Induction of human T lymphocyte cytotoxicity and inhibition of tumor growth by tumor-specific diabody-based molecules secreted from gene-modified bystander cells.
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Infiltrating T cells are found in many malignancies, but they appear to be mostly anergic and do not attack the tumor, presumably because of the absence of activation and/or costimulatory signals. We describe a strategy for cellular antitumor immunotherapy by the in situ production of soluble bifunctional Ab-based molecules that activate and retarget T cells to the tumor. We genetically modified cells to simultaneously secrete two bifunctional molecules, a bispecific diabody directed against the carcinoembryonic Ag (CEA) and the CD3 epsilon chain of the TCR (alphaCEA x alphaCD3), and a fusion protein comprising the extracellular portion of B7-1 fused to a bivalent anti-CEA diabody (B7-alphaCEA). Together, alphaCEA x alphaCD3 and B7-alphaCEA proved potent at inducing the activation, proliferation, and survival of primary human T cells. When producer cells were cocultured with primary T cells and CEA(+) cancer cells, alphaCEA x alphaCD3 and B7-alphaCEA acted in combination to activate and retarget T cell cytotoxicity and completely abrogate tumor growth in the coculture. Furthermore, the introduction of just a few such producer cells at the tumor site efficiently inhibited the growth of established human colon carcinoma xenografts. Despite a cumbersome generation process, the use of autologous gene-modified producer cells opens the way for a new diabody-based gene therapy strategy of cancer. (+info)
CpG-A and B oligodeoxynucleotides enhance the efficacy of antibody therapy by activating different effector cell populations.
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Immunostimulatory CpG oligodeoxynucleotides (ODNs) can enhance the therapeutic effect of monoclonal antibodies (mAbs) by enhancing antibody-dependent cell-mediated cytotoxicity (ADCC). Distinct classes of CpG ODNs have been found recently to stimulate different effector cell populations. We used murine cancer models to explore the role of various effector cell populations in the antitumor activity seen with mAbs combined with CpG ODNs of the A and B classes. In the 38C13 syngeneic murine lymphoma model, both CpG A and CpG B enhanced the efficacy of murine antilymphoma mAb. Depletion of natural killer (NK) cells alone markedly decreased the efficacy of therapy with mAbs plus CpG A. In contrast, depletion of both NK cells and granulocytes was required to decrease the efficacy of mAb plus CpG B. A human (h) Fc gamma receptor I (FcgammaRI)-expressing transgenic (Tg) mouse model was used to explore the role of FcgammaRI in therapy with mAb and CpG ODN. CpG B induced up-regulation of FcgammaRI in hFcgammaRI Tg mice, whereas CpG A did not. In vitro CpG B also enhanced ADCC of HER-2/neu-expressing tumor cells by the FcgammaRI-directed bispecific antibody MDX-H210 using hFcgammaRI-positive effector cells. In a solid tumor model, tumor growth was inhibited in Tg mice treated with a combination of MDX-H210 and CpG B. These data suggest that CpG A enhance ADCC largely by activating NK cells. In contrast, other effector cell populations, including granulocytes, contribute to the antitumor activity of CpG B and mAbs. FcgammaRI plays an important role in this activity. (+info)
Pretargeting with labeled bivalent peptides allowing the use of four radionuclides: (111)In, (131)I, (99m)Tc, and (188)Re.
