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.

Elimination of neuroblastoma and small-cell lung cancer cells with an anti-neural cell adhesion molecule immunotoxin.

BACKGROUND: The development of immunotoxins has been hampered by difficulties, particularly in solid tumors, of finding appropriate target antigens and of linking sufficiently potent toxins. PURPOSE: We evaluated the tissue specificity of an immunotoxin, N901-blocked ricin (N901-bR), and assessed its potential for eliminating neural cell adhesion molecule (NCAM)-positive tumor cells in conditions appropriate for in vitro purging, prior to autologous stem cell transplantation, and its potential for myelosuppression. N901-bR consists of a monoclonal antibody (MAb), N901, directed against CD56, an antigen of the family of NCAMs, covalently linked to blocked ricin as the cytotoxic effector moiety. METHODS: The tissue specificity of the N901 MAb and the N901-bR immunotoxin was tested against a wide array of human tumor tissues and normal human tissues by immunohistochemical staining. The cytotoxic activity of N901-bR was tested against both small-cell lung cancer (SCLC) cells and neuroblastoma cells, either alone or among normal bone marrow mononuclear cells, and the efficacy of this treatment to specifically eliminate these cells was evaluated in a limiting dilution assay. In addition, normal bone marrow mononuclear cells were incubated with N901-bR, and the toxic effects of the immunotoxin on normal hematopoietic progenitors was evaluated. RESULTS: N901 and N901-bR exhibited specificity for several neoplasms of neuroectodermal origin, including SCLC and neuroblastoma. Staining of normal tissues was essentially limited to various neuroendocrine cells, cardiac muscle cells, and cells in peripheral nerve tissue. We observed a time- and dose-dependent elimination of tumor cells in vitro, with three logs (i.e., > 99.9%) of malignant cells being killed following only 5 hours of exposure to 10 nM N901-bR. Unconjugated N901 MAb specifically blocked the elimination of NCAM-positive cells by N901-bR, whereas neither an isotype-matched control MAb nor galactose (the ligand of native ricin) had any effect on the activity of the immunotoxin, confirming the specificity of its cytotoxic activity. Importantly, N901-bR used under optimal conditions for in vitro tumor cell depletion was not toxic to hematopoietic precursors. CONCLUSIONS: N901-bR has the properties required to target CD56, an antigen present not only on cells from a large number of cancers of neuroendocrine origin, but also on some important normal tissues. In addition, treatment with this immunotoxin results in the highly effective and specific elimination of neuroblastoma and SCLC cells and does not affect normal hematopoietic progenitors. IMPLICATIONS: N901-bR may have clinical utility for purging of neuroblastoma cells and SCLC cells before autologous stem cell transplantation. Further toxicology studies are warranted to assess the potential of N901-bR for in vivo administration.

Eradication of large colon tumor xenografts by targeted delivery of maytansinoids.

The maytansinoid drug DM1 is 100- to 1000-fold more cytotoxic than anticancer drugs that are currently in clinical use. The immunoconjugate C242-DM1 was prepared by conjugating DM1 to the monoclonal antibody C242, which recognizes a mucin-type glycoprotein expressed to various extents by human colorectal cancers. C242-DM1 was found to be highly cytotoxic toward cultured colon cancer cells in an antigen-specific manner and showed remarkable antitumor efficacy in vivo. C242-DM1 cured mice bearing subcutaneous COLO 205 human colon tumor xenografts (tumor size at time of treatment 65-130 mm3), at doses that showed very little toxicity and were well below the maximum tolerated dose. C242-DM1 could even effect complete regressions or cures in animals with large (260- to 500-mm3) COLO 205 tumor xenografts. Further, C242-DM1 induced complete regressions of subcutaneous LoVo and HT-29 colon tumor xenografts that express the target antigen in a heterogeneous manner. C242-DM1 represents a new generation of immunoconjugates that may yet fulfill the promise of effective cancer therapy through antibody targeting of cytotoxic agents.

Evaluation of the systemic toxicity and pharmacokinetics of the immunoconjugate anti-B4-blocked ricin in non-human primates. Delivered by multiple bolus injections and by continuous infusion.

