Exotoxin A

By David J. P. FitzGerald

Microbial toxins are potent biological agents that are often capable of killing mammalian cells by enzymatically inactivating normal cellular function. If it were possible to harness the activities of toxins and redirect them to selectively kill or damage discrete populations of cells, powerful reagents might become available for use both as tools for cell biology and cell genetics and as novel drugs for the treatment of cancer or other diseases. Toward this end Pseudomonas exotoxin A (PE) was chemically coupled to a variety of monoclonal antibodies to make hybrid molecules termed immunotoxins (for a review of immunotoxins, see Refs. 1 and 2).

PE is a 68,000 Da bacterial protein toxin produced by Pseudomonas aeruginosa. This toxin inhibits protein synthesis of mammalian cells by virtue of its ability to ADP-ribosylate elongation factor-2 (EF-2). To gain access to EF-2, PE must translocate from outside the cell across a cellular membrane to the cytoplasm. The first step in this process is the binding of PE to a cell surface component that we can term a "PE-receptor." Following binding, PE enters cells by receptor-mediated endocytosis and has been localized by electron microscopy first in coated pits, then in receptosomes (endosomes), the Golgi, and lysosomes.3-5 However, it is not clear yet how or where the toxin traverses one of these membrane-limited compartments to gain access to the cell cytoplasm. Hybrid molecules made using PE should be constructed to deliver PE to a similar intracellular compartment.

PE-immunotoxins have been constructed using antibodies that bind to

11. Pastan, M. C. Willingham, and David J. P. FitzGerald, Cell 47, 641 (1986).

2 J. A. Cumber, J. A. Forrester, B. M. J. Foxwell, W. C. J. Ross, and P. E. Thorpe, this series, Vol. 112, p. 207.

3 D. J. P. FitzGerald, R. E. Morris, and C. B. Saelinger, Cell 21, 867 (1980).

4 R. E. Morris, M. D. Manhart, and C. B. Saelinger, Infect. Immun. 40, 806 (1983).

5 M. D. Manhart, R. E. Morris, P. F. Bonventre, S. Leppla, and C. B. Saelinger, Infec. Immun. 45, 596 (1984).

cell surface antigens of mammalian cells.6-9 Ideally the immunotoxin should bind cells only via the antibody. However, many toxins, including PE, have a cell-binding capacity of their own. This must be inactivated or removed or else the immunotoxin will have two binding domains and will therefore have little or no selectivity. In the case of PE, the binding domain is inactivated following the reaction with 2-iminothiolane (see Ref. 8 and below).

Materials and Methods for Immunotoxin Construction

The following describes a typical conjugation procedure that can be used to couple PE to any monoclonal antibody of the IgG class. No conditions to make immunotoxins with IgM or IgA have yet been worked out.

Purified PE was purchased from Swiss Serum and Vaccine Institute, Berne, Switzerland. Purified monoclonal antibodies were prepared by conventional procedures. Cysteine and NAD were obtained from Sigma and 2-iminothiolane-HCl and 5,5'dithiobis(2-nitrobenzoic acid) (DTNB) were obtained from Pierce Chem. Co.,

1. Typically 5 mg of PE is dissolved in 0.15 M KP04, 1 mM EGTA, pH 8.0 (buffer A). Usually 400 ¡A of buffer A is added to 5 mg of lyophi-lized PE and then desalted on a PD10 (9 ml, bed volume) column (Pharmacia) to remove the lactose which is colyophilized with PE. The running buffer is also buffer A. Three fractions are collected: fraction one is 2.8 ml, fraction two is 2.0 ml, and fraction three is 1.0 ml. Most of the PE is contained in fraction two. The optical density of each fraction is measured at 280 nm (an OD of 1.2 is equivalent to 1 mg/ml of PE). If necessary, additional buffer A is added to fraction two until the OD is 1.7.

2. To PE in fraction two the following are added: 5 fil of NAD (66 mg/ml in buffer A) per ml of PE, and then 5 fA of iminothiolane (IT) (130 mg/ml in buffer A) per ml of PE. The reaction mixture is incubated at 37° for 1 hr.

