B3(Fab)-PE38M

A recombinant immunotoxin in which a mutant form of Pseudomonas exotoxin is fused to the fab fragment of monoclonal antibody B3

MuHyeon Choe, Keith O. Webber, Ira Pastan

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Recombinant immunotoxins were made by fusing the Fab domain of monoclonal antibody (MAb) B3 to PE38M, a truncated mutant form of Pseudomonas exotoxin (PE). The recombinant toxins were made in Escherichia coli by fusing genes encoding the antibody domains to a gene encoding the mutant form of PE. MAb B3 binds to a carbohydrate antigen found on many kinds of carcinomas. Immunotoxins in which MAb B3 has been chemically coupled to recombinant forms of PE have been shown to be very active cytotoxic agents. PE has also been targeted to tumor cells by replacing the cell-binding domain of PE (domain I) with a single-chain antibody to make a single-chain immunotoxin. In the current study, PE38M, a mutant form of PE, with a deletion of the cell-binding domain (amino acids 1-252) as well as mutations in domain III and some nonessential sequences in domain Ib (amino acids 365-380), was fused to the light chain of MAb B3. This protein was renatured in the presence of the Fd fragment of MAb B3 to produce a Fab-toxin fusion protein. Alternatively, the Fd fragment of MAb B3 was fused to PE38M and combined with the light chain. Both types of B3(Fab)-PE38M were just as active on target cells as previously described single-chain immunotoxins. Furthermore, the B3(Fab)-PE38M produced complete remissions of human tumor xenografts growing in nude mice. B3(Fab)-PE38M has two advantages over single-chain immunotoxins. One is that the yield of recombinant Fab-toxin is very high, with 10-22% of the starting protein recovered as cytotoxically active immunotoxin after chromatographic purification. The second is that the B3(Fab)-PE38M has a much longer survival in the circulation of mice with a t1/2β of ∼5 h.

Original languageEnglish
Pages (from-to)3460-3467
Number of pages8
JournalCancer Research
Volume54
Issue number13
Publication statusPublished - 1994 Jul 1
Externally publishedYes

Fingerprint

Immunotoxins
Exotoxins
Immunoglobulin Fab Fragments
Pseudomonas
Monoclonal Antibodies
Light
Amino Acids
Single-Chain Antibodies
Proteins
Cytotoxins
Heterografts
Nude Mice
Genes
Neoplasms
Carbohydrates
Escherichia coli
Carcinoma
Antigens
Mutation
Survival

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

B3(Fab)-PE38M : A recombinant immunotoxin in which a mutant form of Pseudomonas exotoxin is fused to the fab fragment of monoclonal antibody B3. / Choe, MuHyeon; Webber, Keith O.; Pastan, Ira.

In: Cancer Research, Vol. 54, No. 13, 01.07.1994, p. 3460-3467.

Research output: Contribution to journalArticle

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abstract = "Recombinant immunotoxins were made by fusing the Fab domain of monoclonal antibody (MAb) B3 to PE38M, a truncated mutant form of Pseudomonas exotoxin (PE). The recombinant toxins were made in Escherichia coli by fusing genes encoding the antibody domains to a gene encoding the mutant form of PE. MAb B3 binds to a carbohydrate antigen found on many kinds of carcinomas. Immunotoxins in which MAb B3 has been chemically coupled to recombinant forms of PE have been shown to be very active cytotoxic agents. PE has also been targeted to tumor cells by replacing the cell-binding domain of PE (domain I) with a single-chain antibody to make a single-chain immunotoxin. In the current study, PE38M, a mutant form of PE, with a deletion of the cell-binding domain (amino acids 1-252) as well as mutations in domain III and some nonessential sequences in domain Ib (amino acids 365-380), was fused to the light chain of MAb B3. This protein was renatured in the presence of the Fd fragment of MAb B3 to produce a Fab-toxin fusion protein. Alternatively, the Fd fragment of MAb B3 was fused to PE38M and combined with the light chain. Both types of B3(Fab)-PE38M were just as active on target cells as previously described single-chain immunotoxins. Furthermore, the B3(Fab)-PE38M produced complete remissions of human tumor xenografts growing in nude mice. B3(Fab)-PE38M has two advantages over single-chain immunotoxins. One is that the yield of recombinant Fab-toxin is very high, with 10-22{\%} of the starting protein recovered as cytotoxically active immunotoxin after chromatographic purification. The second is that the B3(Fab)-PE38M has a much longer survival in the circulation of mice with a t1/2β of ∼5 h.",
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