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ABSTRACT: BACKGROUND: Advanced or metastatic renal cell carcinoma (RCC) has a poor prognosis, because it is relatively resistant to conventional chemotherapy or radiotherapy. Treatments with human interferon-alpha2b alone or in combination with mammalian target of rapamycin (mTOR) inhibitors have led to only a modest improvement in clinical outcome. One observation made with mTOR inhibitors is that carcinomas can overcome these inhibitory effects by activating the insulin-like growth factor-I (IGF-I) signaling pathway. Clinically, there is an association of IGF-I receptor (IGF-IR) expression in RCC and poor long-term patient survival. We have developed a humanized anti-IGF-IR monoclonal antibody, hR1, which binds to RCC, resulting in effective down-regulation of IGF-IR and moderate inhibition of cell proliferation in vitro. In this work, we evaluate the anti-tumor activity of two novel IGF-1R-targeting agents against renal cell carcinoma given alone or in combination with an mTOR inhibitor. METHODS: hR1 was linked by the DOCK-AND-LOCKTM (DNLTM) method to four Fabs of hR1, generating Hex-hR1, or to four molecules of interferon-alpha2b, generating 1R-2b. Eight human RCC cell lines were screened for IGF-1R expression and sensitivity to treatment with hR1 in vitro. Synergy with an mTOR inhibitor, temsirolimus, was tested in a cell line (ACHN) with low sensitivity to hR1. RESULTS: Hex-hR1 induced the down-regulation of IGF-IR at 10-fold lower concentrations compared to the parental hR1. Sensitivity to growth inhibition mediated by hR1 and Hex-hR1 treatments correlated with IGF-1R expression (higher expression was more sensitive). The potency of 1R-2b to inhibit the in vitro growth of RCC was also demonstrated in two human cell lines, ACHN and 786-O, with EC50--values of 63 and 48 pM, respectively. When combined with temsirolimus, a synergistic growth-inhibition with hR1, Hex-hR1, and 1R-2b was observed in ACHN cells at concentrations as low as 10 nM for hR1, 1 nM for Hex-hR1, and 2.6 nM for 1R-2b. CONCLUSIONS: Both Hex-hR1 and 1R-2b proved to be more potent than parental hR1 in inhibiting growth of RCC in vitro. Synergy was achieved when each of the three hR1-based agents was combined with temsirolimus, suggesting a new approach for treating RCC.
BMC Cancer 04/2013; 13(1):170. · 3.01 Impact Factor
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mAbs 03/2013; 5(3).
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ABSTRACT: We report the in-vivo fusion of two Hodgkin lymphomas with golden hamster cheek pouch cells, resulting in serially-transplanted (over 5-6 years) GW-532 and GW-584 heterosynkaryon tumor cells displaying both human and hamster DNA (by FISH), lymphoma-like morphology, aggressive metastasis, and retention of 7 human genes (CD74, CXCR4, CD19, CD20, CD71, CD79b, and VIM) out of 24 tested by PCR. The prevalence of B-cell restricted genes (CD19, CD20, and CD79b) suggests that this uniform population may be the clonal initiating (malignant) cells of Hodgkin lymphoma, despite their not showing translation to their respective proteins by immunohistochemical analysis. This is believed to be the first report of in-vivo cell-cell fusion of human lymphoma and rodent host cells, and may be a method to disclose genes regulating both organoid and metastasis signatures, suggesting that the horizontal transfer of tumor DNA to adjacent stromal cells may be implicated in tumor heterogeneity and progression. The B-cell gene signature of the hybrid xenografts suggests that Hodgkin lymphoma, or its initiating cells, is a B-cell malignancy.
