for carfilzomib showed no effect on tumor size. However, when MEL was administered first and carfilzomib was added after disease progression, at days 35 and 42 (end of study) post tumor implantation, mice treated with the combination showed a reduction in tumor volume compared to mice that discontinued melphalan (P = 0.0378 and P = 0.0105, respectively) whereas mice treated with carfilzomib alone showed no reduction in tumor size following progression from MEL. Notably, throughout the study there was a trend toward smaller tumors in mice receiving this combination when compared to mice receiving single agent treatment with carfilzomib or MEL alone or vehicle. Similar effects were observed on human IgG levels. Overall, all mice survived combination or single agent treatment with these agents. Conclusions These in vivo studies using our human MM LAGκ–1A SCID–hu model show that animals progressing from initial MEL treatment show a reduction in MM tumor burden when carfilzomib is added to MEL at progression. In contrast, mice progressing from initial carfilzomib treatment did not benefit from the addition of MEL at disease progression. No drug-related deaths occurred in any treatment group. This study demonstrates that using a different MM model (LAGκ-1A), that the PI carfilzomib can produce anti-tumor effects among mice progressing from single-agent MEL treatment, providing further support for the use of this PI as an agent that can help overcome drug resistance in MM. Disclosures: Berenson: Onyx Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau.

scholarly journals Carfilzomib Overcomes Resistance to Bortezomib in the Human Lagk-1A Multiple Myeloma Xenograft Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5720-5720 ◽  
Author(s):  
Eric Sanchez ◽  
Mingjie Li ◽  
Abigail Gillespie ◽  
Nika M Harutyunyan ◽  
Gigi Garzio ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Progression ◽  
Disease Progression ◽  
Tumor Size ◽  
Tumor Volume ◽  
Single Agent ◽  
Xenograft Model ◽  
Scid Mice ◽  
Human Igg ◽  
To Receive

Abstract Introduction: We previously demonstrated that when carfilzomib (CFZ) was administered first to SCID mice bearing our human multiple myeloma (MM) xenografts, followed by the addition of melphalan (MEL) at the time of disease progression, a modest further reduction in tumor size was observed compared with continuing CFZ or adding MEL alone. In addition, substitution of single agent bortezomib (BOR) for CFZ showed no anti-MM effects. However, when MEL was administered first and CFZ was added after disease progression, mice treated with the combination showed a reduction in tumor volume compared with mice that discontinued MEL. Mice treated with CFZ alone showed no reduction in tumor size. Throughout the study, there was a trend toward smaller tumors among mice in which MEL was followed by the addition of CFZ when compared to mice in which MEL was continued or discontinued and treated with single-agent CFZ or vehicle alone. In the current study, we evaluated the anti-MM effects of CFZ, MEL and BOR for severe combined immunodeficient (SCID) mice progressing from BOR+MEL treatment using our human MM xenograft model LAGk-1A. Methods: Each SCID mouse was surgically implanted with a 20 – 40 mm3 LAGk-1A tumor piece into the left hind limb muscle. Seven days post-implantation mice were randomized into treatment groups based on human immunoglobulin (Ig) G levels. Carfilzomib stock solution (2 mg/ml) was diluted to 3 mg/kg using 5% dextrose and administered twice weekly on two consecutive days (Sundays and Mondays) via intravenous (i.v.) injection.Bortezomib stock solution (1 mg/ml) was diluted to 0.25 mg/kg using NaCl and administered twice weekly (Sundays and Tuesdays) via i.v. injection.MEL stock solution (3 mg/ml) was diluted to 1 mg/kg using PBS and administered once weekly (Fridays) via intraperitoneal injection. Mice (n = 10/group) were initially treated with the combination of BOR + MEL until tumor progression. Progression was defined as an increase in paraprotein > 25% confirmed on one consecutive assessment. After tumor progression, mice initially treated with BOR + MEL were randomized to continue to receive BOR + MEL, receive MEL only (discontinue BOR), receive BOR only (discontinue MEL), substitute BOR with CFZ, discontinue BOR + MEL and add in CFZ alone, or discontinue treatment altogether. Tumor size was measured using standard calipers and human IgG levels with an ELISA (Bethyl Laboratories, Montgomery, TX). This study was conducted according to protocols approved by the Institutional Animal Care and Use Committee. Results: Following progression from the combination of BOR + MEL among mice bearing LAGk-1A, substitution of these drugs with single agent CFZ alone did not produce a reduction in tumor volume when compared with mice continuing to receive BOR + MEL. However, significantly smaller tumors were observed when mice progressing from BOR + MEL were switched to CFZ + MEL compared with mice continued on BOR alone (P = 0.0044) or animals switched to CFZ alone (P = 0.05). There was a trend throughout the study toward smaller tumors in mice receiving CFZ + MEL when compared to mice continuing on BOR + MEL, receiving BOR, CFZ or vehicle alone or discontinuing BOR + MEL. Following progression from BOR + MEL, similar anti-MM effects were observed on human IgG (paraprotein) levels among animals treated with CFZ + MEL when compared to mice continuing BOR + MEL, receiving BOR alone switching to CFZ alone or discontinuing both BOR and MEL. Conclusions: These in vivo studies using our human MM xenograft model, LAGk-1A, show that SCID mice progressing from initial BOR + MEL treatment show a reduction in MM tumor burden when BOR is replaced with CFZ at the time of disease progression, and these tumors are significantly smaller than among mice continued on BOR + MEL. These studies demonstrate that once tumors progress from BOR + MEL treatment, CFZ can replace BOR and produce anti-tumor effects. A recent clinical trial confirms our preclinical findings and shows that most MM patients refractory to BOR in combination with other agents, including MEL, will respond to the substitution of CFZ for BOR (Berenson et al., Leukemia 2014). Disclosures No relevant conflicts of interest to declare.

