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Combined low dose radio- and radioimmunotherapy of experimental HeLa Hep 2 tumours.
Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi.
Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi.
Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi.
Umeå universitet, Medicinsk fakultet, Strålningsvetenskaper, Radiofysik.
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2003 (Engelska)Ingår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 30, nr 6, s. 895-906Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Radiation therapy of malignant tumours can be delivered by external beam radiation (RT) or radioimmunotherapy (RIT), using nuclides attached to monoclonal antibodies (mAbs). These treatment modalities have now been combined in order to investigate putative therapeutic advantages and elucidate the biological responses involved. Nude mice were transplanted subcutaneously on the back with human HeLa Hep2 tumour cells. RT (3x5 Gy) and/or 100 microg (131)I-labelled mAb H7, against placental alkaline phosphatase, or (131)I-labelled mAb TS1, against cytokeratin, was administered separately or in combination (specific activity of 120-200 MBq/mg antibody). Significant tumour growth retardation was observed both with RT alone and with RIT alone. Combining these regimens enhanced the therapeutic effects further, and a significant reduction in tumour volume could be demonstrated. The tumours were subjected to extensive histochemical and immunohistochemical investigations in order to elucidate changes in biology and histology within them. The following stainings were used: haematoxylin-eosin (morphology), Ki67 (proliferation), M30 (apoptosis), TUNEL (apoptosis) and endoglin (vascularisation). Tumours in the control group grew fast, with an average tumour doubling time of 9 days. These tumours contained large viable tumour cell masses displaying vast proliferation zones of Ki67-positive tumour cells, as well as necrotic regions and small amounts of connective tissue. Apoptotic cells could be identified both with M30 and TUNEL staining. When RT was applied, the growth rate was significantly reduced (doubling time 19 days) and typical alterations in morphology were seen, with a relative increase in connective tissue and a decrease in necrotic regions. Apoptotic cells were identified and a decrease in cell density was also observed. When RIT alone was applied, the growth parameters indicated a longer lasting growth reduction, especially when TS1 was used separately or in combination with H7. The histological appearances of these tumours were somewhat different from the RT-treated tumours, with a larger portion of intratumoural cysts. These tumours also presented a reduced tumour cell density. Dramatic effects were observed when RT was combined with RIT, with a pronounced growth reduction seen in all combination treatment groups. Pronounced tumour volume reduction was also evident in both the RT + RIT ((131)I-TS1) group and RT + RIT ((131)I-TS1/(131)I-H7) group, and in some animals no tumour remained at all. The morphology of the tumour remnants at day 22 was chaotic with a drastically changed histology, with presence of abundant cysts, low fractions of Ki67-positive cells, reduction in cell density, increased amounts of connective tissue and a decrease in necrotic regions. Again, apoptotic cells could be identified, scattered throughout the viable regions. Combining RT and RIT seems to generate an efficient treatment with convincing and long-lasting tumour growth inhibition, which is reflected in a highly aberrant histology within the tumour. Results obtained in this study indicate that both necrosis and apoptosis may be involved in the process leading to this efficient therapy of epithelially derived tumours.

Ort, förlag, år, upplaga, sidor
2003. Vol. 30, nr 6, s. 895-906
Identifikatorer
URN: urn:nbn:se:umu:diva-4920DOI: 10.1007/s00259-003-1177-2PubMedID: 12721768OAI: oai:DiVA.org:umu-4920DiVA, id: diva2:144202
Tillgänglig från: 2006-01-19 Skapad: 2006-01-19 Senast uppdaterad: 2018-06-09Bibliografiskt granskad
Ingår i avhandling
1. Experimental radioimmunotherapy and effector mechanisms
Öppna denna publikation i ny flik eller fönster >>Experimental radioimmunotherapy and effector mechanisms
2006 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Radioimmunotherapy is becoming important as a new therapeutic strategy for treatment of tumour diseases. Lately monoclonal antibodies tagged with radionuclides have demonstrated encouraging results in treatment of hematological malignancies. The progress in treatment of solid tumours using radioimmunotherapy, however, has been slow. New strategies to improve the treatment response need to be evaluated. Such new strategies include the combination of radioimmunotherapy with other treatment modalities but also elucidation and exploration of the death effector mechanisms involved in tumour eradication.

As the combination of radioimmunotherapy and radiotherapy provides several potential synergistic effects, we started out by optimising a treatment schedule to detect benefits combining these treatment modalities. An anti-cytokeratin antibody labelled with 125I administered before, after, or simultaneously with radiotherapy, indicated that the highest dose to the tumour was delivered when radiotherapy was given prior to the antibody administration. The optimised treatment schedule was then applied therapeutically in an experimental study on HeLa Hep2 tumour bearing nude mice given radiotherapy prior to administration of 131I-labelled monoclonal antibodies. Combining these treatment regimes enhanced the effect of either of the treatment modalities given alone, and a significant reduction in tumour volumes could be demonstrated. This treatment caused a dramatic change in tumour morphology, with increased amounts of connective tissue, giant cells and cysts. Furthermore cellular alterations like heterogeneity of nuclear and cytoplasmic size and shape were observed, and at least a fraction of the tumour cells presented some characteristics of apoptosis.