PLoS ONE 01/2013; 8(2):e55324. · 4.09 Impact Factor
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ABSTRACT: The prospects for using pretargeted immuno-SPECT to monitor the response to pretargeted radioimmunotherapy were examined. In this study, a bispecific anticarcinoembryonic antigen (CEACAM5; CD66e) × antihapten monoclonal antibody, TF2, was used in combination with a small (1.5 kD) peptide, IMP288, labeled with (111)In and (177)Lu. METHODS: First, tumor uptake of (111)In-IMP288 and (177)Lu-IMP288, as determined by immuno-SPECT, was validated by ex vivo counting. Two groups of female BALB/c nude mice had LS174T tumors implanted in the peritoneal cavity. They received intravenous injections of TF2, followed by 10 MBq of (111)In-IMP288 or 90 MBq of (177)Lu-IMP288. A control group of non-tumor-bearing mice received TF2 and (111)In-IMP288. One hour after the radiolabeled IMP288 was given, small-animal SPECT/CT images were acquired, and subsequently animals were dissected. Furthermore, a survival study was performed in 3 groups of 10 mice with intraperitoneal tumors: mice received TF2 and (177)Lu-IMP288 (60 MBq), nonpretargeted (177)Lu-IMP288 (60 MBq), or phosphate-buffered saline. Immuno-SPECT scans were acquired directly after therapy and at 14 and 45 d after therapy. Tumor growth was analyzed in the successive scans in each animal. RESULTS: (111)In- and (177)Lu-labeled IMP288 had similar in vivo distribution. The activity measured in the pretargeted immuno-SPECT images correlated well with the uptake measured in the dissected tumors (Pearson r = 0.99, P < 0.05). In the therapy study, the SPECT images showed rapid and selective tumor targeting with high tumor-to-background contrast (30 ± 12) as early as 1 h after injection. The successive images of the treated mice showed delayed tumor growth in the pretargeted radioimmunotherapy group, corresponding with their prolonged survival. CONCLUSION: Pretargeted immuno-SPECT with TF2 and (111)In- or (177)Lu-IMP288 can be used to predict and confirm tumor targeting and monitor the therapeutic effect of pretargeted radioimmunotherapy.
Journal of Nuclear Medicine 10/2012; · 6.38 Impact Factor
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ABSTRACT: Prevention and treatment of graft-versus-host disease (GVHD) remains a major challenge, given that current T cell depletion and mainstay immunosuppressive therapies compromise preexisting T cell immunity, often leading to severe infections and disease relapse. Thus, there is a critical need for novel anti-GVHD agents that can spare protective T cell memory. Here we show that milatuzumab (hLL1), a humanized anti-CD74 antagonist monoclonal antibody, can moderately reduce the numbers of CD74-expressing B cells and myeloid dendritic cells, but has no effect on the survival of T cells that are CD74(-). Consequently, milatuzumab inhibits allogeneic T cell proliferation in mixed leukocyte reactions. In a human/mouse xenogeneic SCID mouse model in which GVHD is induced and mediated by engrafted human CD4(+) T cells and dendritic cells, milatuzumab effectively prevents the onset and manifestations of acute GVHD, suppresses serum levels of human IFN-γ and IL-5, eliminates the infiltration of human lymphocytes into GVHD target organs (ie, lung, liver, and spleen), and significantly promotes survival (90% versus 20% for controls; P = .0012). Importantly, exposure to milatuzumab does not affect the number of cytomegalovirus-specific, IFN-γ-producing human CD8(+) T cells in allogeneic mixed leukocyte reactions. These encouraging results warrant further exploration of milatuzumab as a possible new therapeutic agent for GVHD.
Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 09/2012; · 3.15 Impact Factor
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Robert M Sharkey,
Catharina M van Rij,
Habibe Karacay,
Edmund A Rossi,
Cathelijne Frielink,
Celeste Regino,
Thomas M Cardillo,
William J McBride, Chien-Hsing Chang,
Otto C Boerman,
David M Goldenberg
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ABSTRACT: RS7 is an internalizing anti-Trop-2 pancarcinoma antibody capable of targeting most epithelial cancers. Because pretargeting strategies could improve the tumor localization of radionuclides, a new anti-Trop-2 × antihapten bispecific antibody for pretargeting, based on humanized RS7, was prepared and evaluated with a radiolabeled hapten-peptide in vitro and in vivo to determine whether its internalization properties would interfere with pretargeting.
