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Recombinant antibodies and tumor targeting
Umeå University, Faculty of Medicine, Clinical Microbiology, Immunology/Immunchemistry. Umeå University, Faculty of Medicine, Radiation Sciences, Diagnostic Radiology.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
2006. , 62 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1051
Research subject
Immunology
Identifiers
URN: urn:nbn:se:umu:diva-875ISBN: 91-7264-165-7 (print)OAI: oai:DiVA.org:umu-875DiVA: diva2:144840
Public defence
2006-10-13, Astrid Fagraeussalen (A 103), 6A, NUS, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2006-09-25 Created: 2006-09-25 Last updated: 2009-10-29Bibliographically approved
List of papers
1. Optimizing the generation of recombinant single-chain antibodies against placental alkaline phosphatase.
Open this publication in new window or tab >>Optimizing the generation of recombinant single-chain antibodies against placental alkaline phosphatase.
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2006 (English)In: Hybridoma, ISSN 0272-457X, E-ISSN 2168-7897, Vol. 25, no 4, 181-192 p.Article in journal (Refereed) Published
Abstract [en]

Recombinant technologies to engineer ordinary hybridoma monoclonal antibodies (MAbs) to single-chain fragment variable (scFv) may cause loss of antibody affinity, increased tendency to aggregate, increased temperature sensitivity, and low yield of active protein. In the present investigation, the well-characterized MAb H7 against placental alkaline phosphatase (PLAP), used as a model antibody, was engineered to improve solubility and stability of scFv with retained high affinity. The original procedure to generate single-chain antibodies with a 10-amino acid linker between VH and VL yielded an almost insoluble product. By site-directed mutagenesis, four selective sequence substitutions were made in the VL fragment and one in the VH fragment to improve solubility. The importance of the linker length was investigated, and a 25/30 amino acid linker was found to improve solubility. In order to further increase the stability of the single-chain antibody, an additional covalent -S-S- bond was introduced between amino acid 100 in the VL fragment and amino acid 44 in the VH region, to make a single-chain disulphide stabilized variable fragment (scdsFv). Altogether five different antibody constructs were produced and compared in terms of solubility, stability, affinity, and production properties. Immunospecificity was tested by enzyme-linked immunosorbent assay (ELISA) against the target antigen, temperature sensitivity by exposing the purified scFv to higher temperatures. All the new constructs retained almost equal activity and high affinity for their target antigen, placental alkaline phosphatase (PLAP), compared to the intact MAb H7, up to +42 degrees C as evaluated by ELISA. The overall affinity K(A) > 10(9) (M(1)) of the new antibodies could be maintained in the same order of magnitude as the original one (H7), when evaluated by Biacore technology. The best final single-chain antibody was obtained by performing the specific site-directed mutations and introducing a linker of 30 amino acids, but not by additional stabilizing disulphide bonds. The yield of the final antibody was improved approximately 10-fold by the modifications. This antibody could easily be expressed in a bacterial system using the PET-32a TrxA vector and the Escherichia coli strain BL21 Origami B (DE3). Purified antibody, which could be kept at concentrations up to 0.8 mg/mL, was obtained, which is sufficient for clinical testing of therapeutic applications.

Identifiers
urn:nbn:se:umu:diva-21198 (URN)10.1089/hyb.2006.25.181 (DOI)16934014 (PubMedID)
Available from: 2009-04-07 Created: 2009-04-07 Last updated: 2017-12-13
2. Construction and purification of a covalently linked divalent tandem single-chain Fv antibody against placental alkaline phosphatase.
Open this publication in new window or tab >>Construction and purification of a covalently linked divalent tandem single-chain Fv antibody against placental alkaline phosphatase.
2006 (English)In: Hybridoma, ISSN 0272-457X, E-ISSN 2168-7897, Vol. 25, no 5, 255-263 p.Article in journal (Refereed) Published
Abstract [en]

Multivalency is a recognized means to increase the functional affinity of single-chain antibody fragments (scFvs) for optimizing tumor uptake at radioimmunotargeting. A unique divalent tandem single-chain Fv antibody [sc(Fv)2], based on the variable regions of the monoclonal antibody (MAb) H7, has now been generated. The antibody differs from other dimeric single-chain constructs in that a linker sequence (L) is introduced between the repeats of VL and VH domains (VL-L-VH-L-VL-L-VH). This construct was expressed as a His-tagged TrxA fusion protein in the Escherichia coli strain Origami B. Following cleavage with AcTev protease, the antibody was obtained in soluble and active form in E. coli and could be purified by Ni-NTA and cation-exchange chromatography. Purity and immunochemical properties were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), Western blot, and Biacore analyses. The [sc(Fv)2] displayed proper stability and could be purified to homogeneity. This antibody also maintained immunoreactivity at 42 degrees C with only slight decrease at 52 degrees C. The high affinity displayed by the original antibody H7, 6.7 x 10(9) M(-1), was only slightly decreased to 1.2 x 10(9) M(-1) as determined by Biacore. The generation of such a divalent single-chain Fv with a molecular weight around 60 kd would be of value for clinical applications such as radioimmunolocalization and radioimmunotherapy.

Identifiers
urn:nbn:se:umu:diva-21197 (URN)10.1089/hyb.2006.25.255 (DOI)17044780 (PubMedID)
Available from: 2009-04-07 Created: 2009-04-07 Last updated: 2017-12-13
3. Tumor radioimmunolocalization in nude mice by mono- and divalent- single-chain Fv antiplacental alkaline phosphatase antibodies.
Open this publication in new window or tab >>Tumor radioimmunolocalization in nude mice by mono- and divalent- single-chain Fv antiplacental alkaline phosphatase antibodies.
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2007 (English)In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 22, no 1, 64-72 p.Article in journal (Refereed) Published
Abstract [en]

One single-chain Fv antibody fragment (scFv) and a new recombinant covalently linked dimeric scFv antibody (sc(Fv)(2)) against placental alkaline phosphatase (PLAP) were investigated for selective tumor targeting. The biological behavior of these new antibodies was compared to that of the original native antibody, H7 MAb. The sc(Fv)(2)) antibody displayed convincing tumor localization properties with a rapid excretion pattern comparable to the scFv, but with a longer retention time in the tumor, and higher tumor-to-nontumor ratio (27:1), compared to the scFv (15:1), at 48 hours. For the sc(Fv)(2) antibody, more than 50% of the remaining activity in the mouse was present in the tumor between 24 and 48 hours after the injection. With this antibody, scintigraphic visualization of the tumor was also possible 1 week after the injection. It is concluded that this sc(Fv)(2) antibody fragment, with two binding sites, displays properties suitable for in vivo targeting of PLAP expressing tumors.

Identifiers
urn:nbn:se:umu:diva-21194 (URN)10.1089/cbr.2007.340 (DOI)17627415 (PubMedID)
Available from: 2009-04-07 Created: 2009-04-07 Last updated: 2017-12-13
4. Combined low dose radio- and radioimmunotherapy of experimental HeLa Hep 2 tumours.
Open this publication in new window or tab >>Combined low dose radio- and radioimmunotherapy of experimental HeLa Hep 2 tumours.
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2003 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 30, no 6, 895-906 p.Article in journal (Refereed) 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.

Identifiers
urn:nbn:se:umu:diva-4920 (URN)10.1007/s00259-003-1177-2 (DOI)12721768 (PubMedID)
Available from: 2006-01-19 Created: 2006-01-19 Last updated: 2017-12-14Bibliographically approved

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