3. The PE reaction mixture is loaded on an HPLC gel filtration column (Bio-Rad, TSK-250, 600 X 21.5 mm). An FPLC (Pharmacia) system is

6 D. J. P. FitzGerald, I. S. Trowbridge, I. Pastan, and M. C. Willingham, Proc. Natl. Acad. Sci. U.S.A. 80, 4134 (1983).

7 D. J. P. FitzGerald, T. A. Waldmann, M. C. Willingham, and I. Pastan, J. Clin. Invest. 74, 966(1984).

8 R. Pirker, D. J. P. FitzGerald, T. C. Hamilton, R. F. Ozols, M. C. Willingham, and I. Pastan, Cancer Res. 45, 751 (1985).

9 R. Pirker, D. J. P. FitzGerald, T. C. Ozols, W. Laird, A. E. Frankel, M. C. Willingham, and I. Pastan, J. Clin. Invest. 76, 1261 (1985).

used to pump the buffer at 4 ml/min. The column is run in 0.15 M KP04, 1 mM EGTA, pH 7.0 (buffer B).

A gel filtration step is preferable to simple desalting, since the addition of IT causes a small amount of PE to aggregate. The aggregates are seen at the void volume and constitute 5% of less of the total amount of PE. PE elutes from this column (at 144 ml) as a single peak, typically collected in 12 ml.

4. The protein concentration is determined by measurement at OD2g0. Then the number of new SH groups per molecule of PE is determined. To the PE fraction 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) is added to a final concentration of 1 mM. A stock solution of DTNB at 100 mM in 1.0 M KP04 (dibasic salt ~ pH 9.3) is made fresh on the day of use. The OD at 412 is determined against a blank, containing buffer and DTNB at 1 mM. The OD at 412 divided by 0.0136 gives the number of moles of SH/ml. Usually the number is exactly 3. However, we have noted values from 2.6 to 3.2.

5. To remove excess DTNB, PE is desalted over a PD10 column. Before this can be accomplished the volume must be reduced from 12 ml to less than 1 ml. This is done using Centricon 30 microconcentrators (Amicon). Typically PE is concentrated to approximately 0.5 ml. PE is desalted on a column equilibrated with buffer A and three fractions collected: of 2.8, 2.5, and 1.0 ml. The majority of the protein is contained in the second 2.5 ml fraction. PE is now ready to be coupled by disulfide exchange to a monoclonal antibody having a free sulfhydryl.

6. A "new" sulfhydryl group is introduced in the monoclonal antibody by reaction with IT. The antibody is desalted into buffer A by passing it over a PD10 column equilibrated with buffer A. Using microconcentrators the antibody is then concentrated to approximately 0.5 ml and sufficient IT added from a 1.3 mg/ml stock solution to give a 2-fold molar excess of reagent over antibody. The reaction is allowed to proceed for 1 hr at 37° after which the antibody is desalted (PD10 column, buffer A). Ideally, there should be one new SH per antibody. This is evaluated by removing an aliquot of antibody and adding DTNB to a final concentration of 1 mM (see step 4).

7. The activated PE and antibody are mixed at either 37° or RT and the reaction followed by the appearance of thionitrobenzoate (TNB) (OD412) The TNB is displaced from PE as disulfide bonds are formed.

8. When the OD412 ceases to increase, the pH is lowered from 8.0 to 7.0 by the addition of 1.0M monobasic KP04—add ^ the volume of the reaction volume. Cysteine (1.2 mg/ml) in buffer B is then added until the OD412 ceases to increase. This displaces any remaining TNB from PE (see Fig. 1 for schematic of how PE-immunotoxins are prepared).

H NH2

H2C-CH2

PE + H2C C = NH2 -► PE - N - C - CH2 - CH2 - CH2 - SH

(Iminothiolane)

.COOH _COOH

Antibody + Iminothiolane

COOH n02

Antibody-SH + PE

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