Hypoxia Induces Pgp-Mediated Carfilzomib Resistance in Multiple Myeloma Cells and HIF Inhibition Significantly Enhances Sensitivity and Response to Carfilzomib In Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1286-1286
Author(s):  
Barbara Muz ◽  
Joseph Abraham ◽  
Feda Azab ◽  
Pilar De La Puente ◽  
Nicholas Potter ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Progression ◽  
Cell Lines ◽  
Induced Resistance ◽  
Tumor Size ◽  
Research Funding ◽  
Scid Mice ◽  
Tumor Initiation

Abstract Introduction: A plasma cell malignancy called multiple myeloma (MM) is an incurable cancer in which a majority of patients with refractory disease become resistant to therapy. The introduction of carfilzomib, a proteasome inhibitor, significantly improved the clinical outcome of the MM treatment. However, MM patients develop resistance to carfilzomib and relapse. In this study, we investigated the role of hypoxia and P-gp in the carfilzomib-resistance in MM cells in vitro and in vivo, and its therapeutic translational potential using a HIF inhibitor, PX-478. Methods: In vitro, MM cell lines were treated with carfilzomib under normoxic (21% O2) or hypoxic (1% O2) conditions and cell survival was analyzed by MTT assay. The activity of P-gp was assessed by testing the efflux of a known P-gp substrate, RhodamineB (RhoB), in the hypoxic and normoxic conditions by measuring the intracellular RhoB content using flow cytometry. Moreover, we have tested the effect of HIF inhibition using PX-478 on the P-gp activity as well as the response to carfilzomib in hypoxic and normoxic cells. In vivo, we tested the effect of HIF inhibition on tumor initiation, where MM1s-Luc-GFP cells were injected intravenously (IV) into SCID mice, which were treated instantaneously with PX-478 (10mg/kg) three times a week. Furthermore, we tested the effect of PX-478 on MM tumor response to carfilzomib (measuring tumor size and mice survival). MM1s-Luc-GFP cells were injected IV into SCID mice, tumors grew for 3 weeks and the mice were then randomly divided into 4 groups treated with (1) vehicle (Captisol), (2) PX-478 (10mg/kg) alone, (3) carfilzomib (5mg/kg) alone, or (4) a combination of PX-478 (10mg/kg) and carfilzomib (5mg/kg). PX-478 or vehicle were administered by oral gavage three times a week (day 1, day 3, day 5), while vehicle and carfilzomib were injected IV twice a week (day 4 and day 5). Tumor size was imaged using bioluminescence imaging (BLI) and mice survival was followed for 70 days. Results: In vitro, we found that hypoxia induced resistance to carfilzomib in five MM cell lines. Moreover, hypoxia also increased activity of P-gp by causing decreased intracellular RhoB content in hypoxic MM cells. The HIF inhibitor, PX-478, as well as the P-gp inhibitor, tariquidar, reversed the activation of P-gp in hypoxic cells, while the combination of PX-478 and tariquidar did not induce further inhibition of P-gp activity. Furthermore, the combination of PX-478 or tariquidar with carfilzomib reversed the hypoxia-induced resistance in MM. However, tariquidar and other P-gp inhibitors have shown low selectivity and high toxicity in clinical trials; therefore, for our in vivo experiments we chose to inhibit HIF in order to reverse the hypoxia-induced P-gp-mediated resistance to carfilzomib in MM cells. In vivo, in the MM tumor-initiation model, our study revealed that the HIF inhibitor, PX-478, significantly delayed the tumor progression and extended survival in which all control mice died between 42-52 days, while the experiment was stopped at 70 days, with all PX-478-treated mice still alive. In the established in vivo tumor model, low dose carfilzomib alone delayed the progression by BLI but did not improve survival (vehicle and carfilzomib-treated mice died between 21-28 days after treatment). Despite the fact that PX-478 did not decrease tumor progression as shown by BLI compared to the vehicle-treated mice, it significantly extended the survival of the mice (animals died between 38-48 days). The combination of carfilzomib and PX-478 significantly decreased the proliferation of tumor shown by BLI (less than 5% of the growth at day 28), as well as considerable increase in survival (the experiment was stopped at 70 days with 100% of the group alive). Conclusions: We identified a novel resistance mechanism to carfilzomib in MM, in which hypoxia induces P-gp-mediated resistance to carfilzomib. Inhibition of the hypoxic response in MM cells by the HIF inhibitor reduced hypoxia-induced P-gp-mediated resistance to carfilzomib in MM cells in vitro, and delayed tumor progression significantly improving survival and response to carfilzomib in MM-bearing mice in vivo. Disclosures Azab: Verastem: Research Funding; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner.

