Cytotoxicity of CD3-ricin A chain immunotoxins in relation to cellular uptake and degradation kinetics.

The cytotoxicity of WT32 (CD3)-ricin A immunotoxin (IT) to the acute lymphoblastic leukemia T-cell line Jurkat was compared with the rate of internalization and intracellular degradation of WT32 and WT32-ricin A during continuous exposure. Moreover, the influence of NH4Cl and monensin on these processes was studied. Based on protein synthesis inhibition ([3H]leucine incorporation), it appeared that cytotoxicity was not fully expressed directly after exposure to IT due to a delay in either the internalization of membrane-bound IT or the action of intracellular ricin A. Varying the duration of incubation and postponing [3H]leucine addition for up to 24 h after initiation showed that cytotoxicity occurred in two phases, rapid internalization of initially bound IT followed by a continuous but slower uptake, possibly due to reexpression of the CD3 antigen. No differences were found in the rate of internalization and degradation of 125I-labeled WT32 and WT32-ricin A. Internalization started rapidly after binding at 37 degrees C, was fastest during the first 12 h (+/- 360,000 molecules/cell), and continued for at least 24 h (+/- 420,000 molecules/cell). Exocytosis of intracellularly degraded molecules became measurable after 1 to 2 h of incubation at 37 degrees C and increased to approximately 400,000 molecules/cell in 24 h. After 4 h of incubation at 37 degrees C the number of internalized molecules exceeded the amount of WT32 that could maximally bind to the cell membrane (+/- 150,000 molecules/cell), confirming reexpression of antigen. The addition of NH4Cl and monensin enhanced the cytotoxicity of WT32-ricin A, probably due to an increased intracellular amount of IT. These agents appeared to reduce strongly the degradation of internalized WT32, resulting in an accumulation of intracellular molecules. NH4Cl was most effective during the first 12 h of incubation, whereas monensin increased the amount of intracellular WT32 molecules after 2 to 24 h. Our observations suggest that incubation conditions for the optimal cytotoxicity of IT treatment can be predicted by studying the internalization and degradation of the IT or respective monoclonal antibody.

Effect of isotype on internalization and cytotoxicity of CD19-ricin A immunotoxins.

We analyzed the effect of isotype variation on effectiveness of B-cell CD19 immunotoxins (IT) by using class switch variants (CLB-B4-IgG1 and CLB-B4-IgG2a) conjugated to ricin A. Notably, IgG1-IT appeared to be approximately 100-fold more potent than IgG2a-IT toward B-cell lines Daudi and KM3. Binding and internalization studies with 125I-labeled monoclonal antibodies (mAbs) revealed a higher cellular uptake of IgG1 compared to IgG2a, despite similar binding affinities. Following removal of the Fc part, both mAbs internalized at the same rate as IgG2a, indicating that the Fc part of IgG1 is involved in enhanced cellular uptake. The involvement of Fc gamma RII (CD32) in this process was demonstrated by a decreased cytotoxicity of IgG1-IT (and not IgG2a-IT) in the presence of Fc gamma RII-blocking mAbs AT10 or IV.3. To identify the isoform responsible for this phenomenon, internalization of IgG1 and IgG2a in 11 B-cell lines and malignant B-cells of 8 patients was compared with expression of Fc gamma RII subclasses. In addition to four cell lines (Daudi, KM3, Nalm6, and Raji), the malignant B-cells of two patients showed enhanced uptake of IgG1 relative to IgG2a. Only the Fc gamma RIIa transcript was found in all B-cells. Furthermore, enhanced uptake of IgG1 correlated with rosetting of erythrocytes sensitized with anti-glycophorin A mAb of IgG1 isotype rather than with Fc gamma RIIa membrane expression levels. These data support the idea that functional Fc gamma RIIa is involved in the enhanced IgG1 uptake observed in a subset of B-cells. Our study, therefore, points to an important role for the Fc region of IT in the delivery of cytotoxic effects.

Influence of cytotoxicity enhancers in combination with human serum on the activity of CD22-recombinant ricin A against B cell lines, chronic and acute lymphocytic leukemia cells.

