EphA3 As a Molecular Target In Multiple Myeloma: Opportunity For a Novel Therapeutic Approach With a Specific Monoclonal Antibody

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3211-3211
Author(s):  
Antonella Caivano ◽  
Francesco La Rocca ◽  
Ilaria Laurenzana ◽  
Alessandra Favole ◽  
Irma Airoldi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Therapeutic Approach ◽  
Molecular Target ◽  
Eph Receptors ◽  
Phase I Clinical Trials ◽  
Protein Levels ◽  
Novel Therapeutic

Abstract Introduction Multiple myeloma (MM) remains an incurable malignancy despite important recent advances in treatments. Neo-vascularization entails a crucial aspect of interactions between neoplastic plasma cells (PCs) and their microenvironment. Without it, MM would be unable to grow and progress, and would probably regress to a low-mass steady-state comparable to monoclonal gammopathy of undetermined significance (MGUS). To overcome drug resistance and improve clinical response to novel therapeutic approaches halting both PC growth and the increased bone marrow (BM) microvascular density are needed. In this setting, monoclonal antibodies against MM-specific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate membrane target structures expressed across all MM cells. The Eph receptors, a large family of receptor tyrosine kinases, have been implicated in many processes involved in malignancy, including alteration of the tumour microenvironment, and in angiogenesis, in both of which EphA3 likely plays an active role. Interestingly, the over-expression of EphA3 is sufficient to confer tumorigenic potential, although probably further mechanisms can occur to abnormally activate the receptor. A first-in-class engineered IgG1 antibody targeting the EphA3 was developed and it is now under phase I clinical trials in USA and Australia for the treatment of EphA3 over-expressing hematological myeloid malignancies refractory to conventional treatment. Methods We investigated the EphA3 role in MM patients in order to define whether it may represent a potential new molecular target for a novel therapeutic approach with a specific anti EphA3 monoclonal antibody. The EphA3 expression was studied through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with MM (MMECs) or with MGUS (MGECs), of control subjects (normal ECs). Moreover, the effects of anti EphA3 antibody in MM were studied in vitro and in vivo in a MM xenograft mouse model. After written informed consent, BM aspirates were collected from 26 MM and 6MGUS patients. Normal ECs were derived from 5 BM aspirates of subjects with anemia due to iron or vitamin B12 deficiency. We analyzed both mRNA and protein levels of EphA3 in normal ECs, MGECs and MMECs and in MM cell lines by absolute RT-PCR and by WB coupled to immunofluorescence and FACS analysis respectively. Immunoistochemistry was also performed on MM BM biopsies. The biological effects of EphA3 targeting were studied in vitro silencing (siRNA) the EphA3 mRNA in MMECs and using the anti EphA3 antibody testing them in series of in vitro functional assays including viability, apoptosis, adhesion, migration, wound healing and angiogenesis tests. We further examined the inhibitory capacity of anti-EphA3 Ab on tumor growth in SCID mice bearing MM tumor cell xenografts. Finally, we assessed morphology, vessel density, and apoptosis of excised xenotransplanted tumors. Results Briefly, our data showed that EphA3 mRNA and protein levels are progressively increased from ECs to MGECs, reaching the highest values in MMECs. EphA3 stained intensely and diffusely MM microvessels and PC in MM BM biopsies. The EphA3siRNA MMECs revealed a protein level reduction of approximately 80% when compared to the control. We not detected viability or apoptotic defects, whereas in vitro adhesion, migration and angiogenesis inhibition was evident when compared to the not silenced counterpart. The anti EphA3 antibody inhibited MMECs migration and reduced in vitro MM angiogenesis. In particular, tumour masses developed in xenograft mice treated with anti-EphA3 Abs were smaller in size and showed foci of ischemic-hemorrhagic necrosis, in association with a significant (P < 0.05) reduction in the number of intact tumor microvessels. The proliferative activity was not significantly different from that observed in tumors from untreated or control isotype treated mice, while the apoptotic index was significantly (P < 0.05) increased in comparison with tumors from both groups of mice. Conclusions In this study we have characterized the role of the EphA3in MM patients, providing in vitro and in vivo experimental evidences that support the possibility of using EphA3 as a new molecular target for MM. Disclosures: No relevant conflicts of interest to declare.