A Clinically Relevant SCID-hu in Vivo Model of Human Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2455-2455
Author(s):  
Pierfrancesco Tassone ◽  
Paola Neri ◽  
Daniel R. Carrasco ◽  
Renate Burger ◽  
Laurence Catley ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Fluorescence Imaging ◽  
Scid Mice ◽  
In Vivo Model ◽  
Murine Models ◽  
Fetal Bone ◽  
Investigational Drugs ◽  
Human Multiple Myeloma

Abstract Human multiple myeloma (MM) xenografts in immunodeficient mice have limitations as a model for the human disease since they lack the human bone marrow (huBM) microenvironment. In contrast, murine models harboring a huBM microenvironment with implantation of patient MM cells in the huBM recapitulate the in vivo pathophysiology of MM and have significant advantages over conventional murine models for pre-clinical evaluation of investigational drugs. However, there are significant limitations in using patient MM cells in such models since i) not all patient MM samples engraft in the huBM; ii) only a fraction of engrafted specimens produce measurable paraprotein and/or osteolytic lesions; and iii) a limited number of MM cells can be harvested from an individual patient, thus limiting the number of mice that can be injected with cells from the same patient. To overcome these limitations, we have developed a novel murine model of MM by engrafting INA-6, a cytokine-dependent human MM cell line into SCID mice previously implanted with a human fetal bone chip (SCID-hu mice). INA-6 cells require either exogenous IL-6 or interaction with the bone marrow stromal cells (BMSCs) to proliferate in vitro. In this model, we monitored the in vivo growth of INA-6 cells stably transfected with a green fluorescent protein (GFP) expression vector (INA-6GFP+). Serum soluble human IL-6 receptor (shuIL-6R) and fluorescence imaging of host animals were sensitive indicators of tumor burden with time dependent increase. Fluorescence imaging was able to detect the myeloma cell growth earlier than measurement of sIL-6R levels. INA-6 MM cells grew in SCID-hu mice, but not in SCID mice injected subcutaneously or intravenously without the human fetal bone. We have further confirmed the feasibility of this model in monitoring the response to therapeutic agents such as dexamethasone by detecting reduction in the intensity of the fluorescent lesions as well as shuIL-6R in SCID-hu mice following anti-MM treatment. This highly reproducible model therefore allows for evaluation of investigational drugs targeting MM cells in the huBM milieu.

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.