The induced sequential events in Hela Hep2 cells exposed to 2.5-10 Gy of ionizing radiation were studied further, with special emphasis on cell cycle arrest, mitotic aberrations and finally cell death. Following radiation HeLa Hep2 cells initiated a transient G2/M arrest trying to repair cellular damage. This arrest was followed by a sequence of disturbed mitoses with anaphase bridges, lagging chromosomal material, hyperamplification of centrosomes and multipolar mitotic spindles. These mitotic disturbances produced multinuclear polyploid cells and cells with multiple micronuclei, cells that were destined to die via mitotic catastrophes and delayed apoptosis.

Induction of apoptosis in HeLa Hep2 cells following radiation doses and dose-rates equivalent to those delivered at radioimmunotherapy was concurrently studied in vitro. Significant induction of apoptosis was obtained and found to be induced relatively slowly, peaking 72-168 hours post irradiation. Caspases from the intrinsic pathway as well as the extrinsic pathway were found to be activated in response to ionizing radiation. Furthermore caspase-2, which has recently been acknowledged for its role as an initiator caspase was found to be activated following radiation and seems to play an important role in this delayed apoptosis.

Ort, förlag, år, upplaga, sidor
Umeå: Klinisk mikrobiologi, 2006. s. 72
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1002
Nyckelord
radioimmunotherapy, apoptosis, mitotic catastrophe, caspases, cell cycle disturbances, combination treatment.
Nationell ämneskategori
Immunologi
Identifikatorer
urn:nbn:se:umu:diva-673 (URN)91-7264-013-8 (ISBN)
Disputation
2006-02-10, A 103, byggnad 6A, våning 1, 90185 NUS, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2006-01-19 Skapad: 2006-01-19 Senast uppdaterad: 2009-10-07Bibliografiskt granskad
2. Recombinant antibodies and tumor targeting
Öppna denna publikation i ny flik eller fönster >>Recombinant antibodies and tumor targeting
2006 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Different antibody derived constructs are rapidly advancing as putative tools for treatment of malignant diseases. Antibody engineering has added significant new technologies to modify size, affinities, solubility, stability and biodistribution properties for immunoconjugates.

In the present thesis, the aim was to increase our knowledge on how new recombinant antibodies could be tailored to optimize localization to experimental tumors in mice.

One hybridoma, producing the monoclonal antibody H7, against placental alkaline phosphatase was used to provide CDR-containing regions for new recombinant constructs. Both single-chain variable fragments (scFv) antibodies or divalent tandem monospecific scFv [sc(Fv)2] antibodies with properties suitable for targeting were produced. By site directed mutagenesis four selective sequence substitutions were made in the VL fragment and one in the VH fragment of the antibody to improve solubility. To increase the stability of the antibody an extra -S-S- bond was introduced between the VL fragment and the VH region to create single-chain disulphide stabilized variable fragments (scdsFv) antibodies. Altogether four different recombinant scFv/scdsFv antibody constructs were produced and compared in terms of solubility, stability, affinity and production properties. The overall affinity could be maintained at the same order of magnitude as the wild type antibody (KA >109 (M-1) as determined by Biacore measurements. The antibodies could easily be expressed in the E.Coli strain BL21 Origami B (DE3). The purified recombinant antibodies could be maintained stable in concentrations up to 0.8 mg/ml.

Similarly a bivalent recombinant scFv antibody was generated from the same basic scFv, in form of a divalent tandem single-chain antibody [sc(Fv)2] by introduction of a short linker sequence between the two scFvs. The recombinant antibody was characterized by SDS-PAGE, ELISA, Western blot and Biacore analyses and was found to retain a similar high affinity as the original H7 antibody. All recombinant antibody derivates were subsequently investigated for targeting ability in vivo. Nude mice, inoculated with HeLa Hep2 tumor cells were exposed in vivo to the 125I-labelled recombinant constructs. All recombinant antibodies were found to localize and display characteristic differences in retention time within the tumor, tumor to non-tumor ratios and biodistribution properties. The tandem antibody, with a molecular weight around 60 kDa, was found to be superior compared to the scFv/scdsFv and intact antibodies in terms of discriminating properties at localization.

The immunotherapeutic potential of the intact antibodies was tested both alone with radiolabeled antibody (radioimmunotherapy, RIT) and in combination with external radiotherapy (RT). The combined effects of RT and RIT were found to cause significant growth retardation of the tumor with dramatic changes in morphology within the tumors and appearance of both necrosis and apoptosis. Increase in amount of connective tissue and cysts and decrease in cell density were observed. These result support the notion that antibodies have the potential of becoming significant tools in the arsenal of therapeutics for treatment of malignancies.

Förlag
s. 62
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1051
Forskningsämne
immunologi
Identifikatorer
urn:nbn:se:umu:diva-875 (URN)91-7264-165-7 (ISBN)
Disputation
2006-10-13, Astrid Fagraeussalen (A 103), 6A, NUS, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2006-09-25 Skapad: 2006-09-25 Senast uppdaterad: 2009-10-29Bibliografiskt granskad

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Johansson, LennartÅhlström-Riklund, Katrine

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