Somatic Cell Mutants Resistant to Ricin, Diphtheria Toxin, and to Immunotoxins*

The human B-cell line Namalwa expresses the common acute lymphoblastic leukemia antigen (CALLA). Frame-shift mutants in Namalwa cell cultures were generated with ICR-191, and mutants were then se- lected for resistance to ricin or resistance to a conjugate of ricin with the anti-CALLA antibody 55 in the pres- ence of lactose. Three mutants were found that were resistant to ricin and were in addition shown to be resistant to diphtheria toxin, to a J5-ricin conjugate, and to a conjugate between ricin B-chain and gelonin. The mutants, however, were sensitive to a J5-gelonin conjugate. These mutants expressed high levels of CALLA and/or receptors for ricin, and their cell-free translation systems appeared to be as sensitive to the inhibitory action of ricin A-chain and of gelonin as the translation system of wild-type Namalwa cells. The behavior of these mutants was consistent with the hypothesis that these cells possess an alteration of their surface that impedes the passage of ricin and diphtheria toxin across the plasma membrane. A fourth mutant was found to bind reduced quantities of ricin and was resistant to ricin but was sensitive to J5-ricin. The properties of this cell line provide evidence that the binding of antibody-ricin conjugates to cells via the ricin moiety may be prevented without impeding the cytotoxicity of the conjugates.

Ricin, a lectin isolated from the seeds of Ricinus communis L. is extremely toxic to most eucaryotic cells (1). This toxin can be directed to kill target cells by conjugating it to monoclonal antibodies as targeting agents (Z), and the hope is that conjugates of ricin with antibodies will be developed that are highly toxic and selective in their action (3).
In order to study the pathway(s) of the internalization of ricin and of ricin-antibody conjugates by cells, we employed the techniques of biochemical genetics: the analysis of somatic cell mutants that are resistant to ricin or ricin-antibody conjugates.
This paper describes the isolation and general characterization of somatic cell mutants that are resistant to ricin or to a ricin-anti-CALLA1 antibody 55 conjugate. ICR-191, apotent and moderately toxic mutagen for human cells was used for * This work was supported by a grant from ImmunoGen, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be addressed. The abbreviations used are: CALLA, common acute lymphoblastic leukemia antigen; BSA, bovine serum albumin; J5-ricin, a conjugate of ricin with the 55 antibody; P, resistant to ricin; J5p, resistant to J5-ricin; PBS, 10 mM potassium phosphate buffer, pH 7.2, containing NaCl (145 mM); TCA, trichloroacetic acid. the generation of the mutants (4). The CALLA-expressing human B-lymphoblast cell line Namalwa was used as the parental line for generation of mutants.

EXPERIMENTAL PROCEDURES AND RESULTS*
Resistance of Mutants to Protein Toxins, Zmmunotoxins, and Some Other Cytotoxic Drugs-All clones that are listed in Table I1 were resistant to both ricin and J5-ricin, when compared to the wild type cells. In the case of J5-ricin, this resistance was observed either with or without iactose. In order to understand better the mechanisms of resistance, the cytotoxicities of ricin (Fig. 3a), J5-ricin ( Fig. 3b), diphtheria toxin ( Fig. 3c), gelonin, and J5-gelonin (Table 111) as well as three other cytotoxic agents (Table 111) toward representative cell lines p23, J5p6, J5rR11, and J5P1'2 were studied in detail. Gelonin is a protein which inhibits protein synthesis in cell-free translation systems as efficiently as ricin A-chain but is not very toxic to intact cells because it apparently cannot penetrate cell membranes (10,18). The rR23 cell line appeared to be 65-fold more resistant to ricin than the Namalwa line ( Fig. 3a) but showed no increased resistance to J5-ricin. J5p6, J5rR11, and J5p12 cell lines appeared to be resistant to ricin and to J5-ricin (Fig. 3, a and b). In addition, these three lines were resistant to diphtheria toxin (Fig. 3c). J5p6 cells were resistant to ricin B-gelonin, and J5r% cells were resistant to J5-gelonin (Table 111). The mutant J5rR11 was resistant to ricin B-gelonin but sensitive to J5-gelonin (Table 111). In their sensitivity to free gelonin, all four of these cell lines were similar to the parental Namalwa line (Table 111). We also tested whether the mutants acquired the phenotype of so-called "multidrug resistance," which is associated with an alteration of the plasma membrane that impedes the internalization of a number of cytotoxic drugs (19). However, none of the four mutants were resistant to actinomycin D, adriamycin, or colchicine (Table III), the agents to which the cells that acquired the multidrug resistance phenotype would become resistant (19).