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PURPOSE: The therapeutic effect of directly labeled antibodies in solid tumors is limited, mainly due to the relatively low uptake of the radiolabeled antibody in tumors as compared with their blood level. In previous studies, we have shown that renal cell carcinoma (RCC) can be targeted very effectively with the (111)In-labeled bivalent peptide di-diethylenetriamminepentaacetic acid diDTPA-FKYK, after pretargeting the tumor with a bispecific antibody. In this study, we further developed this pretargeting approach for radioimmunotherapy of renal cell cancer. EXPERIMENTAL DESIGN: Pretargeting with the biologically produced anti-RCC x anti-DTPA bispecific monoclonal antibody (bsMAb G250xDTIn1) was tested in mice with SK-RC-52 RCC tumors. Tumors were pretargeted with 15 micro g of bispecific monoclonal antibody G250xDTIn1, and 24 h later, mice received 6 ng of the radiolabeled bivalent peptide. Two different peptides were used: (a) diDTPA-FKYK labeled with (111)In or (131)I; and (b) thiosemicarbonylglyoxylcysteinyl-diDTPA(In)-KYKK labeled with (99m)Tc or (188)Re. Mice were killed 6, 24, 48, and 72 h postinjection (p.i.), and biodistribution of the radiolabel was determined. RESULTS: The (111)In-labeled peptide showed excellent tumor uptake [42.6 +/- 7.3% injected dose/gram (ID/g) at 6 h p.i. and 25.6 +/- 7.7% ID/g at 72 h p.i.] and tumor:blood ratios (700 at 72 h p.i.). The specific tumor targeting of (188)Re- and (99m)Tc-labeled peptides was similar (20-25% ID/g, 6 h p.i.). However, the uptake and the retention in the tumor of the (99m)Tc- and (188)Re-labeled peptide were significantly lower than those of the (111)In-labeled peptide. Tumor uptake of the (131)I-labeled peptide was significantly lower as compared with the other three radiolabeled peptides; furthermore, an almost complete washout of the radiolabel from the tumor over time was observed (14.5 +/- 4.9% ID/g at 6 h p.i. and 0.33 +/- 0.15% ID/g at 72 h p.i.). CONCLUSIONS: Using a newly developed bivalent peptide, this pretargeting approach can now be used for targeting with the matched pair (188)Re and (99m)Tc. (+info)
Development of new multivalent-bispecific agents for pretargeting tumor localization and therapy.
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PURPOSE: Two bispecific diabodies (BS1.5 and BS1.5H) and two bispecific trivalent proteins (BS6 and BS8) were produced and tested as potential agents for pretargeted delivery of radiolabeled bivalent haptens to tumors expressing carcinoembryonic antigen. EXPERIMENTAL DESIGN: Each of the four proteins was expressed in Escherichia coli and purified from the soluble fraction. BS1.5 and BS1.5H (a humanized version of BS1.5) were evaluated in the GW-39 human colonic tumor-nude mouse model using a di-HSG-1,4,7,10-tetra-azacyclododecane-N,N',N" N"'-tetraacetic acid peptide (IMP-241) radiolabeled with (111)In. The biodistribution and T/NT ratios were compared with those of hMN-14 x m679 (Fab' x Fab') prepared chemically. RESULTS: In animals, both BS1.5 and BS1.5H cleared more rapidly than hMN-14 x m679 and showed tumor to nontumor ratios far superior to those of hMN-14 x m679. For example, with BS1.5 injected 8 h before (111)In-IMP-241, the tumor uptake of (111)In was 10.3 +/- 2.7 and 6.3 +/- 2.2% ID/g at 3 and 24 h, respectively, with the tumor to blood ratios being 167 +/- 35 at 3 h and 631 +/- 231 at 24 h. In comparison, the tumor to blood ratios of (111)In observed for hMN-14 x m679 given 24 h earlier were 8 +/- 2 at 3 h and 16 +/- 3 at 24 h. CONCLUSIONS: These results indicate that BS1.5 and BS1.5H are promising candidates for use in a variety of pretargeting applications, including tumor therapy with radionuclides and drugs. BS6 and BS8 may be even more attractive because of their potential to achieve higher levels of tumor uptake because of divalent carcinoembryonic antigen binding. (+info)
Optimizing bispecific antibody pretargeting for use in radioimmunotherapy.