Efficacy of NK-92 Against Human Multiple Myeloma in a NOD/SCID Gamma Null Mouse Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1854-1854
Author(s):  
Brenna E. Swift ◽  
Brent A. Williams ◽  
Yoko Kosaka ◽  
Joaquín Martínez-López ◽  
Xinghua Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Mouse Model ◽  
Disease Progression ◽  
Cell Line ◽  
Xenograft Model ◽  
Tumor Burden ◽  
Clinical Disease ◽  
Nsg Mice

Abstract Abstract 1854 Introduction: Human NK cell lines NK-92 and KHYG-1 exhibit cytotoxicity against a broad range of tumor types in vitro, including multiple myeloma (MM). To further test efficacy of the NK lines against MM, we developed a bioluminescent mouse model that recapitulates clinical MM using the human U266 MM cell line transduced to express GFP and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow (BM) engraftment. Results: In a pilot study in which 2×106 U266 cells were injected intravenously into NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice, CD138+ MM cells engrafted in BM, with no detectable engraftment in the liver, lungs, spleen, heart, or kidneys by anti-CD138 immunohistochemistry staining at 10 weeks. We used the U266eGFPluc bioluminescent NSG mouse model to evaluate efficacy of NK-92 cell therapy in vivo. We gave 10×106 NK-92 cells every 5 days to a total dose of 50×106 cells 7 days after MM injection. Tumor burden was monitored weekly by bioluminescence imaging 4 weeks after MM inoculation using the IVIS Imaging System, and LivingImage™ Software was used to acquire images and quantify bioluminescence. We showed that U266eGFPluc cells localized to BM and spine, reflecting MM pathophysiology. Disease burden in the NK-92 treated group was consistently lower than controls over time and significantly lower at 8 weeks (Dorsal and Ventral Mann-Whitney p=0.0381) whereas for KHYG-1, the signal increased slightly over control, but was not significant at 8 weeks (Mann-Whitney Dorsal p=0.540 and Ventral p=0.247). Clinical disease progression in MM control mice correlated with IVIS signal intensity at week 11 (r2=0.4; F test p=0.0279). Engraftment was determined by sacrificing mice at 10 weeks and analyzing BM for GFP+ cells by flow cytometry. Engraftment of MM cells in BM was as follows (mean+/− SEM): control (5 +/− 1.9%), NK-92 (0.24 +/− 0.19%) and KHYG-1 (5.2 +/− 1.6%) showing a trend toward a significant decrease in mean engraftment for the NK-92 group versus control (unpaired student's t test p=0.055), but not for KHYG-1 (p=0.939). One of 6 control mice had low engraftment with U266eGFPluc at 10 weeks increasing the variance of the control mean. There was a statistically significant decrease in median engraftment in the NK-92 group (Mann-Whitney p=0.019), but not for KHYG-1 (p =0.792) (Figure). GFP BM engraftment corresponded with bioluminescence detected in R and L BM by IVIS. Presence of NK cells in BM was detected in only 1/3 NK-92 mice tested (0.2%) and in none of the KHYG-1 mice at 10 weeks. To assess biodistribution of KHYG-1 we injected 10×106 CFSE-labeled KHYG-1 via tail vein into healthy NSG mice. Blood and organ samples were collected 8 and 24 hours later and analyzed by flow cytometry. We detected CFSE-labeled KHYG-1 primarily in liver, blood and lung, less in kidney, and none in heart, spleen or BM. Conclusion: We have established a human MM cell line xenograft model in NSG mice comparable to clinical disease. Treatment efficacy can be monitored in live NSG mice by IVIS imaging technology and tumor burden at sacrifice can be determined by GFP detection. MM progression was reduced by NK-92, but not KHYG-1 as measured by bioluminescence and reduction of engrafted U266eGFPluc cells. We have shown that a MM xenograft model can screen for in vivo efficacy of immune therapies for MM. Our results indicate that NK-92 is a potentially effective therapy for MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Disruption of Src function potentiates Chk1-inhibitor–induced apoptosis in human multiple myeloma cells in vitro and in vivo

Blood ◽  
2011 ◽  
Vol 117 (6) ◽  
pp. 1947-1957 ◽  
Author(s):  
Yun Dai ◽  
Shuang Chen ◽  
Rena Shah ◽  
Xin-Yan Pei ◽  
Li Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Xenograft Model ◽  
Mitogen Activated Protein Kinase ◽  
Loss Of Function ◽  
Compensatory Response ◽  
Human Multiple Myeloma ◽  
Chk1 Inhibitor ◽  
Induced Apoptosis

