Immunotoxin therapy of small-cell lung cancer: a phase I study of N901-blocked ricin.

PURPOSE: Immunotoxins could improve outcome in small-cell lung cancer (SCLC) by targeting tumor cells that are resistant to chemotherapy and radiation. N901 is a murine monoclonal antibody that binds to the CD56 (neural cell adhesion molecule [NCAM]) antigen found on cells of neuroendocrine origin, including SCLC. N901-bR is an immunoconjugate of N901 antibody with blocked ricin (bR) as the cytotoxic effector moiety. N901-bR has more than 700-fold greater selectivity in vitro for killing the CD56+ SCLC cell line SW-2 than for an antigen-negative lymphoma cell line. Preclinical studies suggested the potential for clinically significant cardiac and neurologic toxicity. We present a phase I study of N901-bR in relapsed SCLC. PATIENTS AND METHODS: Twenty-one patients (18 relapsed, three primary refractory) with SCLC were entered onto this study. Successive cohorts of at least three patients were treated at doses from 5 to 40 microg/kg/d for 7 days. The initial three cohorts received the first day's dose (one seventh of planned dose) as a bolus infusion before they began the continuous infusion on the second day to observe acute toxicity and determine bolus pharmacokinetics. Toxicity assessment included nerve-conduction studies (NCS) and radionuclide assessment of left ventricular ejection fraction (LVEF) before and after N901-bR administration to fully assess potential neurologic and cardiac toxicity. RESULTS: The dose-limiting toxicity (DLT) of N901-bR given by 7-day continuous infusion is capillary leak syndrome, which occurred in two of three patients at the dose of 40 microg/kg (lean body weight [LBW])/d. Detectable serum drug levels equivalent to effective in vitro drug levels were achieved at the 20-, 30-, and 40-microg/kg(LBW)/d dose levels. Specific binding of the immunotoxin to tumor cells in bone marrow, liver, and lung was observed. Cardiac function remained normal in 15 of 16 patients. No patient developed clinically significant neuropathy. However, a trend was noted for amplitude decline in serial NCS of both sensory and motor neurons. One patient with refractory SCLC achieved a partial response. CONCLUSION: N901-bR is an immunotoxin with potential clinical activity in SCLC. N901-bR is well tolerated when given by 7-day continuous infusion at the dose of 30 microg/kg(LBW)/d. Neurologic and cardiac toxicity were acceptable when given to patients with refractory SCLC. A second study to evaluate this agent after induction chemoradiotherapy in both limited- and extensive-stage disease was started following completion of this study.

Anti-CD6-blocked ricin: an anti-pan T-cell immunotoxin.

We report the development of a potent anti-pan T-cell immunotoxin capable of killing cells in an antigen dependent manner. The immunotoxin is composed of a high affinity anti-CD6 antibody (IgG2a, Kd = 1.3 x 10(-11) M) conjugated to blocked ricin that is a chemically modified ricin molecule wherein the lectin binding sites of the B-chain have been blocked by covalent attachment of affinity ligands. Conjugation of blocked ricin to the antibody has minimal effect on the apparent affinity of the antibody and no effect on the ribosome-inactivating activity of the ricin A-chain moiety. Anti-CD6-blocked ricin is a specific and highly toxic immunoconjugate killing the antigen-positive Molt-4 cell line with an IC37 of 4 x 10(-12) M after a 24 h exposure of cells to the immunotoxin. Nonspecific cytotoxicity of anti-CD6-blocked ricin for the antigen-negative Namalwa cell line was more than 750-fold lower with an IC37 > 3 x 10(-9) M. The cytotoxicity of anti-CD6-blocked ricin is dependent on the length of the incubation of cells with the conjugate ranging from an IC37 of 1.5 x 10(-11) M leaving a surviving fraction of Molt-4 cells of 0.03 after a 2.5 h exposure to an IC37 of 5 x 10(-13) M and leaving a surviving fraction of 3 x 10(-6) after a continuous (3 weeks) exposure. Anti-CD6-blocked ricin is also capable of killing CD6 positive cells in human peripheral blood lymphocyte populations. Systemic toxicity of anti-CD6-blocked ricin in mice is similar to the toxicity of other immunotoxins containing blocked ricin that were found to be tolerated well by patients. An application of this immunoconjugate for the prevention and treatment of graft versus host disease or tissue graft rejection is suggested.

Immunotoxins recognising a new epitope on the neural cell adhesion molecule have potent cytotoxic effects against small cell lung cancer.