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PURPOSE: With increasing interest in pretargeting procedures for improving the delivery of radionuclides for cancer imaging and therapy, this investigation was undertaken to examine how to optimize a bispecific monoclonal antibody (bsMAb) pretargeting procedure for therapeutic applications. EXPERIMENTAL DESIGN: The model system examined was a bsMAb composed of two Fab' fragments, one from a humanized anti-carcinoembryonic antigen antibody (hMN-14), and the other a murine antibody (679) against histamine-succinyl-glycine. These Fab' fragments were chemically conjugated to form a F(ab')(2) that is joined by a stable thioether bond. The peptide used for these studies (IMP-241) contained two histamine-succinyl-glycine moieties for binding to the 679 portion of the bsMAb and a single 1,4,7,10-tetra-azacyclododecane N,N',N",N"'-tetraacetic acid chelate for radiolabeling with (111)In. RESULTS: The bsMAb cleared rapidly in nude mice bearing the GW-39 human colonic cancer xenograft. Administration of a radiolabeled peptide 1 day after the bsMAb, using a bsMAb/peptide mole injection ratio of 10:1, allowed for higher tumor accretion than if delayed by 2 days. Tumor uptake measured 3 h after the peptide injection given 1 day after the bsMAb was 11.3 +/- 2.2% percentage of injected dose/gram (%ID/g), with just 2.9 +/- 0.4% ID/g of the bsMAb in the tumor at this time. Tumor/blood ratios were 8.1 +/- 2.1. Peptide uptake was highest in the kidneys, but even so, the tumor/kidney ratio was 2.5 +/- 1.9 just 3 h after the peptide injection. Although low bsMAb/peptide mole injection ratios allow for greater concentrations of the peptide in the tumor, kidney uptake is increased at a proportionally higher amount than in the tumor. Therefore, a bsMAb/peptide injection ratio of 10:1 with a 24-h interval was preferred for pretargeting. Increasing the bsMAb dose, and thereby increasing the bsMAb/peptide injection ratio, further enhanced the delivery of the radiolabeled peptide to the tumor, but the interval spacing between the bsMAb and peptide had to be increased. Despite having a lower %ID/g of the bsMAb in the tumor, with a bsMAb/peptide injection ratio of 50:1 and a 48-h interval, tumor uptake of the (111)In-peptide was nearly 30% ID/g, a 1.6-fold improvement over that seen with the 10:1/24-h interval pretargeting group, and tumor/blood was 35:1, and tumor/kidney ratio was 8:1. Two fractionation strategies were also examined. Giving two equal fractions of peptide after a single injection of bsMAb loaded more moles of peptide into the tumor but would not permit higher radioactivity delivery than what could be achieved with a single injection. However, area under the curve analysis indicated that giving repeated cycles of the bsMAb followed by the peptide would enable improvements in the amount of radioactivity delivered to the tumor without increasing the amount delivered to normal tissues, but the timing of the bsMAb/peptide cycles was important to optimize this process. Finally, it was noted that larger tumors (e.g. those > 0.3 g) were more likely to have higher peptide uptake in a pretargeting procedure than smaller tumors (e.g., those of approximately 0.1 g), perhaps due to the greater mass of the bsMAb localized in the larger tumors, but also possibly because of better blood supply in these tumors. CONCLUSIONS: These studies reveal principles that might be applied generally to other pretargeting procedures and demonstrate how a bsMAb pretargeting method could potentially exceed a directly radiolabeled antibody in its ability to deliver radionuclides for cancer therapy. (+info)
Pharmacokinetics and dosimetry studies for optimization of anti-carcinoembryonic antigen x anti-hapten bispecific antibody-mediated pretargeting of Iodine-131-labeled hapten in a phase I radioimmunotherapy trial.