Identification of EpHA3 As a New Potential Molecular Target in Multiple Myeloma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2926-2926
Author(s):  
Antonella Caivano ◽  
Francesco La Rocca ◽  
Alessandra Favole ◽  
Sonia Carturan ◽  
Enrico Bracco ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Cell Lines ◽  
Plasma Cells ◽  
Molecular Target ◽  
Over Expression ◽  
In Vitro Angiogenesis ◽  
Novel Therapeutic

Abstract Abstract 2926 Introduction Angiogenesis plays a central role in the progression of both solid and hematological tumors. In particular, in multiple myeloma (MM) the critical role of bone marrow (BM) microenvironment and angiogenesis has been well documented. The past decade has witnessed a dramatic improvement in the therapeutic options in MM. However, the disease remains incurable, underscoring the need for continued efforts towards understanding MM biology and exploitation of novel therapeutic approaches. In this setting, monoclonal antibodies against myeloma-specific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate target structures expressed across all pathogenic myeloma cells. The Eph receptors, a large family of receptor tyrosine kinases (RTKs) activated by ephrins binding, have been implicated in many processes involved in malignancy, including alteration of the tumor microenvironment and in angiogenesis, in both of which EpHA3 likely plays an active role. Aberrant expression of EpHA3 is seen in many types of hematolologic malignancies (some leukemic cell lines, T-cell lymphoma, acute lymphoblastic leukemia, myeloproliferative neoplasms) although it is not expressed ubiquitously. Finally, the over-expression of Eph is believed to be sufficient to confer tumorigenic potential although probably further mechanisms can occur to abnormally activate the receptor. Basing on the role of EpHA3 in haematological malignancies, a first-in-class engineered IgG1 antibody targeting the EpHA (KB004) was developed and it is now under phase I clinical trials in USA and Australia for the treatment of EpHA3 overexpressing hematological myeloid malignancies refractory to conventional treatment. We investigated the EpHA3 role and its preferential membrane–bound by GPI linker ligand EFNA5, in MM patients in order to define EpHA3 as new molecular target for a novel therapeutic approach with a specific anti EpHA3 monoclonal antibody. The EpHA3 expression has been studied through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with MM (MMECs) or with monoclonal gammopathy of undetermined significance (MGECs), of control subjects (normal ECs) and in MM cell lines. Methods After written informed consent, BM aspirates have been collected from 20 MM and 4 MGUS patients. Normal ECs were derived from 3 BM aspirates of subjects with anemia due to iron or vitamin B12 deficiency. We analyzed the expression levels of EpHA3 in normal ECs, MGECs and MMECs and MM cell lines evaluating the mRNA and protein levels by RT-qPCR and by WB coupled to ImmunoFluorescence analysis. The biological effects of EpHA3 targeting in MMECs have been studied silencing the EpHA3 mRNA in MMECs and testing them at 72h after silencing in series of functinal assays including viability assay by trypan blue exclusion staining and by in vitro angiogenesis assay followed by measurement of mesh areas and vessel length. Moreover, we studied EFNA5 mRNA expression levels in Normal ECs, MGECs and MMECs and in MM cell lines by PCR. Results Our data showed that EpHA3 mRNA levels are progressively increased from ECs to MGECs reaching the highest values in MMECs. Subsequent analysis by WB and immunofluorescence confirmed EpHA3 protein upregulation among the different EC types. The MMECs in which EpHA3 has been silenced revealed a protein level reduction of approximately 60% when compared to the control. We could not detect major viability defects. Furthermore, in vitro angiogenesis inhibition was marginal when compared to the not silenced counterpart. To know whether EpHA3 may impact not only MM angiogenesis but also plasma cells, three MM cell lines were studied for the EpHA3 expression. We found the plasma cell lines gave constant over expression of EpHA3. Finally, the preliminary data regarding EFNA5 mRNA expression level showed it is expressed in either MMECs and MM plasma cell lines. The evaluation of KB004 effect on MMECs in term of apoptosis induction and in vitro tube formation inhibition, as well as the analysis of EpHA3 levels in primary MM plasma cells are in progress. Conclusions From this study we expect to characterize the role of the EpHA3in MM patients and to provide experimental evidences supporting the possibility of using EpHA3 as a new molecular target for MM by proving the in vitro efficacy of a monoclonal antibody to target the angiogenesis of MM. Disclosures: No relevant conflicts of interest to declare.