The anti-Trop-2 × antihapten bispecific antibody, TF12, was prepared using the modular dock-and-lock method. TF12 and humanized RS7 binding was assessed by cell binding assays and fluorescence-activated cell sorting analysis in a variety of human carcinoma cell lines. The internalization of TF12 was evaluated in vitro using a fluorescent TF12 conjugate or hapten-peptide and (111)In-labeled TF12 and RS7. The biodistribution of TF12 and its use as a pretargeting agent with an (111)In-labeled hapten-peptide were assessed in several human epithelial cancer xenografts. Dose optimization was examined in 2 tumor models.
TF12 internalizes, but a substantial fraction remained accessible on the tumor surface. Fluorescence-activated cell sorting analysis showed only a minor change in fluorescent signal when the tumor was probed with a fluorescent hapten-peptide over 4 h, and microscopy showed substantial membrane staining when reassessed at 24 h after TF12 exposure. Only 40.1% of (111)In-TF12 was internalized after 24 h. In vivo, excellent tumor localization of the (111)In-labeled peptide was observed in several tumor models.
TF12 was retained sufficiently on the cell surface in several epithelial cancers, thereby making it suitable for pretargeted imaging and therapy of various Trop-2-expressing carcinomas.
Journal of Nuclear Medicine 09/2012; 53(10):1625-32. · 6.38 Impact Factor
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Pierre-Yves Salaun,
Loïc Campion,
Claire Bournaud,
Alain Faivre-Chauvet,
Jean-Philippe Vuillez,
David Taieb,
Catherine Ansquer,
Caroline Rousseau,
Françoise Borson-Chazot,
Stéphane Bardet,
Aurore Oudoux,
Bertrand Cariou,
Eric Mirallié, Chien-Hsing Chang,
Robert M Sharkey,
David M Goldenberg,
Jean-François Chatal,
Jacques Barbet,
Françoise Kraeber-Bodéré
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ABSTRACT: The prognosis of medullary thyroid carcinoma (MTC) varies from long- to short-term survival based on such prognostic factors as serum calcitonin and carcinoembryonic antigen (CEA) doubling times (DTs). This prospective phase II multicenter trial evaluated the efficacy and safety of anti-CEA pretargeted radioimmunotherapy (pRAIT) in rapidly progressing metastatic MTC patients and also how serum biomarker DTs correlate with clinical outcome.
From June 2004 to January 2008, 42 patients were treated with anti-CEA × anti-diethylenetriaminepentaacetic acid (DTPA) bispecific antibody (hMN-14 × m734) (40 mg/m(2)), followed by (131)I-di-DTPA-indium bivalent hapten (1.8 GBq/m(2)) 4-6 d later.
The disease control rate (durable stabilization plus objective response) was 76.2%. Grade 3-4 hematologic toxicity was observed in 54.7% of patients and myelodysplastic syndrome in 2, including 1 heavily treated previously. After pRAIT, 21 of 37 assessed patients (56.7%) showed a significant impact on DT (≥100% increase of pre-pRAIT calcitonin or CEA DT or prolonged decrease of the biomarker concentration after pRAIT). Pre-pRAIT DT and post-pRAIT DT were significant independent predictors for overall survival (OS) from pRAIT (pre-pRAIT: hazard ratio [HR], 0.46; 95% confidence interval [CI], 0.24-0.86; P = 0.016; and post-pRAIT: HR, 5.32; 95% CI, 1.63-17.36; P = 0.006) and OS from diagnosis (pre-pRAIT: HR, 0.21; 95% CI, 0.08-0.51; P = 0.001; and post-pRAIT: HR, 6.16; 95% CI, 1.81-20.98; P = 0.004).
pRAIT showed antitumor activity, with manageable hematologic toxicity in progressive MTC. Increased biomarker DT after treatment correlated with increased OS.
Journal of Nuclear Medicine 06/2012; 53(8):1185-92. · 6.38 Impact Factor
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ABSTRACT: Protein engineering technologies involving site-specific conjugation of two or more modular components can expand the existing repertoires that can be produced recombinantly or by chemical conjugation. The dock-and-lock (DNL) method combines recombinant engineering with site-specific conjugation, allowing the construction of various complex, yet defined, biostructures with multivalency and multispecificity. The technology platform exploits the natural interaction between two interactive human protein binding domains that are modified to provide covalent fusion. It has been validated by the production of various bioconjugates, including trivalent, tetravalent, pentavalent, and hexavalent antibodies of monospecificity or bispecificity; polyvalent immnocytokines; and site-specifically PEGylated cytokines. Here we assert that the DNL method is a useful tool in the development of novel therapeutic and diagnostic agents from both proteins and non-proteins for unmet medical needs.