Despite the strong in vitro activity of some immunotoxins (ITs), clinical application did not result in complete cure. The outcome of therapy may be improved by combining ITs with IT-cytotoxicity enhancing agents. We studied the effect of various agents that influence the intracellular routing of ITs on the activity of the anti-B cell IT CD22-recombinant (rec) ricin A. In protein synthesis inhibition assays the carboxylic ionophores monensin and nigericin enhanced the activity of the IT 117- and 382-fold, respectively, against the cell line Daudi, and 81- and 318-fold, respectively, against the cell line Ramos. IT activity to Daudi and Ramos was enhanced to a lesser extent by the lysosomotropic amines chloroquine (14- and 11-fold, respectively) and NH4Cl (nine- and 10-fold, respectively). However, the combination of NH4Cl and chloroquine induced more than an additive effect (145- and 107-fold, respectively). Cytotoxicity was not influenced by brefeldin A, all-trans retinoic acid (ATRA), verapamil and perhexiline maleate. Bacitracin enhanced the IT cytotoxicity in contrast to the other protease inhibitors aprotinin, leupeptin and soybean trypsin inhibitor, albeit enhancement was weak (two-fold). The enhancers exerted only a negligible effect on bone marrow progenitor cells. We recently developed a flow cytometric cytotoxicity assay in which cell elimination can be assessed. In order to detect enhancement in this assay, we used 5 x 10(-11) M IT (approximately the 50% protein synthesis inhibiting dose (ID50)). This concentration killed 41% of the Daudi cells and 42% of the Ramos cells. In the presence of 10 nM monensin the IT killed 74% and 99% and in the presence of 10 nM nigericin 96% and 99% of the Daudi and Ramos cells, respectively. At 10(-8) M, CD22-rec ricin A eliminated malignant cells originating from three patients with B-CLL (0.42 log) and two with B-ALL (0.19 log) patients. Cytotoxicity to malignant cells was enhanced by NH4Cl, chloroquine, monensin and nigericin. The combination of NH4Cl and chloroquine enhanced the activity most effectively (up to 2.06 log). To determine the applicability of the IT in combination with enhancers in vivo we investigated the effect of human serum. Human serum inhibited IT activity which could not be restored by monensin and nigericin because of complete inhibition of these enhancers by serum. In contrast, chloroquine partially restored the activity of CD22-rec ricin A in the presence of human serum. We conclude that monensin, nigericin and the combination of NH4Cl and chloroquine can be used instead of NH4Cl to potentiate CD22-rec ricin A activity in purging autologous bone marrow transplants contaminated with malignant B cells. Chloroquine might be a promising enhancer of CD22-rec ricin A for treating patients in vivo.

Relationship of the CD22 immunotoxin dose and the tumour establishment in a SCID mice model.

Immunotoxins (ITs) may be very potent to erradicate tumour growth in vivo. We investigated the influence of the IT-dose, in relation to the establishment of the tumour, on the anti-tumour activity of CD22-recombinant (rec) ricin A for a disseminated tumour (Ramos) in SCID mice. Furthermore, the enhancement of the IT cytotoxicity in vivo by chloroquine was assessed. CD22-rec ricin A appeared to be highly effective. Paralysis of the hind legs was significantly delayed by a very low IT-dose of 2 microg administered intravenously (i.v.) 7 days after i.v. inoculation of the tumour cells. Even a dose of 30 microg administered 21 days after inoculation of the target cells significantly delayed the onset of paralysis up to 8 days compared with the median paralysis time (MPT) of the control group. The efficacy of treatment was obviously influenced by the establishment of the tumour, the tumour load and localisation. The anti-tumour activity of 10 and 30 microg IT diminished when the IT was administered after increasing the time lag following inoculation of tumour cells. Delaying IT administration resulted in growth of solid tumours. This implies that cells migrate to sanctuaries protected from the IT indicating that the anti-tumour activity was influenced by the accessibility of the IT to the target cells. The in vivo anti-tumour activity of CD22-rec ricin A could not be enhanced by simultaneously administered chloroquine, despite the continuous infusion with an intraperitoneally (i.p.) implanted mini-osmotic pump. Ex vivo experiments revealed that the maximally tolerated serum concentration (3.9 microM) was too low to be effective. In conclusion, CD22-rec ricin A is highly effective for in vivo treatment of B-cell malignancies, in particular if treatment is started when the tumour load is low and before migration takes place to poorly accessible sanctuaries.

Production of anti-CD3 and anti-CD7 ricin A-immunotoxins for a clinical pilot study.

This report describes the preparation of an immunotoxin-combination, consisting of an anti-CD3 and anti-CD7 monoclonal antibody (MoAb) both conjugated to the A-chain of plant toxin ricin, for the experimental treatment of graft-versus-host disease. MoAbs and toxin were conjugated by conventional biochemical and chromatographic techniques. Raw materials, intermediate and final products were evaluated in accordance with the relevant 'points to consider' of the FDA. Yields, purity and sterility of the two final products were all satisfactory. Preservation of MoAb-affinity and toxin-activity were confirmed in biological assays. The LD50, 25-45 mg immunotoxin-combination/kg mouse, equalled that of similar immunotoxins already in clinical use. Because in vitro cross-reactivity screening revealed an unexpected binding of the CD3-MoAb to the esophagus epithelium, human doses of immunotoxin-combination were administered to two cynomolgus monkeys. Clinically relevant serum concentrations were obtained without irreversible toxicities occurring. The T(1/2) varied between approximately 6 and 9 h and the C(max) ranged from 1.8 to 3.9 microg/ml. The main side effect was a transient rise of serum creatine kinase. Importantly, neither damage nor binding of the CD3-immunotoxin to the monkey esophagus epithelium could be demonstrated. It was concluded that sufficient material of proper quality and with an acceptable toxicity profile was produced, warranting the evaluation in a clinical pilot-study.

Cytotoxic potency of CD22-ricin A depends on intracellular routing rather than on the number of internalized molecules.