A Vascular Targeted Pan PI-3 Kinase Inhibitor, SF1126 with Activity Against Multiple Myeloma In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 244-244 ◽  
Author(s):  
Pradip De ◽  
Qiong Peng ◽  
Nandini Dey ◽  
Breanne McDermitt ◽  
Xiaodong Peng ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Tissue ◽  
Kinase Inhibitor ◽  
Surrogate Marker ◽  
Phase I Clinical Trials ◽  
Lymphoid Malignancies ◽  
Antiangiogenic Activity ◽  
Density Analysis

Abstract Background: Considerable evidence suggests an important role for the PI-3 kinase and AKT signaling pathways in survival and chemoresistance in multiple myeloma (MM) and other lymphoid malignancies. Our group and others have demonstrated that downregulation of p-AKT with combination therapy (bortezomib + lonafarnib; David et al, Blood, 2005) is a surrogate marker for myeloma apoptosis. It has been demonstrated that the compound, LY294002 has significant pan PI-3 kinase inhibitory properties but is not suitable for clinical use due to PK issues. SF1126 is a novel RGD targeted derivative of LY294002 that has been shown to have activity in a number of different tumor models. Herein, we evaluated the activity of SF1126 against the MM.1S and MM.1R MM cell lines in vitro and in vivo for sensitivity to PI-3 kinase inhibition. The results demonstrate that MM.1S and MM.1R tumor cell growth is sensitive to SF1126 with IC50 of 7.5 and 10.8 uM, respectively. The effects of SF1126 on MM.1R signaling in vitro was examined with profound inhibition of HIF1a induction under hypoxia, the suppression of phosphorylation states of MDM2, ERK and RS6 kinase. The IC50 for inhibition of p-AKT in MM.1S and MM.1R cells was determined to be 2.4 and 2.8 uM, respectively. SF1126 treatment (50 mg/kg/dose sc given every other day) inhibited MM.1R tumor growth in nude mouse xenografts 95% as compared to untreated controls on day 38 (p < .01). Microvessel density analysis of MM.1R tumor tissue demonstrated that SF1126 had significant antiangiogenic activity in vivo. Conclusion: The results provide preclinical data to support SF1126 as a clinically viable antiangiogenic, pan PI-3 kinase inhibitor for Phase I clinical trials in the treatment of multiple myeloma. Further studies in primary myeloma cells and in combination with conventional agents will be presented.

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.

scholarly journals The Phenoxyalkyltriazine Antagonists for 5-HT6 Receptor with Promising Procognitive and Pharmacokinetic Properties In Vivo in Search for a Novel Therapeutic Approach to Dementia Diseases

2021 ◽  
Vol 22 (19) ◽  
pp. 10773
Author(s):  
Sylwia Sudoł ◽  
Agnieszka Cios ◽  
Magdalena Jastrzębska-Więsek ◽  
Ewelina Honkisz-Orzechowska ◽  
Barbara Mordyl ◽  
...  
Keyword(s):  
Therapeutic Approach ◽  
Cognitive Disorders ◽  
Drug Candidates ◽  
Piperazine Derivatives ◽  
Pharmacokinetic Properties ◽  
Chemical Class ◽  
Novel Therapeutic ◽  
Dementia Diseases

Among the serotonin receptors, one of the most recently discovered 5-HT6 subtype is an important protein target and its ligands may play a key role in the innovative treatment of cognitive disorders. However, none of its selective ligands have reached the pharmaceutical market yet. Recently, a new chemical class of potent 5-HT6 receptor agents, the 1,3,5-triazine-piperazine derivatives, has been synthesized. Three members, the ortho and meta dichloro- (1,2) and the unsubstituted phenyl (3) derivatives, proved to be of special interest due to their high affinities (1,2) and selectivity (3) toward 5-HT6 receptor.. Thus, a broader pharmacological profile for 1–3, including comprehensive screening of the receptor selectivity and drug-like parameters in vitro as well as both, pharmacokinetic and pharmacodynamic properties in vivo, have been investigated within this study. A comprehensive analysis of the obtained results indicated significant procognitive-like activity together with beneficial drug-likeness in vitro and pharmacokinetics in vivo profiles for both, (RS)-4-[1-(2,3-dichlorophenoxy)propyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (2) and (RS)-4-(4-methylpiperazin-1-yl)-6-(1-phenoxypropyl)-1,3,5-triazin-2-amine (3), but insensibly predominant for compound 2. Nevertheless, both compounds (2 and 3) seem to be good Central Nervous System drug candidates in search for novel therapeutic approach to dementia diseases, based on the 5-HT6 receptor target.