Trends in Pharmacological Sciences 06/2012; 33(9):474-81. · 10.93 Impact Factor
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ABSTRACT: Bispecific antibody pretargeting is a two-step procedure for selectively delivering radionuclides to tumors. The procedure was developed to solve a number of problems encountered when radionuclides are directly coupled to an IgG, such as slow blood clearance and delayed tumor accretion. While various forms of antibody fragments can reduce blood pool activity and provide faster tumor localization, tumor uptake is reduced considerably. In pretargeting procedures, the radionuclide is attached to a small molecule that quickly traverses the vascular barrier to reach the tumor cells, achieving maximum accretion within 0.5 to 1.0 h. Just as quickly, it is eliminated from the body, thereby minimizing tissue exposure and developing high tumor/tissue ratios very early. In order to capture the radionuclide in the tumor, a bispecific antibody (bsMAb) that binds to the tumor and to the isotope carrier (e.g., a hapten-peptide) is pre-administered some time earlier. The pretargeting procedure has been shown repeatedly to improve tumor localization as compared to directly radiolabeled antibodies, thereby enhancing both imaging and therapy. In this article, we review the progress our group has made toward developing and testing bsMAb pretargeting systems for cancer detection and therapy.
Tumor Biology 03/2012; 33(3):591-600. · 1.94 Impact Factor
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ABSTRACT: In this study, pretargeted immuno-positron-emission tomography [PET] with a bispecific monoclonal anti-carcinoembryonic antigen [CEA] (CEACAM5) × anti-hapten antibody (bispecific monoclonal antibody [bsmAb]) and a small (1.5 kD) peptide labeled with 68Ga was compared to fludeoxyglucose [18F-FDG]-PET for detecting intraperitoneal [i.p.] CEA-expressing human colonic tumor xenografts in nude mice.
Two groups of female BALB/c nude mice were inoculated with LS174T human colonic tumor cells i.p. One group received 5 MBq 18F-FDG, and the other received intravenous injections of the bsmAb, followed 16 h later with 5 MBq of 68Ga-labeled peptide. One hour after the radiolabeled peptide or FDG was given, micro-PET/computed tomography images were acquired. Thereafter, the uptake of the 68Ga or 18F in dissected tissue was determined.
Within 1 h, high uptake of the 68Ga-labeled peptide in the tumor lesions (23.4 ± 7.2% ID/g) and low background activity levels were observed (e.g., tumor-to-intestine ratio, 58 ± 22). This resulted in a clear visualization of all intra-abdominal tumor lesions ≥ 10 μL and even some tumors as small as 5 μL (2 mm diameter). 18F-FDG efficiently localized in the tumors (8.7 ± 3.1% ID/g) but also showed physiological uptake in various normal tissues (e.g., tumor-to-intestine ratio, 3.9 ± 1.1).
Pretargeted immuno-PET with bsmAb and a 68Ga-labeled peptide could be a very sensitive imaging method for imaging colonic cancer, disclosing occult lesions.
EJNMMI research. 01/2012; 2:5.
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ABSTRACT: We describe the use of novel bispecific hexavalent Abs (HexAbs) to enhance anticancer immunotherapy. Two bispecific HexAbs [IgG-(Fab)(4) constructed from veltuzumab (anti-CD20 IgG) and milatuzumab (anti-CD74 IgG)] show enhanced cytotoxicity in mantle cell lymphoma (MCL) and other lymphoma/leukemia cell lines, as well as patient tumor samples, without a crosslinking Ab, compared with their parental mAb counterparts, alone or in combination. The bispecific HexAbs have different properties from and are more potent than their parental mAbs in vitro. The juxtaposition of CD20 and CD74 on MCL cells by the HexAbs resulted in homotypic adhesion and triggered intracellular changes that include loss of mitochondrial transmembrane potential, production of reactive oxygen species, rapid and sustained phosphorylation of ERKs and JNK, down-regulation of pAkt and Bcl-xL, actin reorganization, and lysosomal membrane permeabilization, culminating in cell death. They also displayed different potencies in depleting lymphoma cells and normal B cells from whole blood ex vivo and significantly extended the survival of nude mice bearing MCL xenografts in a dose-dependent manner, thus indicating stability and antitumor activity in vivo. Such bispecific HexAbs may constitute a new class of therapeutic agents for improved cancer immunotherapy, as shown here for MCL and other CD20(+)/CD74(+) malignancies.