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PURPOSE: Pharmacokinetics and dosimetry of hMN-14 x m734 bispecific monoclonal antibody (BsMAb) and (131)I-labeled di-diethylenetriaminepentaacetic acid-indium ((131)I-hapten) were studied to optimize pretargeted radioimmunotherapy. EXPERIMENTAL DESIGN: Thirty-five patients with carcinoembryonic antigen-expressing tumors were included. In a first group of 12 patients, (131)I-trace-labeled BsMAb doses were escalated from 10 to 100 mg/m(2), and 3.7 GBq of (131)I-hapten were administered 7 days later. In a second group, 12 patients received 75 mg/m(2) BsMAb and 2.6-4.2 GBq of (131)I-hapten 5 days later. The BsMAb dose was then reduced to 40 mg/m(2), and 10 patients received 1.9-5.5 GBq of (131)I-hapten. Blood samples were collected. Biodistribution was monitored by quantitative scintigraphy. RESULTS: Directly labeled BsMAb pharmacokinetics was described by two exponentials: half-lives were 8.1 h (2.0-18.1 h) and 48.2 h (22.8-79.4 h); blood clearance was 123 ml/h (64-195 ml/h). With a 7-day interval, 10 or 30 mg/m(2) BsMAb resulted in fast elimination and very low tumor uptake of hapten, whereas 50 or 100 mg/m(2) resulted in favorable tumor accretion. With 75 mg/m(2) BsMAb and a 5-day interval, hapten clearance was 152 ml/h (81-298 ml/h). Calculated radiation dose to tumor was 3.9 Gy/GBq (0.4-22.4 Gy/GBq) for the hapten, compared with 2.0 Gy/GBq (0.3-3.8 Gy/GBq) for the BsMAb, but hematological toxicity prevented dose escalation. Reduction of the BsMAb dose to 40 mg/m(2) accelerated hapten clearance to 492 ml/h (113-2544 ml/h) and reduced hematological toxicity without compromising tumor uptake [5.2 Gy/GBq (0.5-12.6 Gy/GBq)]. CONCLUSIONS: Optimized BsMAb doses and time interval will allow for the administration of higher, tumoricidal, activity doses. (+info)
Simultaneous blockade of both the epidermal growth factor receptor and the insulin-like growth factor receptor signaling pathways in cancer cells with a fully human recombinant bispecific antibody.
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Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR) have been implicated in the tumorigenesis of a variety of human cancers. Effective tumor inhibition has been achieved both experimentally and clinically with a number of strategies that antagonize either receptor activity. Here we constructed and produced two fully human recombinant bispecific antibodies (BsAb) that target both EGFR and IGFR, using two neutralizing human antibodies originally isolated from a phage display library. The BsAb not only retained the antigen binding capacity of each of the parent antibodies, but also were capable of binding to both targets simultaneously as demonstrated by a cross-linking enzyme-linked immunosorbent assay. Furthermore, the BsAb effectively blocked both ligands, EGF and IGF, from binding to their respective receptors, and inhibited tumor cell proliferation as potently as a combination of both the parent antibodies. More importantly, the BsAb were able to completely block activation of several major signal transduction molecules, including Akt and p44/p42 MAP kinases, by both EGF and IGF, whereas each individual parent antibody was only effective in inhibiting those signal molecules activated by the relevant single growth factor. The BsAb molecules retained good antigen binding activity after incubation with mouse serum at 37 degrees C for up to 6 days. Taken together, our results underscore the benefits of simultaneous targeting multiple growth factor receptor pathways for more efficacious cancer treatment. This report describes the first time use of a recombinant BsAb for targeting two tumor-associated molecules on either a single or adjacent tumor cells for enhanced antitumor activity. (+info)
Potentiation of ionising radiation by targeting tumour necrosis factor alpha using a bispecific antibody in human pancreatic cancer.
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The aim of this study was to treat carcinoembryonic antigen (CEA)-expressing pancreatic carcinoma cells with tumour necrosis factor alpha (TNFalpha) and simultaneous radiation therapy (RT), using a bispecific antibody (BAb) anti-TNFalpha/anti-CEA. TNFalpha used alone produced a dose-dependent inhibition of the clonogenic capacity of the cultured cells. Flow cytometry analysis of cell cycle progression confirmed the accumulation of cells in G(1) phase after exposure to TNFalpha. When TNFalpha was added 12 h before RT, the surviving fraction at 2 Gy was 60% lower than that obtained with irradiation alone (0.29 vs 0.73, respectively, P<0.00001). In combination treatment, cell cycle analysis demonstrated that TNFalpha reduced the number of cells in radiation-induced G(2) arrest, blocked irreversibly the cells in G(1) phase, and showed an additive decrease of the number of cells in S phase. In mice, RT as a single agent slowed tumour progression as compared with the control group (P<0.00001). BAb+TNFalpha+RT combination enhanced the delay for the tumour to reach 1500 mm(3) as compared with RT alone or with RT+TNFalpha (P=0.0011). Median delays were 90, 93, and 142 days for RT alone, RT+TNFalpha, and RT+BAb+TNFalpha groups, respectively. These results suggest that TNFalpha in combination with BAb and RT may be beneficial for the treatment of pancreatic cancer in locally advanced or adjuvant settings. (+info)