A Novel Bcma-Specific, Centyrin-Based CAR-T Product for the Treatment of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2127-2127 ◽  
Author(s):  
David L. Hermanson ◽  
Burton Earle Barnett ◽  
Srinivas Rengarajan ◽  
Rebecca Codde ◽  
Xinxin Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
M Protein ◽  
Manufacturing Processes ◽  
Protein Levels ◽  
Car T

Abstract Chimeric-antigen receptor (CAR)-T cell immunotherapies have been remarkably effective in treating acute lymphoblastic leukemia. However, current strategies generally suffer from difficult, inefficient and costly manufacturing processes, significant patient side effects and poor durability of response in some patients. Here, we report for the first time a CAR-T cell therapeutic comprising a non-immunoglobulin alternative scaffold Centyrin molecule (a "CARTyrin") manufactured with a novel non-viral piggyBacTM (PB) transposon-based system. Our lead candidate, P-BCMA-101, encodes a CARTyrin that targets the B cell maturation antigen (BCMA) for the treatment of multiple myeloma (MM) and has several unique aspects that improve upon earlier CAR-T products. First, P-BCMA-101 is manufactured using only in vitro transcribed mRNA and plasmid DNA without the need for lentivirus or g-retrovirus, resulting in time and cost savings. Importantly, PB is also safer than viral systems due to a less mutagenic insertional profile and is non-oncogenic. Furthermore, PB can efficiently deliver transgenes as large as several hundred kilobases, and, once inserted, transgenes demonstrate more stable, prolonged and higher expression when compared to those delivered by virus. Second, a mutein of the dihydrofolate reductase (DHFR) gene is included in the P-BCMA-101 transgene that can be used in combination with the non-genotoxic drug methotrexate (MTX) to provide a simple and effective method of CARTyrin+ cell enrichment and reduces variability in patient product material. Third, P-BCMA-101 incorporates a safety switch for optional depletion in vivo in case of adverse events. Lastly, the CARTyrin is comprised of a BCMA-specific Centyrin, which are based on a human tenascin fibronectin type III (FN3) consensus sequence. Centyrins have similar binding affinities to the antibody-derived single chain variable fragments (scFv), but are smaller, more thermostable and predicted to be less immunogenic. Importantly, no signs of tonic signaling leading to T cell exhaustion have been observed with CARTyrins unlike scFv-based CAR molecules, which can interact with each other on the surface causing non-specific CAR signaling. The manufacture process of P-BCMA-101 from primary human T cells is straightforward, employs no cytokines, and easily produces enough CARTyrin+ cells to treat patients. Within 18 days of electroporation of purified T cells, we demonstrate > 95% of the cell product is positive for CARTyrin expression and ready to be administered. Notably, our manufacturing process results in > 60% of CARTyrin+ T cells exhibiting a stem-cell memory phenotype (i.e. CD45RA+ CD62L+). P-BCMA-101 cells exhibit specific and robust in vitro activity against BCMA+ tumor targets, ranging from high to very low levels of BCMA, as measured by target-cell killing and CARTyrin-T cell proliferation. Importantly, proliferating P-BCMA-101 cells were highly sensitive in vitro to activation of the safety switch. Finally, we have evaluated the anti-tumor efficacy of P-BCMA-101 in a model of human MM. NSG™ mice were injected IV with 1.5x106 luciferase+ MM.1S cells, an aggressive human MM-derived cell line. After the tumor cells were allowed to grow for 21 days, animals received a single IV administration of 5x106 P-BCMA-101 cells. All untreated control animals demonstrated a marked increase in serum M-protein levels, rapid growth of tumor cells demonstrated by bioluminescent imaging (BLI), and death within four weeks. In stark contrast, 100% of animals that received P-BCMA-101 rapidly eliminated tumors within 7 days as measured by BLI and serum M-protein levels and improved survival out to at least 60 days post-treatment. P-BCMA-101 is the first-in-class of Centyrin-based CAR therapeutics. The CARTyrin, combined with our advanced manufacturing processes, represents a significant improvement over first generation, immunoglobulin-based and virally-transduced CAR-T products. P-BCMA-101 exhibited an advantageous stem-cell memory phenotype and demonstrated specific and potent anti-tumor efficacy against BCMA+ myeloma cells both in vitro and in vivo. Based on these results, we plan to initiate a phase I clinical trial of P-BCMA-101 for the treatment of patients with relapsed and/or refractory MM. Disclosures Hermanson: Poseida Therapeutics: Employment. Barnett:Poseida Therapeutics: Employment. Rengarajan:Poseida Therapeutics: Employment. Codde:Poseida Therapeutics: Employment. Wang:Poseida Therapeutics: Employment. Tan:Poseida Therapeutics: Employment. Martin:Poseida Therapeutics: Employment. Smith:Poseida Therapeutics: Employment. Osertag:Poseida Therapeutics: Employment, Equity Ownership. Shedlock:Poseida Therapeutics: Employment.