The present study describes a comparison of two potent immunotoxins which utilise an identical targeting component, a monoclonal antibody (SEN7) specific for small cell lung cancer (SCLC), conjugated to two different effector components, blocked ricin (bR) and Pseudomonas exotoxin A (PE). SEN7 recognises a novel epitope on the neural cell adhesion molecule (NCAM) which is highly associated with SCLC. The immunotoxins SEN7-PE and SEN7-bR were selectively and potently active against a number of SCLC cell lines, of both classic and variant morphologies, inhibiting the incorporation of [3H]leucine with IC50 values ranging between 22 pM and 85 pM and between 7 pM and 62 pM for SEN7-PE and SEN7-bR respectively. Intoxication by both immunotoxins proceeded rapidly following short 2 h lag phases; the initial rates of protein synthesis inhibition occurred with t50 values of 6.5 h for SEN7-PE and 5.5 h for SEN7-bR. Monensin drastically enhanced the cytotoxic activity of the weakly active SEN7-ricin A-chain by 2,100-fold and of SEN7-bR by 80-fold but had no effect on SEN7-PE. In limiting dilution assays, four and more than 4.5 logs of clonogenic SW2 tumour cells were selectively eliminated from the cultures during continuous exposure to the immunotoxins SEN7-PE and SEN7-bR respectively, while antigen-negative cells required up to 1,000-fold more drug for a similar cell kill. SW2 cells surviving SEN7-bR treatment in the cultures did not express NCAM and consequently were not selectively killed by SEN7 immunotoxins. SW2 cells surviving continuous exposure to SEN7-PE showed no alteration in NCAM expression but were more resistant to intoxication mediated by PE. These cells were still sensitive to SEN7-bR.

An amplified assay for thiols based on reactivation of papain.

A sensitive spectrophotometric assay has been developed for thiol (sulfhydryl) groups using an inactive disulfide derivative of papain (papain-S-SCH3). The thiol-disulfide interchange reaction of a thiol with papain-S-SCH3 results in the stoichiometric formation of active papain (papain-SH). The reactivated papain catalyzes the hydrolysis of a chromogenic substrate, resulting in an amplified spectrophotometric signal proportional to the initial amount of thiol. A variety of thiols, e.g., cysteine, glutathione, penicillamine, cysteine methyl ester, and cysteamine, yield similar linear plots for the activity of papain vs the initial amount of thiol. An unknown concentration of a thiol is measured using a standard plot for the activity of papain vs the amount of thiol, obtained for the same thiol or for a similar thiol. Thiol groups on proteins and thiol groups of high values of pKa (2-mercaptoethanol, 3-mercaptopropanoic acid) can also be assayed using papain-S-SCH3 in the presence of excess cystamine. The assay is about 100-fold more sensitive than that using Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid)]. A 0.4 microM solution of cysteine produces an absorbance change of 0.55 at 410 nm after 30 min in the assay, compared to a predicted change in absorbance of 0.0054 using Ellman's assay.

Anti-tumor activity of a blocked ricin immunotoxin with specificity against the cluster-5A antigen associated with human small-cell lung cancer.

The monoclonal antibody (MAb SEN31, a mouse IgG1 which recognizes the cluster-5a antigen on small-cell lung cancer (SCLC) cells, was used to prepare a selective and potent blocked ricin immunotoxin. In a series of experiments in vitro and in a SCLC xenograft model in nude mice, the tumor localization potential of the radiolabeled antibody SEN31 and the anti-tumor activity of the immunotoxin SEN31-bR, the non-specific binding activity of which had been greatly reduced by blocking of the galactose binding domains of the B-chain, was determined. Radiolabeling of SEN31 was performed by linking a 67Ga-labeled desferrioxamine moiety to the oligosaccharide side chains of the antibody in order to preserve the specific cell-binding activity. 67Ga-SEN31 bound to the antigenic sites on cells of the SW2 SCLC cell line, with a dissociation constant of 3.5 nM and, when injected i.v., selectively localized at the site of s.c.-growing SW2 tumor xenografts in nude mice, with a tumor-to-blood ratio of 3.5. The immunotoxin SEN31-bR was potently and selectively active against SCLC cell lines both of classic and of variant morphologies. At a concentration of 300 pM the immunotoxin selectively eliminated 4.5 logs of clonogenic tumor cells. In nude mice, SEN31-bR was cleared from the blood with biphasic kinetics following i.v. injection and maintained a stable serum level during continuous i.p. infusion. The growth of s.c. SW2 solid-tumor xenografts was delayed following a single i.v. injection or a continuous i.p. infusion, each at a non-toxic dose.

Blocked ricin-conjugated T cell immunotoxins: effect of anti-CD6-blocked ricin on normal T cell function.

