Different antibody derived constructs are speedily evolving as putative tools for management of cancerous diseases. Antibody engineering has added substantial new technologies to modify size, affinities, solubility, stability and biodistribution properties for immunoconjugates. In this dissertation, the goal was to enhance our knowledge on how new recombinant antibodies might be tailored to optimize localization to experimental tumors in mice. One hybridoma, producing the monoclonal antibody H7, against placental alkaline phosphatase was adopted to offer 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…
Contents
INTRODUCTION
Basic principals of tumor targeting
Tumor antigens
Placental alkaline phosphatase
Cytokeratins
IMMUNOGLOBULINS AS TARGETING AGENTS
Recombinant antibodies
scFv, scdsFv, [sc(Fv)2] and scFv-fusion proteins
Radionuclides in use at tumor targeting
TREATMENT OF MALIGNANT DISEASES
Immunotherapy and radioimmunotherapy
Therapeutical antibodies in use
Animal models and human tumor xenografts
AIMS
MATERIAL & METHODS
MONOCLONAL ANTIBODIES, PRODUCTION AND MOLECULAR STUDIES
Antigens and antibodies in this thesis
Radiolabeling
mRNA isolation, cDNA production and DNA amplification
DNA purification, cloning and sequencing
In vitro mutagenic modifications of scFvs
Construction of scFv/scdsFv and [sc(Fv)2] recombinant antibodies
Expression and purification of recombinant antibodies
Protein quantification
SDS and native gel electrophoresis
Immunoblotting of native and SDS exposed proteins
ELISA
ANIMAL STUDIES
Human tumor xenografts
Radioimmunolocalization experiments
Radioimmunotherapy with intact antibodies
Histological examination
Immunohistochemistry
Ki67 and M30 staining
In situ cell death detection-POD staining…
Source: Umea University
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