Blood 01/2012; 119(16):3767-78. · 9.90 Impact Factor
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ABSTRACT: We constructed novel HIV-1 fusion inhibitors that may overcome the current limitations of enfuvirtide, the first such therapeutic in this class. The three prototypes generated by the Dock-and-Lock (DNL) technology to comprise four copies of enfuvirtide tethered site-specifically to the Fc end of different humanized monoclonal antibodies potently neutralize primary isolates (both R5-tropic and X4-tropic), as well as T-cell-adapted strains of HIV-1 in vitro. All three prototypes show EC(50) values in the subnanomolar range, which are 10- to 100-fold lower than enfuvirtide and attainable whether or not the constitutive antibody targets HIV-1. The potential of such conjugates to purge latently infected cells was also demonstrated in a cell-to-cell viral inhibition assay by measuring their efficacy to inhibit the spread of HIV-1(LAI) from infected human peripheral blood mononuclear cells to Jurkat T cells over a period of 30 days following viral activation with 100 nM SAHA (suberoylanilide hydroxamic acid). The IgG-like half-life was not significantly different from that of the parental antibody, as shown by the mean serum concentration of one prototype in mice at 72 h. These encouraging results provide a rationale to develop further novel anti-HIV agents by coupling additional antibodies of interest with alternative HIV-inhibitors via recombinantly-produced, self-assembling, modules.
PLoS ONE 01/2012; 7(7):e41235. · 4.09 Impact Factor
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Chien-Hsing Chang,
Yang Wang,
Preeti Trisal,
Rongxiu Li,
Diane L Rossi,
Anju Nair,
Pankaj Gupta,
Michele Losman,
Thomas M Cardillo,
Edmund A Rossi,
David M Goldenberg
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ABSTRACT: A major mechanism of monoclonal antibodies that selectively target the insulin-like growth factor type 1 receptor (IGF-1R) to inhibit tumor growth is by downregulating the receptor, regardless whether they are capable (antagonistic) or incapable (agonistic) of blocking the binding of cognate ligands. We have developed and characterized a novel agonistic anti-IGF-1R humanized antibody, hR1, and used the Dock-and-Lock (DNL) method to construct Hex-hR1, the first multivalent antibody comprising 6 functional Fabs of hR1, with the aim of enhancing potency of hR1. Based on cross-blocking experiments, hR1 recognizes a region of cysteine-rich domain on the α-subunit, different from the epitopes mapped for existing anti-IGF-1R antibodies, yet hR1 is similar to other anti-IGF-1R antibodies in downregulating IGF-1R and inhibiting proliferation, colony formation, or invasion of selected cancer cell lines in vitro, as well as suppressing growth of the RH-30 rhabdomyosarcoma xenograft in nude mice when combined with the mTOR inhibitor, rapamycin. Hex-hR1 and hR1 are generally comparable in their bioactivities under the in-intro and in-vivo conditions investigated. Nevertheless, in selective experiments involving a direct comparison of potency, Hex-hR1 demonstrated a stronger effect on inhibiting cell proliferation stimulated by IGF-1 and could effectively downregulate IGF-1R at a concentration as low as 20 pM.
