Vijay K. Chaudhary

Single-chain immunotoxins directed at the human transferrin receptor containing Pseudomonas exotoxin A or diphtheria toxin: anti-TFR(Fv)-PE40 and DT388-anti-TFR(Fv).

Two single-chain immunotoxins directed at the human transferrin receptor have been constructed by using polymerase chain reaction-based methods. Anti-TFR(Fv)-PE40 is encoded by a gene fusion between the DNA sequence encoding the antigen-binding portion (Fv) of a monoclonal antibody directed at the human transferrin receptor and that encoding a 40,000-molecular-weight fragment of Pseudomonas exotoxin (PE40). The other fusion protein, DT388-anti-TFR(Fv), is encoded by a gene fusion between the DNA encoding a truncated form of diphtheria toxin and that encoding the antigen-binding portion of antibody to human transferrin receptor. These gene fusions were expressed in Escherichia coli, and fusion proteins were purified by conventional chromatography techniques to near homogeneity. In anti-TFR(Fv)-PE40, the antigen-binding portion is placed at the amino terminus of the toxin, while in DT388-anti-TFR(Fv), it is at the carboxyl end of the toxin. Both these single-chain immunotoxins kill cells bearing the human transferrin receptors. However, anti-TFR(Fv)-PE40 was usually more active than DT388-anti-TFR(Fv), and in some cases it was several-hundred-fold more active. Anti-TFR(Fv)-PE40 was also more active on cell lines than a conjugate made by chemically coupling the native antibody to PE40, and in some cases it was more than 100-fold more active.

A proper amino terminus of diphtheria toxin is important for cytotoxicity

A series of deletions and substitutions were made at the 5 end of the gene fusion between the first 388 codons of diphtheria toxin (DT) and a cDNA encoding human IL2. The chimeric protein (DT388-IL2) was expressed and purified from E. coli and found to be very cytotoxic to a human T cell line, HUT 102, that expresses a large number of IL2 receptors. Deletion of the first five amino acids of DT resulted in a non-cytotoxic chimeric protein that had both ADP-ribosylation activity and IL2 receptor binding activity. Deletion of the first two amino acids of DT had little effect on cytotoxicity, while deletion of the first four amino acids or of two acidic residues at positions 3 and 4 greatly reduced cytotoxicity. Unexpectedly, a mutant containing a single leucine in place of the first two amino acids (gly, ala) was 2-3 fold more active. The amino terminus of DT may participate in the translocation of the A chain to the cytosol in a manner similar to Pseudomonas exotoxin (PE) in which a specific C-terminal sequence has been proposed to be involved in its cytotoxicity.

Insertion of constant region domains of human IgG, into CD4-PE40 increases its plasma half-life

CD4-PE40 is a recombinant toxin containing the binding domain of CD4 and a mutant form of Pseudomonas exotoxin A from which the cell binding domain has been removed. To increase the serum half-life of CD4-PE40, we have inserted various portions of the constant domain of human IgG1 into CD4-PE40. The constructs made include CD4-CH2-PE40, CD4-CH3-PE40, CD4-CH1-CH2-PE40 and CD4-CH2-CH3-PE40. The fusion proteins were expressed and purified from E. coli. Insertion of various domains from the constant region of IgG1 did not alter the cytotoxic activity of CD4-PE40; all these molecules were equally cytotoxic to cells expressing gp120 on their surface. However, there was a marked increase in the serum mean residence time of CD4-CH2-PE40 which was 115 min as compared to 47 min for CD4-PE40. Insertion of other domains also increased the half-life of CD4-PE40, however, CD4-CH2-PE40 was found to have the longest mean residence time in the circulation. One possible explanation for the increase in plasma half-life is diminished susceptibility of proteins to proteolysis. It was found that CD4-CH2-PE40 was much more resistant to proteolysis by trypsin than CD4-PE40. We proposed that insertion of the CH2 domain into CD4-PE40 covers up the protease sensitive sites in the molecule, thereby making the molecule less susceptible to degradation. The increase in size and reduced sensitivity to proteases could both be responsible for the increased plasma half-life of CD4-CH2-PE40.

TGFα-anti-Tac(Fv)-PE40 : a bifunctional toxin cytotoxic for cells with EGF or IL2 receptors

Abstract Conventional immunotoxins and chimeric toxins made in bacteria are directed to only one receptor or antigen on target cells. In this report we describe the construction of a chimeric molecule TGFα-antiTac(Fv)-PE40 which is composed of human transforming growth factor type α attached to anti-Tac(Fv) which is in turn attached to PE40, a form of pseudomonas exotoxin, devoid of its cell recognition domain. TGFα-anti-Tac(Fv)-PE40 is a bifunctional toxin that is produced in E. coli and is active on cells bearing either IL2 or EGF receptors.