The biological properties of an immunotoxin composed of an anti-CD6 monoclonal antibody conjugated to whole ricin, which had been modified so that the galactose-binding sites of the B chain were blocked ("blocked ricin"), were examined. Treatment of peripheral blood lymphocytes with anti-CD6-blocked ricin for a 24-h period prevented T cell proliferation induced by phytohemagglutinin in a dose-dependent manner with concentrations causing 50% inhibition (IC50) ranging from 5 pM to 30 pM. In contrast, treatment with either blocked ricin alone or with a control immunotoxin prepared with a B-cell-lineage-restricted monoclonal antibody gave IC50 values of approximately 2 nM. Although shortening the duration of the anti-CD6-blocked ricin treatment to as little as 3 h had little significant effect on the observed inhibition, T cell viability experiments demonstrated that the magnitude of immunotoxin-induced killing after a given time period is significantly higher when the target cells become activated. Thus, from the initial concentration of cells treated with anti-CD6-blocked ricin placed in culture, 40%-45% viable cells remained after 2 days yet only 3%-9% remained if phorbol ester and Ca2+ ionophore were added; activation of T cells after mock treatment using blocked ricin plus nonconjugated anti-CD6 demonstrated that this effect was not the result of activation alone. The toxicity of anti-CD6-blocked ricin was also measured by inhibition of PHA-induced clonogenic growth of normal T cells. Continuous treatment of the cells using anti-CD6-blocked ricin at 0.1 nM resulted in a surviving fraction of about 3.5 x 10(-3); when immunotoxin treatment was for 24 h or less, the surviving fraction was only about 10(-1). As an indication of the unique specificity of anti-CD6-blocked ricin, immunotoxin pretreatment of potential responder cells prevented the generation of allogeneic cytolytic T lymphocytes in mixed lymphocyte cultures yet had little effect on the generation of interleukin-2-induced lymphokine-activated killer cell activity. We conclude that anti-CD6-blocked ricin demonstrates a cellular specificity and potency that make it a highly promising anti-T cell reagent.

An immunotoxin prepared with blocked ricin: a natural plant toxin adapted for therapeutic use.

Ricin, the cytotoxic protein isolated from castor beans, is composed of two subunits, A-chain and B-chain. Ricin intoxicates cells by binding through its B-chain to galactose-terminated oligosaccharides found on the surface of all eukaryotic cells and then transferring its A-chain to the cytosol where it disrupts protein synthesis by inactivating ribosomes. In addition to binding, the B-chain plays an important, but not yet understood, role in the translocation of the A-chain through a cellular membrane to the cytosol. Blocking the two galactose-binding sites of native ricin by chemical modification with affinity ligands created an altered toxin, called blocked ricin, that has at least a 3500-fold lower binding affinity and is more than 1000-fold less cytotoxic than native ricin for Namalwa cells (a Burkitt's lymphoma line) but that has maintained the translocation function of the B-chain and the catalytic activity of the A-chain. Conjugation of blocked ricin to monoclonal antibodies that bind to cell surface antigens creates new cytotoxins that approach the potency of native ricin. These cytotoxins incorporate the three essential functions of natural toxins, i.e., binding to cells, transport through a membrane, and catalytic inactivation of an essential cellular process; but in addition they possess a defined cellular target specificity. Such potent immunotoxins may play an important therapeutic role in cancer treatment. Clinical trials with an anti-CD19-blocked ricin and an anti-CD33-blocked ricin conjugate against B-cell cancers and acute myeloblastic leukemia have begun.

Regulation of soluble 5'-nucleotidase I from rabbit heart.

Rabbit heart contains two soluble 5'-nucleotidases, termed N-I and N-II, which can be separated using phosphocellulose chromatography. N-I prefers AMP over IMP as substrate, in contrast to N-II which prefers IMP over AMP. Both enzymes require Mg2+, but the optimum Mg2+ concentrations for the two enzymes are different. High concentrations of NaCl inhibit N-I and activate N-II. Purified N-I is activated by ADP but not by ATP. According to Itoh et al. (1986), purified N-II is activated by both ADP and ATP. N-I has been purified approximately 1000-fold to a specific activity of approximately 100 mumol/mg protein/min. The properties of N-I suggest that it is the enzyme responsible for the release of adenosine from AMP under conditions of hypoxia or increased work load.

5'-Nucleotidases in rat heart. Evidence for the occurrence of two soluble enzymes with different substrate specificities.

Chromatography of soluble proteins from rat heart on phosphocellulose columns separates two 5'-nucleotidases. The first to emerge from the column shows a preference for AMP over IMP as substrate, whereas the second shows a preference for IMP over AMP. The properties of the IMP-preferring enzyme, including the conditions under which it is eluted from phosphocellulose columns, show it to be the enzyme studied by Itoh, Oka & Ozasa [Biochem. J. (1986) 235, 847-851]. The kinetic properties of the AMP-preferring enzyme indicate that it is likely to be the enzyme responsible for the production of adenosine under conditions of hypoxia and increased work load, and with metabolic stresses such as a high load of acetate.