Arthur E. Frankel

Therapy of patients with T-cell lymphomas and leukemias using an anti-CD7 monoclonal antibody-ricin A chain immunotoxin.

Anti-CD7-dgA, DA7, consists of deglycosylated ricin A chain coupled to a mouse monoclonal anti-human CD7 antibody. This study determined the maximally tolerated dose (MTD) of this immunotoxin administered as a one hour infusion over five days to 11 patients with T-cell lymphoma (>30% CD7+ malignant cells). The MTD was 0.2 mg/kg/day or 1 mg/kg/120 hours (maximal toxicity grade 3) with vascular leak syndrome (VLS) as dose-limiting toxicity (DLT). Predictors of severe VLS included age and absence of circulating lymphoma cells. Two partial responses and one minimal response were seen. Patients with minimal lymphoma burden or T-cell large granular lymphocyte (LGL) leukemia showed the best responses. The mean maximal serum concentration of immunotoxin at the MTD was 2.5 ug/ml. The mean alpha-phase half-life was 1.5 hours and the mean beta-phase half-life was 8 hours. Repeated dosing had minimal effects on either peak serum immunotoxin concentrations or serum half-lives. While human antimouse antibodies were observed, they were low in concentration (<55 ng/ml). Human anti-ricin antibody was elevated in one patient (190 ng/ml). VLS presented with hypoalbuminemia, dyspnea, pulmonary edema, aphasia, and peripheral edema and cleared over a two week period. Serum fibronectin levels were measured in three patients and were very low in one patient who developed VLS. No specific binding of DA7 immunotoxin was seen with vascular endothelium in various human tissues.

DAB389IL2 diphtheria fusion toxin produces clinical responses in tumor stage cutaneous T cell lymphoma

Four patients with late stage cutaneous T cell lymphoma (IB‐IVA) who had failed at least two previous therapies were treated with DAB389IL2 at 9 or 18 μ/kg as 15‐min intravenous infusions daily for 5 days every 3 weeks for eight cycles. Mild vascular leak syndrome (VLS) with transient edema, hypoalbuminemia, weight gain, and myalgias was observed in two of the patients lasting 7–10 days and only occurring on the first cycle. One stage IB patient had a pathologic complete remission (CR) lasting 11+ months from treatment initiation, one stage IIB patient had a complete clinical remission (CCR) lasting >6 months with complete clearing of a large tumor lasting >18 months, and one stage IIB and the one stage IVA patient had partial remissions (80–99% reduction in tumor masses) lasting 5 months. While IL2 receptor (IL2R) was expressed on 20–50% of tumor cells prior to therapy, recurrent tumor was IL2R negative in three of the patients. DAB389IL2 at tolerable doses decreased tumor burden in each of these four standard treatment refractory CTCL patients and may offer an important alternative to standard palliative chemotherapy regimens. Am. J. Hematol. 58:87–90, 1998.

Toxicology and Pharmacokinetics of DT388IL3, a Fusion Toxin Consisting of a Truncated Diphtheria Toxin (DT388) Linked to Human Interleukin 3 (IL3), in Cynomolgus Monkeys

The fusion toxin DT388IL3 composed of the catalytic and translocation domains of diphtheria toxin (DT388) linked to interleukin-3 (IL3) was administered to 6 cynomolgus monkeys which possessed cross-reactive IL3 receptors. Groups of 2 animals (1 male and 1 female) received up to 6 every other day slow intravenous infusions of 40, 60, or 100 microg/kg DT388IL3. Monkeys given 40 or 60 microg/kg showed mild or moderate transient malaise and anorexia, respectively, without evidence of organ damage by blood tests or histopathology. Animals treated at 100 microg/kg showed severe malaise and anorexia. The female monkey had moderate to severe vasculitis in multiple tissues. Necropsies were performed on the 40 microg/kg monkeys on day 14 and the 100 microg/kg monkeys on days 6 and 7. DT388IL3 plasma half-life was approximately 30 min with a peak concentration of 0.45 microg/ml or 10,000 pM (IC50 for AML blasts treated in vitro was 6 pM). Immune responses were minimal in 4 animals tested at 12 days and 2 animals tested at 30 days post treatment with anti-DT388IL3 levels < 1 microg/ml. Bone marrow aspirates were obtained on all animals at day 19 or at necropsy and revealed myeloid suppression in the females and myeloid hyperplasia in the males irrespective of dose groups. The maximal tolerated dose of 60 microg/kg for 6 doses is markedly higher than other recombinant diphtheria toxins and provides a dose level sufficient for anti-leukemic activity in vitro and in rodent models. Thus, we propose this agent is a promising drug for AML patients.

Lectin-deficient ricin toxin intoxicates cells bearing the D-mannose receptor.