The systemic toxicity of an immunoconjugate of blocked ricin and the anti-CD19 monoclonal antibody, anti-B4, was studied in cynomolgus monkeys to evaluate its safety for use in humans. Anti-B4-blocked Ricin (Anti-B4-bR) is a highly cytotoxic immunoconjugate which can kill up to 5 logs of antigen positive target cells at concentrations easily achievable in blood. Subacute toxicity studies with Anti-B4-bR were performed in 20 cynomolgus monkeys and 4 rhesus monkeys, which, unlike humans, do not express the CD19 epitope recognized by the anti-B4 antibody on their B-lymphocytes. Anti-B4-bR was administered to cynomolgus monkeys by 5 daily intravenous bolus injections of 10 or 100 micrograms/kg/day, and non-conjugated blocked ricin was administered by 5 daily intravenous bolus injections of 30 micrograms/kg/day. Total doses of the conjugate of 200, 500, 1000 or 1500 micrograms/kg were also delivered to rhesus monkeys by continuous intravenous infusion over seven days. The clinical signs of toxicity, clinical pathology parameters, and gross and microscopic tissue changes associated with Anti-B4-bR were minimal to moderate where present, and primarily hepatic. In monkeys treated with 5 x 10 micrograms/kg of Anti-B4-bR, lesions were noticeable on day 7 after the start of the treatment but were less severe or absent on day 14, suggesting that the toxic effects were reversible. Clearance of the conjugate from the serum after bolus injections of Anti-B4-bR was evaluated by ELISA and demonstrated an initial t 1/2(alpha) of 1.4-2.0 h and a secondary t 1/2(beta) of about 14 h. Serum concentrations of Anti-B4-bR were about 10-20-fold lower at 24 h as compared to 1 h after each of the 5 bolus injections in monkeys. Continuous infusion of Anti-B4-bR in primates achieved plateau levels of the immunotoxin in blood for almost the entire duration of the infusion. The therapeutic utility of the Anti-B4-bR is currently being evaluated in patients with B-cell malignancies.

Expression of the receptor-linked protein tyrosine phosphatase LAR: proteolytic cleavage and shedding of the CAM-like extracellular region.

The human transmembrane molecule LAR is a protein tyrosine phosphatase (PTPase) with a cell adhesion molecule-like extracellular receptor region. The structure of LAR hinted at its involvement in the regulation of tyrosine phosphorylation through cell-cell or cell-matrix interactions. We show here that LAR is expressed on the cell surface as a complex of two non-covalently associated subunits derived from a proprotein. The LAR E-subunit contains the cell adhesion molecule-like receptor region, while the LAR P-subunit contains a short segment of the extracellular region, the transmembrane peptide and the cytoplasmic PTPase domains. Proprotein processing occurs intracellularly. Analysis of LAR mutants suggested that cleavage occurs in the LAR extracellular region at a paired basic amino acid site by a subtilisin-like endoprotease. A single amino acid substitution at this site blocked LAR proprotein cleavage. The LAR E-subunit is shed during cell growth, suggesting that LAR receptor shedding may be a mechanism for regulating PTPase function. The use of immunohistochemistry techniques on human tissues demonstrated the expression of LAR by various cell lineages, including epithelial cells, smooth muscle cells and cardiac myocytes. The LAR gene is mapped to chromosome 1, region p32-33, which contains candidate tumor suppressor genes.

Divalent hapten-induced intestinal anaphylaxis in the mouse enhances macromolecular uptake from the stomach.

The capacity of the stomach to participate in anaphylaxis induced by the hapten N,N'-di-2,4,dinitrophenyllysine (di-DNP-lysine) was examined in BDF1 female mice immunized with dinitrophenylated Ascaris suum extract. Immunized animals underwent laparotomy and nontraumatic pyloric occlusion using a microvascular clamp. Following wound closure, animals were gavage-fed ovalbumin together with di-DNP-lysine. Other mice were subjected to systemic anaphylaxis by intravenous injection of di-DNP-lysine administered 1 min after gavage feeding of ovalbumin. The intravenous and intragastric administration of di-DNP-lysine led to a sixfold or greater increase in serum immunoreactive ovalbumin. Examination of 1-micron sections of gastric tissue from DNP-Asc-immunized and unimmunized mice showed an intact mucosal and submucosal architecture. A 75% increase in the number of mast cells below the muscularis mucosa was seen in immunized compared with unimmunized BDF1 mice. Gastric tissue sections from immunized mice challenged orally or intravenously with di-DNP-lysine showed compaction of erythrocytes in blood vessels, degranulation of mast cells, degenerative changes in the gastric epithelium, expulsion of mucus from gastric glands, and edema in the lamina propria. The present model may be useful for further defining the consequences of anaphylaxis on the development of immune responses to dietary antigens.

Divalent hapten-induced intestinal anaphylaxis in the mouse: uptake and characterization of a bystander protein.

We examined the mucosal barrier function during anaphylaxis induced by the hapten N,N'-di-2,4,dinitrophenyl-lysine (di-DNP-lysine) in BDF1 female mice immunized with dinitrophenylated Ascaris suum extract. Immunized mice were gavaged with 10 mg or 50 mg of ovalbumin (OVA) with or without N,N'-di-2,4,-DNP-lysine (di-DNP-lysine). Animals that received di-DNP-lysine underwent anaphylaxis and were observed to have significantly greater serum concentrations of immunoreactive OVA (iOVA) than control mice. The severity of anaphylaxis, which varied with the dose of di-DNP-lysine administered, influenced the uptake of OVA; greater amounts of iOVA were detected in serum of mice undergoing more severe anaphylaxis. On gel permeation of serum from both groups of mice, immunoreactive OVA was found to have a molecular size similar to native OVA. Di-DNP-lysine is a synthetic hapten that reliably induced anaphylaxis in sensitized animals challenged by gavage. Anaphylaxis resulted in the uptake into the circulation of greater quantities of an unrelated protein antigen present in the intestinal lumen. The protein antigen that was taken up into the circulation appeared to be intact and thus may have an influence on the development of the immune response, or lack thereof, to this bystander antigen.