scholarly journals Plexin C1 Marks Liver Cancer Cells with Epithelial Phenotype and Is Overexpressed in Hepatocellular Carcinoma

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Gorkem Odabas ◽  
Metin Cetin ◽  
Serdar Turhal ◽  
Huseyin Baloglu ◽  
A. Emre Sayan ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Monoclonal Antibody ◽  
Cell Lines ◽  
Tissue Microarrays ◽  
Mesenchymal Phenotype ◽  
Protein Levels ◽  
Epithelial Phenotype ◽  
Well Differentiated

Background and Aims. Hepatocellular carcinoma is an aggressive malignancy of the liver and is ranked as the sixth most common cancer worldwide. There is still room for novel markers to improve the diagnosis and monitoring of HCC. Our observations in cancer databases that PLXNC1 is upregulated in HCC led us to investigate the expression profile of Plexin C1 mRNA and protein in HCC cell lines and tissues. Methods. A recombinant protein encompassing part of the extracellular domain of Plexin C1 was used as an antigen for monoclonal antibody development. Transcript and protein levels of Plexin C1 in HCC cell lines were determined by RT-qPCR and Western blotting, respectively. In vivo evaluation of Plexin C1 expression in HCC tissues was accomplished by immunohistochemistry studies in tissue microarrays. Results. A monoclonal antibody, clone PE4, specific to Plexin C1, was generated. In silico and in vitro analyses revealed a Plexin C1-based clustering of well-differentiated HCC cell lines. Staining of HCC and nontumoral liver tissues with PE4 showed a membrane-localized overexpression of Plexin C1 in tumors (p=0.0118). In addition, this expression was correlated with the histological grades of HCC cases. Conclusions. Plexin C1 distinguishes HCC cells of epithelial characteristics from those with the mesenchymal phenotype. Compared to the nontumoral liver, HCC tissues significantly overexpress Plexin C1. The newly generated PE4 antibody can be evaluated in larger HCC cohorts and might be exploited for the examination of Plexin C1 expression pattern in other epithelial malignancies.

scholarly journals Targeting Bclxl Mitigates Mcl1 Chemoresistance in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2656-2656
Author(s):  
Ioanna Savvidou ◽  
Tiffany Khong ◽  
Irena Carmichael ◽  
Jaynish S Shah ◽  
Sridurga Mithraprabhu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Research Funding ◽  
Ex Vivo ◽  
Combined Treatment ◽  
Live Imaging ◽  
Synergistic Activity ◽  
Protein Levels ◽  
Bax Protein