Ricin toxin with genetic or chemical modification of lectin sites has been previously reported to show markedly reduced cytotoxicity to cells following uptake by several receptors including the mannose receptor. Investigators have hypothesized that an intracellular galactoside-binding function was required for optimal intracellular targeting of ricin for these receptors. We have prepared insect-derived mutant ricin toxin B chain (RTB) with modifications of three lectin side domains (1 alpha, 1 beta, and 2 gamma) yielding a 1000-fold reduced galactoside avidity. After reassociation with plant RTA, the recombinant heterodimer and plant ricin were tested for cytotoxicity on mammalian cells expressing (mouse peritoneal macrophages, J774E cells, and MMR61 cells) or not expressing (KB cells) the D-mannose receptor. Receptor expression was confirmed by immunofluorescence microscopy. Lactose was included in the media to block cell-surface galactoside binding, and mannan was added as a control in each experiment to confirm mannose receptor-specific targeting. Plant ricin A chain (RTA) and E. coli-derived RTA were also tested for cytotoxicity on J774E and KB cells. Both wild-type and lectin-deficient ricin displayed mannose-receptor mediated cell cytotoxicity. This is the first report of a genetically modified ricin showing that RTB intracellular galactose binding activity is not required for ricin cytotoxicity. Sensitivity of mannose-receptor bearing cells, but not control cells, to mannosylated RTA, but not unglycosylated RTA, confirmed these observations. These results imply fusion toxins employing ricin can be prepared with maximal reductions in normal tissue binding.

Ricin toxin contains three lectin sites which contribute to its in vivo toxicity.

Ricin intoxication of mammalian cells is initiated by B chain (RTB) binding to cell surface galactosides. Recombinant insect-derived RTB mutants with modifications in lectin-site subdomains 2 gamma, 1 alpha/2 gamma, and 1 alpha/1 beta/2 gamma were reassociated with plant RTA and tested for lethality in C57B1/6 6-8 weeks old mice. The LD50 of intraperitoneally injected castor bean ricin was 75 ng per 18 g mouse. The LD50 of single-site 2 gamma mutant heterodimer was 100 ng: the LD50 of the double-site 1 alpha/2 gamma mutant heterodimer was 500 ng, and the LD50 of the triple-site 1 alpha/1 beta,2 gamma mutant heterodimer was > 10 micrograms. Plant RTA alone had an LD50 of 300 micrograms. Animals died between 1 and 10 days post-injection. Histopathological examination of morbid animals receiving an LD50 dose of each toxin revealed only apoptosis in the thymus and spleen. The present data provide clear evidence for participation of three lectin sites in ricin in vivo toxicity. These results suggest an origin for some of the normal tissue toxicities observed with clinical trials of doubly blocked ricin conjugates and suggest modification of at least three RTB subdomains will be necessary in genetically engineered ricin fusion proteins.

Laboratory preparation of a deglycosylated ricin toxin A chain containing immunotoxin directed against a CD7 T lineage differentiation antigen for phase I human clinical studies involving T cell malignancies

An immunotoxin consisting of a monoclonal antibody specific for CD7, a cell surface determinant expressed on T acute lymphocytic leukemia (T-ALL) blast cells, was linked to the potent plant toxin deglycosylated ricin toxin A chain (dgRTA) and is currently under evaluation in phase I clinical trials. Scale-up production of this immunotoxin, called DA7, was simplified using a two-step purification protocol that resulted in a highly purified immunotoxin meeting FDA criteria for IND approval. The anti-CD7 antibody, 3Ale, an IgG2b, was coupled to toxin using two different heterobifunctional cross-linkers, (1) N-succinimidyl-3-(2-pyridyl-dithiolproprionate) (SPDP), considered a standard croslinker and (2) 4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)tolu ene (SMPT), designed to hinder the in vivo breakdown of the toxin/antibody disulfide bond. Since experiments revealed that SPDP-DA7 had similar pharmacokinetics and biodistribution in mice and higher yields than DA7 made with a hindered cross-linker, SPDP-DA7 was scaled up for clinical study. Yield of SPDP-DA7 was 25% relative to starting material. Fractions were collected containing a toxin: antibody ratio of 1:1 to 4:1 rather than only a 1:1 ratio since studies showed that this heterogenous fraction was just as toxic to proliferating CD7-expressing leukemia cells as a homogeneous 1:1 fraction. In vitro, the concentration of heterogenous SPDP-DA7 selectively inhibiting 50% activity (IC50) of the CD7+ CEM cell line was 0.01 microgram/ml to 0.05 microgram/ml for inhibiting activated T cells or T cell lines. In vivo, SPDP-DA7 showed a significant anti-tumor effect against CEM cells administered to scid/scid mice, but even more importantly was effective against primary T cell leukemias taken from patients and injected into scid/scid mice.