PLoS ONE 01/2012; 7(8):e44235. · 4.09 Impact Factor
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ABSTRACT: Pretargeting is a multi-step process that first has an unlabeled bispecific antibody (bsMAb) localize within a tumor by virtue of its anti-tumor binding site(s) before administering a small, fast-clearing radiolabeled compound that then attaches to the other portion of the bsMAb. The compound's rapid clearance significantly reduces radiation exposure outside of the tumor and its small size permits speedy delivery to the tumor, creating excellent tumor/nontumor ratios in less than 1 hour. Haptens that bind to an anti-hapten antibody, biotin that binds to streptavidin, or an oligonucleotide binding to a complementary oligonucleotide sequence have all been radiolabeled for use by pretargeting. This review will focus on a highly flexible anti-hapten bsMAb platform that has been used to target a variety of radionuclides to image (SPECT and PET) as well as treat tumors.
Theranostics. 01/2012; 2(5):523-40.
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ABSTRACT: Cell fusion in vitro has been used to study cancer, gene mapping and regulation, and the production of antibodies via hybridomas. However, in-vivo heterosynkaryon formation by cell-cell fusion has received less attention. This investigation describes the spontaneous fusion of a human glioblastoma with normal hamster cells after xenogeneic transplantation, resulting in malignant cells that express both human and hamster genes and gene products, and retention of glioblastoma traits with an enhanced ability to metastasize. Three of 7 human genes found showed translation of their proteins during serial propagation in vivo or in vitro for years; namely, CD74, CXCR4 and PLAGL2, each implicated with malignancy or glioblastoma. This supports the thesis that genetic hybridization of cancer and normal cells can transmit malignancy and also, as first described herein, regulatory genes involved in the tumor's organotypic morphology. Evidence also is increasing that even cell-free human cancer DNA can induce malignancy and transfer genetic information to normal cells. Hence, we posit that the transfer of genetic information between tumor and stromal cells, whether by cell-cell fusion or other mechanisms, is implicated in the progression of malignancy, and may further define the crosstalk between cancer cells and their stromal neighbors.
International Journal of Cancer 07/2011; 131(1):49-58. · 5.44 Impact Factor
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ABSTRACT: Biotechnology products are being designed increasingly as multivalent, multifunctional structures based on recombinant fusion
proteins. To augment the existing repertoires of such agents as produced by genetic engineering alone, we envisioned and developed
the Dock-and-Lock (DNL) method, which enables site-specific conjugation of two modular components only with each other, producing
a covalent construct of defined composition with retained bioactivity. The basic strategy of DNL involves the generation of
two types of modules, one containing the dimerization and docking domain (DDD) of cAMP-dependent protein kinase A and the
other containing the anchoring domain (AD) of an interactive A-kinase anchoring protein. Docking a DDD module with an AD module
occurs spontaneously, and the resulting complex is locked with disulfide bonds to enhance in vivo stability. This new platform
technology provides a versatile approach to create novel bispecific antibodies of multiple valencies, with potential applications
in the detection and therapy of diverse diseases.
07/2011: pages 199-216;
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ABSTRACT: The short circulating half-life and side effects of IFNα affect its dosing schedule and efficacy. Fusion of IFNα to a tumor-targeting mAb (mAb-IFNα) can enhance potency because of increased tumor localization and improved pharmacokinetics. We used the Dock-and-Lock method to generate C2-2b-2b, a mAb-IFNα comprising tetrameric IFNα2b site-specifically linked to hL243 (humanized anti-HLA-DR). In vitro, C2-2b-2b inhibited various B-cell lymphoma leukemia and myeloma cell lines. In most cases, this immunocytokine was more effective than CD20-targeted mAb-IFNα or a mixture comprising the parental mAb and IFNα. Our findings indicate that responsiveness depends on HLA-DR expression/density and sensitivity to IFNα and hL243. C2-2b-2b induced more potent and longer-lasting IFNα signaling compared with nontargeted IFNα. Phosphorylation of STAT1 was more robust and persistent than that of STAT3, which may promote apoptosis. C2-2b-2b efficiently depleted lymphoma and myeloma cells from whole human blood but also exhibited some toxicity to B cells, monocytes, and dendritic cells. C2-2b-2b showed superior efficacy compared with nontargeting mAb-IFNα, peginterferonalfa-2a, or a combination of hL243 and IFNα, using human lymphoma and myeloma xenografts. These results suggest that C2-2b-2b should be useful in the treatment of various hematopoietic malignancies.