Mutants with Reduced Expression of CALLA andlor of
Receptors of Ricin-The resistance of mutants to ricin or to J5-ricin could originate from several factors: (i) reduced binding to the cell surface; (ii) reduced endocytosis; (iii) reduced * Portions of this paper (including "Experimental Procedures," p" to Protein Toxins experiments with wild-type Namalwa cells also suggest that the level of binding of a toxin or of an immunotoxin to the cell surface may determine the cytotoxicity of a toxin. Namalwa cells bind less 55 than ricin B-chain (Fig. 2) and, accordingly, J5-ricin in the presence of lactose (ie. under conditions where conjugate can bind only to the cell's CALLA) is 15-fold less cytotoxic than ricin and 11-fold less cytotoxic than J5-ricin in the absence of lactose. Similarly, J5-gelonin was 7-fold less toxic to the Namalwa cells than ricin B-gelonin. The rR23 cell line appeared to be 65-fold more resistant to ricin than the Namalwa line (Fig. 3a) but was equally sensitive to J5-ricin. This mutant expresses less receptors for ricin but more CALLA than do Namalwa cells (Table 11). Ribosomes of this cell line are as sensitive as the ribosomes of the wild-type cells toward ricin A-chain. These data suggest that reduced expression of receptors for ricin was the main factor responsible for the resistance of rR23 cells to ricin, which is consistent with previous reports that the binding of ricin-antibody conjugates to cells via the ricin moiety could be blocked without significant loss of cytotoxicity of the conjugates (2, 3, 38-40). Similar data were obtained in our experiments with J5-ricin and Namalwa cells (Fig. 1). These experiments showed that the cytotoxicity of this CALLAdirected immunotoxin was for the most part retained in the presence of lactose while the cytotoxicity of ricin was abolished by lactose.
Mutants with Reduced Transport of Toxins-Mutants J5rR6, J5rR11, and J5rR12 expressed levels of CALLA and/or of receptors for ricin that are comparable with those of wildtype Namalwa cells, and these mutants might, therefore, possess another type of resistance.
J5rR12 cells internalized only about 40% as much ricin Bchain as Namalwa cells. This fact may indicate a deficiency in endocytosis of ricin and could contribute toward the resistance of these cells. In contrast, J5rR11 cells endocytosed 80% as much ricin B-chain and 120% as much diphtheria toxin as did Namalwa cells. No indication was found that the mutants had elevated levels of cytoplasmic proteases that might cause faster degradation of toxins than in the cytoplasm of the Namalwa cells. The cell-free translation systems of the four mutants rR23, J5P6, J5rR11, J5rR12 and of the Namalwa cells are equally sensitive to ricin A-chain and to gelonin, and these mutants are, therefore, different from the mutants described by Ono et al. (35), which were resistant to ricin because of an alteration in their ribosomes. An explanation for the resistance of J5rR11 cells (and a possible contributing factor for the resistance of J5rR6 and J5rR12 cells) toward ricin and J5-ricin is that these cells may have an alteration on their surface that diminishes the transport of ricin, J5-ricin, and of ricin Bgelonin across the plasma or an intracellular membrane. This hypothesis is consistent with the finding that all three J5rR Assayed by back-extrapolation from growth curves.
ND. not done.
*Assayed by inhibition of 3H-labeled thymidine incorporation into cell DNA. penetrate a cellular membrane in order to reach the cytoplasm. The J5rR11 cells may possess an alteration in the plasma membrane that impedes penetration of both ricin and diphtheria toxin. While J 5 p l l cells acquired resistance to ricin, JS-ricin, and ricin B-gelonin, the cells were sensitive to J5-gelonin. The common feature of the first three substances that distinguishes them from J5-gelonin is that they all possess ricin B-chain and may interact with cell surface oligosaccharides through this moiety. This argument suggests that the mechanism of the resistance may be linked to an alteration in the composition of the cell surface glycoproteins or an alteration in the plasma membrane area situated in the vicinity of these glycoproteins. J5rR Mutants Are Resistant to Ricin-All J5rR mutants that are listed in Table I1 appeared to be resistant not only to J5ricin in the presence of lactose, the agents they were selected with, but also to ricin and JS-ricin in the absence of lactose. Six out of eight mutants had decreased levels of receptors for ricin on their surface (Table 11). Similar selection of ricinresistant cells occurred in the presence of a mixture of ~( 6 -phosph0)pentamannose-ricin conjugate with 0.1 M lactose, i.e. under conditions where binding of the conjugate to the cell surface occurred solely through the mannose 6-phosphate receptor (46) but contrary to our finding, these mutants did not show a reduced level of surface receptors for ricin. A mechanism for ricin-resistance selection mav be as follows.