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Formation of Herceptin-PE38 and e23-PE38 multimerized antibody immunotoxin Utilizing Repeats of the Fab binding Domain of Protein G

  • Lee jineui (대학원 생명과학과 생화학전공)

초록/요약

Recombinant abtibody-toxins were made by chemically or genetically connecting toxin to specific antibody which selectively binds to specific cancer cells (1). To make dimeric recombinant antibody fusion proteins we used tandem repeat proteins (up to 20 repeat) of Fab binding domain of Streptococcal protein G. The tandem-repeat constructs were used as scaffolds for the formation of dimeric recombinant antibody fusion protein. Trastuzumab(trade names Herceptin) is antibody that interferes with the HER2/neu receptor and is widely used for the treatment of breast cancer(2,3). [Herceptin(Fab)-PE38] was used as a monomeric recombinant antibody fusion protein and also [e23(Fab)-PE38]. [Herceptin(Fab)-PE38] and [e23(Fab)-PE38] are recombinant antibody-toxin fusion protein that is constructed by fusion the Fab domain of monoclonal antibody. Hercepin and e23 are the truncated form of pseudomonas exotoxin(4). The previous study showed that the tandem repeats formed dimeric complexes of [Herceptin(Fab)-PE38] and [e23(Fab)-PE38]. To check the functionality of the two complexes cytotoxicity assay on human cancer cell lines were performed. The tested dimeric complexes were obviously more cytotoxic than monomer about 20 times. Because of simple and fast way of making dimeric recombinant antibody fusion protein, it is anticipated that this method of complex between Fab domain and tandem-repeats should be generally applicable to multimerization of any protein that have Fab domain of immunoglobulin G, and this multimeric protein should be used as more sensitive analytical and diagnostic purposes by means of increased avidity of the protein that has low affinity to its antigen.

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초록/요약

Fab-PE38 used in this study is [Herceptin(Fab)-ext-PE38] and it is an antibody-toxin that is made by fusing the Pseudomonas exotoxin A (1-3). The Fd-ext-PE38 and light chain are covalently linked by a disulfide bond. The Fab-ext-PE38 made in this study is against the extracellular domain of HER2, showed a significant anti-tumor effect. As a result, it inhibits the protein synthesis of cancer cells and finally induces apoptosis. Previously studied divalent antibody-toxins are dimerized form of Fab-monomer molecule by disulfide-bond located in the modified hinge regions. To increase the yield of disulfide-dimer antibody toxin, we used streptococcal protein G which has affinity to CH1 domain of the Fab fragment. Association purified Fab-monomer and GR were used for redox shuffling to initiate disulfide-dimer antibody-toxin by reduction and oxidation with 2-mercaptoethanol and glutathione oxidized form (4). The yield of disulfide-dimer was increased through Fab-monomer with streptococcal protein G association by redox suffling experiment. It is considered that Protein G makes close intermolecular interactions between monomers possible, and the use of it greatly enhances the yield of disulfide-bridged dimer.

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목차

TABLE OF CONTENTS

List of tables …………………………………...………………...…… i
List of Figures ………………………………….………………..….. iii



Chapter I. Comparision of Cytotoxic effect
between e23 and Herceptin Antibody toxin

1. Abstract ………………………………………………………2
2. Introduction ……………………………………….................3
3. Materials and Method ………………………………..............5
4. Result …………………………………………………..….. 11
5. Discussion……………………………………………………15
6. References ……………………………………………….….46

Chapter II. Disulfide-dimer formation of
Fab-monomer antibodytoxin by redox suffling

1. Abstract ……………………………………………..………51
2. Introduction …………………………………………………52
3. Materials and Method ………………………………………54
4. Result ……………………………………………………… 58
5. Discussion ………………………………………………… 61
6. References …………………………………………………..76

Acknowledgement …………………………………………….…….79

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