Cytotoxicity of KDEL-terminated ricin toxins correlates with distribution of the KDEL receptor in the Golgi

DNAs encoding ricin toxin A chain (RTA), with or without a C-terminal endoplasmic reticulum retention signal KDEL, were subcloned into pGEX2T bacterial expression plasmid. After transformation of JM105 E. coli cells and induction with isopropylthio-beta-galactoside (IPTG), fusion proteins were bound to an immobilized glutathione matrix and recombinant ricin A chains released with thrombin. Both recombinant wild-type RTA and RTA with KDEL had immunological reactivity and catalytic activity indistinguishable from plant RTA. The bacterial RTA products reassociated with plant ricin B chain (RTB) similarly to plant RTA. Cell cytotoxicities were measured on seven cell lines for each A-chain and heterodimer. Although KDEL sequences enhanced cytotoxicity in most cases, significant variability was observed. In each case, addition of KDEL enhanced A-chain cytotoxicity more than holotoxin cytotoxicity. Three cell lines showed reduced KDEL enhancement of both RTA and ricin cytotoxicity. The concentration of KDEL receptor was examined on each cell line by immunofluorescence microscopy with an antireceptor monoclonal antibody. Differences in sensitivity to KDEL-containing toxins correlated with altered distribution of KDEL receptor between endoplasmic reticulum (ER) and Golgi compartments.

Ricin fusion toxin targeted to the human granulocyte-macrophage colony stimulating factor receptor is selectively toxic to acute myeloid leukemia cells

Treatment failure of patients with acute myelogenous leukemia (AML) is frequently due to the development of multidrug resistance phenotype blasts. We have expressed a fusion protein consisting of human granulocyte-macrophage colony stimulating factor (GMCSF) fused to the N-terminus of a lectin-deficient ricin toxin B chain (RTB) in Spodoptera frugiperda insect cells. The fusion protein was purified by immunoaffinity chromatography and reassociated with chemically deglycosylated ricin toxin A chain (RTA). The resulting fusion toxin was found to react with antibodies to GMCSF, RTB and RTA and had the predicted molecular mass of 80 kDa. GMCSF-ricin bound poorly to asialofetuin (Kd = 10(6) M-1) and receptor negative cells indicating loss of lectin activity, but bound strongly to GMCSF receptor positive HL60 cells. Ligand displacement assays showed fusion toxin affinity 2.6-fold less than native GMCSF. Selective inhibition of protein synthesis was observed on receptor positive cells. Induction of apoptosis was also observed on receptor positive cells. Cells expressing multidrug resistance gene products (P-gp, Bcl2 and BclXL) were also sensitive to fusion toxin. These results suggest that GMCSF-ricin deserves further preclinical development.

IL2 fused to lectin-deficient ricin is toxic to human leukemia cells expressing the IL2 receptor

Interleukin-2 (IL2) fused to ricin B chain (RTB) with modifications of amino acid residues in each of three galactose-binding subdomains (1α, 1β and 2γ) was expressed in insect cells, purified by immunoaffinity chromatography and reassociated with ricin A chain (RTA). The fusion toxin-bound human leukemic cells with IL2 receptors and the binding was competed with IL2 but not asialofetuin. In contrast, binding was not observed with receptor negative human cell lines, and the fusion molecule very weakly bound asialofetuin (Kd = 10−6 M), indicating lectin-deficient RTB. The IL2–lectin-deficient RTB-RTA intoxicated IL2 receptor bearing cells as well as ricin or IL2–wild-type RTB-RTA. While ricin and IL2–wild-type RTB-RTA were equally toxic to receptor negative cell lines, the IL2–lectin-deficient RTB-RTA was two-two and one half logs less cytotoxic to these cell lines. The sensitivity of receptor-positive cells to the lectin-deficient fusion protein suggests that high avidity intracellular galactose binding may not be required for ricin intoxication, at least in the case of IL2 receptor-targeted molecules. Furthermore, the potent selective cytotoxicity of the fusion protein suggests that the IL2–lectin-deficient RTB-RTA and similar ricin fusion molecules directed against other leukemic cell surface receptors provide a novel class of fusion toxins for therapy of human leukemias.

Immunotoxin Therapy of Chronic Lymphocytic Leukemia

There are approx 8000 new cases of chronic lymphocytic leukemia (CLL) a year in the United States (1). Most patients eventually progress to an advanced stage or develop rapidly progressive disease with a median survival of only 18–36 mo (2). Although allogeneic bone marrow transplantation (including nonmyeloablative conditioning regimens) offers the possibility of longterm remission or cure (3), most patients either lack suitable donors and have comorbidities that prevent such therapy. Monoclonal antibodies, radiolabeled antibody conjugates, and antibody-drug conjugates have been used in CLL and have produced clinical remissions, but most of the responses have been partial and have not been durable. Hence, additional novel agents that may significantly impact on the natural history of this disease are needed. One such class of agents is the immunotoxins consisting of monoclonal antibodies, antibody fragments, or cytokines linked either chemically or genetically to peptide toxins. The antibody or cytokine directs the molecule to the CLL cell surface. The toxin moiety is internalized and translocated to the cytosol, where it enzymatically inactivates protein synthesis inducing programmed cell death. In this review, we discuss the unique biology of the CLL cell that makes it an attractive candidate for immunotoxin therapy. We describe potential CLL cell surface receptors for targeting with immunotoxins as well as the structure and function of selected CLL immunotoxins. Finally, the clinical results of previous and on-going clinical trials with these agents in CLL are analyzed.