Cytotoxicity of immunotoxins (ITs) varies considerably depending on factors like the capability of the target antigen to internalize IT molecules, intracellular processing and routing of the IT. We studied factors that may influence cytotoxicity of CD22-ricin A IT to several B cell lines. The antigen density varied from 5.9 x 10(3) to 6.0 x 10(4) molecules/cell. The ID50, determined by protein synthesis inhibition, varied from 2.1 x 10(-12) to 3.8 x 10(-11) M IT in absence and from 2.8 x 10(-14) M to 5.2 x 10(-12) M IT in presence of the cytotoxicity enhancer NH4Cl (6 mM). In absence as well as in presence of NH4Cl no correlation could be found between antigen density and ID50. No relation was observed either with the rate of cytotoxicity. Even in cell lines with a low antigen density, such as KM3, protein synthesis was quickly inhibited. In order to investigate whether the cytotoxicity was dependent on the number of internalized molecules the kinetics of internalization and exocytosis of degraded 125I-labelled CD22 molecules were studied. After 24 h the number of internalized CD22 molecules was highest in Ramos (154,500), followed by Daudi (110,300) and KM3 (69,900). However, despite the higher internalization rate of Daudi the rate of cytotoxicity of 10(-8) M IT was comparable with KM3. NH4Cl did not influence the number of internalized molecules but postponed degradation of CD22. In conclusion, CD22-ricin A is a very potent and fast acting IT even for elimination of target cells that express low numbers of antigen. These results may have implication for treatment of different B cell malignancies with CD22-ricin A.

Highly potent CD22-recombinant ricin A results in complete cure of disseminated malignant B-cell xenografts in SCID mice but fails to cure solid xenografts in nude mice.

The highly specific cytotoxic action of ribosome-inactivating protein (RIP) containing immunotoxins (ITs) makes IT therapy a promising approach to eliminating residual malignant cells. We investigated the cytotoxicity of the IT CD22-recombinant ricin A to the B-cell line Ramos in vitro and in vivo. Cytotoxicity of CD22-recombinant ricin A in vitro was very high as expressed by the very low 50% inhibition dose (ID50) of 3.5 x 10(-11) M. Cytotoxicity was increased 7 times in the presence of the cytotoxicity enhancer NH4Cl. The ultimate kill of Ramos cells by CD22-recombinant ricin A was high (2.7-log kill) and was increased strongly in the presence of NH4Cl (4.2-log kill). Anti-tumor activity in vivo was investigated by i.v. treatment of solid s.c. Ramos xenografts in nude BALB/c mice. A single dose did not inhibit tumor growth. Treatment on 5 consecutive days resulted in evident tumor reduction. In one mouse, tumor could no longer be detected on the 6th day after starting treatment. However, after 8 days tumor volumes increased again. Antitumor activity was more pronounced in a disseminated tumor model in SCID mice. IT treatment (i.v.) 7 days after i.v. inoculation with Ramos cells resulted in cure of all mice. Non-specific toxicity was low. Alanine aminotransferase (ALAT) levels in serum were elevated temporarily. Serum values of gamma-glutamyl transferase (gamma-GT), bilirubin and creatinin did not change. Body weight was also transiently reduced. The LD50 in SCID mice after i.v. administration was high (0.626 mg IT per mouse). The clearance rate in SCID mice, as determined by ELISA, was biphasic.

A combination of anti-CD3 and anti-CD7 ricin A-immunotoxins for the in vivo treatment of acute graft versus host disease.

This study evaluated the anti-graft versus host disease (GVHD) potential of a combination of immunotoxins (IT), consisting of a murine CD3 (SPV-T3a) and CD7 (WT1) monoclonal antibody both conjugated to deglycosylated ricin A. In vitro efficacy data demonstrated that these IT act synergistically, resulting in an approximately 99% elimination of activated T cells at 10(-8 )mol/L (about 1.8 microg/mL). Because most natural killer (NK) cells are CD7(+), NK activity was inhibited as well. Apart from the killing mediated by ricin A, binding of SPV-T3a by itself impaired in vitro cytotoxic T-cell cytotoxicity. Flow cytometric analysis revealed that this was due to both modulation of the CD3/T-cell receptor complex and activation-induced cell death. These results warranted evaluation of the IT combination in patients with refractory acute GVHD in an ongoing pilot study. So far, 4 patients have been treated with 3 to 4 infusions of 2 or 4 mg/m(2) IT combination, administered intravenously at 48-hour intervals. The T(1/2) was 6.7 hours, and peak serum levels ranged from 258 to 3210 ng/mL. Drug-associated side effects were restricted to limited edema, fever, and a modest rise of creatine kinase levels. One patient developed low-titer antibodies against ricin A. Infusions were associated with an immediate drop of circulating T cells, followed by a more gradual but continuing elimination of T/NK cells. One patient mounted an extensive CD8 T-cell response directly after treatment, not accompanied with aggravating GVHD. Two patients showed nearly complete remission of GVHD, despite unresponsiveness to the extensive pretreatment. These findings justify further investigation of the IT combination for treatment of diseases mediated by T cells. (Blood. 2000;95:3693-3701)