Abstract Ras/MEK/ERK pathway activation represents an important compensatory response of human multiple myeloma (MM) cells to checkpoint kinase 1 (Chk1) inhibitors. To investigate the functional roles of Src in this event and potential therapeutic significance, interactions between Src and Chk1 inhibitors (eg, UCN-01 or Chk1i) were examined in vitro and in vivo. The dual Src/Abl inhibitors BMS354825 and SKI-606 blocked Chk1-inhibitor–induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation, markedly increasing apoptosis in association with BimEL up-regulation, p34cdc2 activation, and DNA damage in MM cell lines and primary CD138+ MM samples. Loss-of-function Src mutants (K297R, K296R/Y528F) or shRNA knock-down of Src prevented the ERK1/2 activation induced by Chk1 inhibitors and increased apoptosis. Conversely, constitutively active Ras or mitogen-activated protein kinase/ERK kinase 1 (MEK1) significantly diminished the ability of Src inhibitors to potentiate Chk1-inhibitor lethality. Moreover, Src/Chk1-inhibitor cotreatment attenuated MM-cell production of vascular endothelial growth factor and other angiogenic factors (eg, ANG [angiogenin], TIMP1/2 [tissue inhibitor of metalloproteinases 1/2], and RANTES [regulated on activation normal T-cell expressed and secreted]), and inhibited in vitro angiogenesis. Finally, coadministration of BMS354825 and UCN-01 suppressed human MM tumor growth in a murine xenograft model, increased apoptosis, and diminished angiogenesis. These findings suggest that Src kinase is required for Chk1-inhibitor–mediated Ras → ERK1/2 signaling activation, and that disruption of this event sharply potentiates the anti-MM activity of Chk1 inhi-bitors in vitro and in vivo.

scholarly journals Engineering an Optimized Trimeric APRIL-Based CAR to Broaden Targetability of Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2059-2059 ◽  
Author(s):  
Andrea Schmidts ◽  
Maria Ormhoj ◽  
Allison O. Taylor ◽  
Selena J. Lorrey ◽  
Irene Scarfò ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Research Funding ◽  
Antigen Expression ◽  
Tumor Burden ◽  
Monomeric Form ◽  
Car T Cells ◽  
Car T

Abstract Background: Targeting BCMA (B-cell maturation factor) with chimeric antigen receptor (CAR) T cells has shown great success in the treatment of multiple myeloma (MM), but is limited by heterogeneous antigen expression and imminent antigen escape of tumor cells. Combinatorial antigen targeting may help address these challenges. Taking the naturally occurring receptor-ligand pairs as a model, we designed monomeric and trimeric APRIL- (A proliferation-inducing ligand) based CARs targeting BCMA and TACI (transmembrane activator and CAML interactor) simultaneously. Methods: The following 2nd generation CARs were designed to target BCMA and TACI concurrently: membrane-tethered truncated APRIL monomer ("APRIL-CAR") and three truncated and fused APRIL monomers ("TriPRIL-CAR"). A single chain variable fragment-based anti(α)-BCMA CAR served as control. CAR multimerization and binding affinity to BCMA and TACI were characterized. In vitro effector function was compared by cytotoxic potency, activation (CD69), degranulation (CD107a), cytokine production and proliferation in response to target antigens. In vivo anti-tumor efficiency was assessed in a xenograft mouse model of MM. Results: CAR T cell manufacturing of all three constructs was accomplished successfully (transduction efficiency 46-78%) from three different donors. Western blot analysis of CARs showed multimerized forms of the TriPRIL and α-BCMA CAR, while only the monomeric form of the APRIL CAR was detected. Binding affinity to soluble BCMA and TACI was higher for the TriPRIL CAR compared to the APRIL CAR. Evaluating the cytotoxic potential, activation and degranulation kinetics as well as long-term proliferation against a panel of BCMA and/or TACI positive target cells, the TriPRIL CAR T cells outperformed the APRIL CAR T cells. All three CAR constructs demonstrated robust antigen-specific production of Th1-type cytokines, like Il-2, IFNƔ, GM-CSF and TNFα. Next, we performed an in vivo stress test, engrafting NSG mice with high tumor burden of MM.1s myeloma cells. The TriPRIL and α-BCMA CAR T cells were able to eradicate the tumors while the APRIL CAR T cells only led to a stabilization of tumor burden. In vivo studies with a mixed antigen population aiming at modeling heterogeneous antigen expression and antigen escape are ongoing. Conclusion: Our APRIL-based chimeric antigen receptors were able to redirect T cell cytotoxicity to both BCMA and TACI positive tumor cells. Since both these receptors are consistently up-regulated on malignant plasma cells this is an attractive method to target MM. Furthermore, we found that using a trimeric form of APRIL rather than monomeric form as the CAR binding domain increased recognition of MM antigens in vitro and in vivo. Disclosures Maus: crispr therapeutics: Consultancy, Research Funding; adaptimmune: Consultancy; novartis: Consultancy; kite therapeutics: Consultancy, Research Funding; windmil therapeutics: Consultancy; agentus: Consultancy, Research Funding.

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