Blood 06/2011; 118(7):1877-84. · 9.90 Impact Factor
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ABSTRACT: Bispecific antibody pretargeting is highly sensitive and specific for cancer detection by PET. In this study, the preparation of a high-specific-activity (68)Ga-labeled hapten-peptide, IMP288, was evaluated.
IMP288 (DOTA-D-Tyr-D-Lys(histamine-succinyl-glycine [HSG])-D-glu-D-Lys(HSG)-NH(2)) was added to buffered (68)Ga and then heated in boiling water and purified on a reversed-phase cartridge. Tumor-bearing nude mice were used for biodistribution and tumor localization studies.
(68)Ga-IMP288 was prepared at a starting specific activity up to 1.78 GBq/nmol, with final yields of 0.74 GBq/nmol (decay-corrected) and less than 1% unbound (68)Ga. Purification was essential to remove unbound (68)Ga and (68)Ge breakthrough. Pretargeted animals showed a high (68)Ga-IMP288 uptake (27.5 ± 5.8 percentage injected dose per gram), with ratios of 13.6 ± 4.8, 66.8 ± 14.5, and 325.9 ± 61.9 for the kidneys, liver, and blood, respectively, at 1.5 h after peptide injection.
High-specific-activity labeling of DOTA-hapten-peptide was obtained from the (68)Ga/(68)Ge generator for approximately 1 y, yielding products suitable for immunoPET.
Journal of Nuclear Medicine 03/2011; 52(4):555-9. · 6.38 Impact Factor
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ABSTRACT: With over 25 monoclonal antibodies (mAbs) currently approved and many more in development, there is considerable interest in gaining improved productivity by increasing cell density and enhancing cell survival of production cell lines. In addition, high costs and growing safety concerns with use of animal products have made the availability of serum-free cell lines more appealing. We elected to transfect the myeloma cell line Sp2/0-Ag14 with Bcl2-EEE, the constitutively active phosphomimetic mutant of Bcl2, for extended cell survival. After adaptation of the initial transfectants to serum-independent growth, a clone with superior growth properties, referred to as SpESF, was isolated and further subjected to iterative rounds of stressful growth over a period of 4 months. The effort resulted in the selection of a promising clone, designated SpESFX-10, which was shown to exhibit robust growth and resist apoptosis induced by sodium butyrate or glutamine deprivation. The advantage of SpESFX-10 as a host for generating mAb-production cell lines was demonstrated by its increased transfection efficiency, culture longevity, and mAb productivity, as well as by the feasibility of accomplishing the entire cell line development process, including transfection, subcloning, and cryopreservation, in the complete absence of serum.
Biotechnology Progress 03/2011; 27(3):766-75. · 2.34 Impact Factor
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ABSTRACT: We have generated hexavalent antibodies (HexAbs) comprising 6 Fabs tethered to one Fc of human IgG1. Three such constructs, 20-20, a monospecific HexAb comprising 6 Fabs of veltuzumab (humanized anti-CD20 immunoglobulin G1κ [IgG1κ]), 20-22, a bispecific HexAb comprising veltuzumab and 4 Fabs of epratuzumab (humanized anti-CD22 IgG1κ), and 22-20, a bispecific HexAb comprising epratuzumab and 4 Fabs of veltuzumab, were previously shown to inhibit pro-liferation of several lymphoma cell lines at nanomolar concentrations in the absence of a crosslinking antibody. We now report an in-depth analysis of the apoptotic and survival signals induced by the 3 HexAbs in Burkitt lymphomas and provide in vitro cytotoxicity data for additional lymphoma cell lines and also chronic lymphocytic leukemia patient specimens. Among the key findings are the significant increase in the levels of phosphorylated p38 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) by all 3 HexAbs and the notable differences in the signaling events triggered by the HexAbs from those incurred by crosslinking veltuzumab or rituximab with a secondary antibody. Thus, the greatly enhanced direct toxicity of these HexAbs correlates with their ability to alter the basal expression of various intracellular proteins involved in regulating cell growth, survival, and apoptosis, with the net outcome leading to cell death.
Blood 10/2010; 116(17):3258-67. · 9.90 Impact Factor