Abstract Aim: Despite the adoption of novel therapeutic modalities, Multiple Myeloma (MM) remains incurable. The Bcl2 inhibitor Venetoclax is active in several haematologic malignancies, but the benefits in MM patients are limited to those with the t(11;14) and/or high Bcl2 expression. These results underscore the significance of Bcl2 alternative anti-apoptotic proteins (Mcl1 and BclxL) for the survival of myeloma cells. Method: We validated the anti MM effect of the Mcl1 inhibitor S63845 both in vitro utilising 11 human myeloma cell lines (HMCL) and ex vivo against n=30 primary MM tumours. Comparative analysis of RNAseq between S63845 sensitive and resistant HMCL was undertaken to identify candidate proteins that potentially modulate resistance to S63845. Treatment with S63845 and rationally selected combination partners was further evaluated in vitro, ex vivo and in vivo with flow cytometry, immunoblotting and live imaging mitochondria fitness monitoring. Results: RNAseq identified BclxL as potential mediator of resistance to S63845 in HMCL. Immunoblotting confirmed high BclxL expression and high BclxL/BclS in S63845 resistant HMCL. Five S63845 resistant HMCL (U266, ANBL6, KMS28PE, EJM, MM1R) and primary tumours were treated with S63845 combined with the BclxL inhibitor A1331852 . Combined treatment of the HMCL demonstrated a high Bliss synergy score for all the HMCL tested (54, 42, 24, 47, 45 for U266, EJM, KMS28PE, MM1R and ANBL6 respectively) and induced synergistic killing of 80% of the primary tumours treated. Dual inhibition in U266 induced an 80% drop in intracellular ATP at 4h with an increase in active Caspases 9 and 8 (4.5 and 5 fold, respectively). Similarly, the combination induced a 78% drop in mitochondrial transmembrane potential (TMRE intensity) by 4h with live imaging revealing striking mitochondrial damage as early as 40 minutes after exposure (figure). These changes were associated with a reduction of both Mcl1 and, BclxL proteins and Bim and Bid protein levels. No changes were seen in the level of Bcl2, Bak or Bax protein expression. The combination of S63845 and A1331852 in healthy NSG mice at 12.5mg/kg proved lethal due to hepatotoxicity, arguing against the clinical utility of such an approach. However, this observed anti-MM synergistic activity was recapitulated when S63845 was combined with the already approved anti-MM therapeutic panobinostat, with the induction of a significant reduction in both BclxL and Myc protein levels at 24h, and synergistic killing of 56% of primary tumours. Conclusion: High BclxL expression and BclxL/BclxS ratio correlates with resistance to the Mcl1 inhibitor S63845. A combinatorial approach targeting Mcl1 and BclxL induced immediate and significant anti-MM effect both in vitro and ex vivo but proved to be toxic in vivo. Combination of the anti-MM therapeutic panobinostat in combination with S635845 recapitulated the anti-MM activity seen with A1331852 and warrants further evaluation. Figure 1 Figure 1. Disclosures Spencer: Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Takeda: Honoraria, Research Funding, Speakers Bureau; STA: Honoraria.

A Monoclonal Antibody Specific for Free Human Kappa Light Chains Induces Apoptosis of Multiple Myeloma Cells and Exhibits Anti-Tumor Activity In Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2416-2416 ◽  
Author(s):  
Parisa Asvadi ◽  
Darren R. Jones ◽  
Rosanne D. Dunn ◽  
Andre B.H. Choo ◽  
Matthew J. Raison ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Light Chains ◽  
High Dose ◽  
Mouse Tumor ◽  
Cell Transplant ◽  
Kappa Light Chains ◽  
Anti Tumor Activity

Abstract Despite high dose chemotherapy and autologous stem cell transplant, multiple myeloma (MM) remains an incurable malignancy, with a median 5 year survival of less than 20%. With the exception of idiotype, few antigen targets have been identified that would facilitate specific immunotherapy of MM. We have previously described a murine monoclonal antibody that recognizes a conformation-dependent epitope on free human kappa light chains and a cell surface antigen, KMA, expressed on kappa MM plasma (MMκ) cells (Raison, RL and Boux, HA Mol Immunol 1985 22:1393). Here we show that the murine antibody, designated mKap, bound specifically to a range of MMκ cell lines and inhibited the in vitro growth of these cells. Flow cytometric analysis (Annexin-V and PI staining) of MMκcell lines incubated with mKap demonstrated a dose dependent induction of apoptosis. Furthermore, the presence of activated caspases in mKap treated cells was detected using the CaspACE™ FITC-VAD-FMK reagent. The induction, by mKap, of apoptosis in MMκ cells occured in the absence of cross-linking second antibody or effector cells. In vivo, anti-tumor activity by mKap was demonstrated in a SCID mouse tumor xenograft model. Tumor growth was measured by quantitation of secreted myeloma Ig over a period of 6 weeks. From week 4 onwards, significantly lower serum concentrations of myeloma Ig were detected in animal groups receiving 3.0, 1.5, 0.3 and 0.15 mg total mKap compared to the untreated control (P<0.005 at week 4; P<0.0001 at week 5). The tumor-restricted specificity of mKap, coupled with its ability to inhibit MMκ cell growth in vitro and in vivo, suggests the potential use of either a chimeric or humanised version of this antibody, alone or in combination with chemotherapy, for the treatment of kappa MM.

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