Selective cytotoxic effects of a ricin A chain immunotoxin made with the monoclonal antibody SWA11 recognising a human small cell lung cancer antigen

The potential of mouse monoclonal antibodies for recognising different antigens associated with human small cell lung cancer (SCLC) to form active immunotoxins was assessed by an indirect in vitro screening assay. The screening agent used was a conjugate made by linking ricin A chain to a sheep anti-mouse IgG Fab' fragment via a disulphide bond. The monoclonal antibodies SWA11 and SWA20 both mediated the toxic effects of ricin A chain against the HC12 classic SCLC cell line in dose-dependent fashion. The SWA11 antibody was the more effective; in combination with the screening agent at a concentration of 1 x 10(-7) M, it inhibited the incorporation of [3H] leucine into HC12 cells by 94% compared with only 44% inhibition in the case of SWA20. An immunotoxin made by the direct chemical conjugation of ricin A chain to SWA11 exhibited selective toxic effects upon HC12 cells in tissue culture inhibiting the incorporation of [3H] leucine by 50% at a concentration (IC50) of 6.2 x 10(-10) M and by 98% at 1 x 10(-7) M. SWA11-ricin A chain had an IC50 of 4.4 x 10(-10) M against the NCI-H69 classic SCLC cell line but showed no cytotoxic activity against the human lung adenocarcinoma cell line NCI-H23 at a concentration of 1 x 10(-8) M.     

Refinement of an indirect immunotoxin assay of monoclonal antibodies recognising the human small cell lung cancer cluster 2 antigen.

Monoclonal antibodies (Mabs) from the Second International Workshop on Small Cell Lung Cancer (SCLC) Antigens that recognise the cluster 2 SCLC-associated antigen mediated potent and selective cytotoxic effects in an indirect assay of immunotoxin cytotoxicity. In this assay, the NCI-H69 cell line was treated with each Mab at 4 degrees C, washed to remove unbound Mab, and then incubated at 37 degrees C in the presence of a fixed concentration, 1 x 10(-8) M, of the screening agent, sheep anti-mouse IgG-ricin A chain. The use of a fixed high concentration of screening agent led to a 300-fold overestimate of the potency of a cluster 2-directed immunotoxin, MOC-31-ricin A chain. In contrast, when the concentration of the screening agent was identical to the Mab concentration, a precise match to immunotoxin potency was obtained. MOC-31-ricin A chain selectivity inhibited the incorporation of [3H]leucine by the NCI-H69, SW2 and GLC-8 SCLC cell lines by 50% at a concentration between 3 x 10(-11) M and 3 x 10(-10) M, and by the NCI-H125 lung adenocarcinoma cell line at 7 x 10(-11) M, but exerted no selective toxic effects upon human lung and non-lung tumour cell lines lacking surface expression of the cluster 2 antigen.     

Molecular and biological properties of an abrin A chain immunotoxin designed for therapy of human small cell lung cancer.

An immunotoxin (IT) comprising abrin A chain attached to the mouse monoclonal antibody SWA11, recognising a cell surface antigen highly associated with human small cell lung cancer (SCLC), was synthesised using a hindered disulphide crosslinker, N-succinimidyl 3-(2-pyridyldithio) butyrate (SPDB), and purified by Blue Sepharose CL-6B affinity chromatography. The IT preparation contained monomeric conjugate, composed of one abrin A chain molecule linked to one SWA11 molecule, and was free from unconjugated A chain or antibody. The IT fully retained the cell-binding capacity of the antibody component and the ribosome-inactivating activity of the A chain. In cytotoxicity assays using the SW2 SCLC cell line in tissue culture, SWA11-SPDB-abrin A chain inhibited the incorporation of 3H-leucine by 50% at a concentration of 10 pM and by 99% at a concentration of 1 nM. The anti-tumour efficacy of the IT was tested in nude mice bearing established s.c. solid SW2 tumour xenografts. A single i.v. injection of SWA11-SPDB-abrin A chain at a non-toxic dose induced a significant 7 to 10 day growth delay that could not be matched by administration of equivalent doses of either unconjugated SWA11 or abrin A chain alone. The results of this study indicate that the antigen recognised by SWA11 is an effective target for therapy of SCLC with A chain ITs in vivo.     

An anti-mucin immunotoxin BrE-3-ricin A-chain is potently and selectively toxic to human small-cell lung cancer.

Monoclonal antibodies (MAbs) known to recognize epithelial mucin or defined carbohydrate structures present on mucin molecules were screened for their ability to form cytotoxic agents with ricin A-chain active against human small-cell lung cancer (SCLC) in an indirect assay of immunotoxin cytotoxicity. Anti-X hapten and anti-Y hapten antibodies binding to a high proportion of SCLC cells mediated only weak to moderate effects on 3H-leucine incorporation in combination with the screening agent, sheep anti-mouse IgG F'ab-ricin A-chain. In contrast, the mouse MAb BrE-3, recognizing the polypeptide core of the MUCI mucin gene product, exerted potent and selective cytotoxic effects in the assay. An immunotoxin made by the direct attachment of ricin A-chain to BrE-3 was selectively toxic to SCLC cell lines in tissue culture. The cytotoxic activity of BrE-3-ricin A-chain was enhanced 100-fold in the presence of monensin but not by lysosomotropic amines or calcium antagonists. Our findings suggest that anti-mucin immunotoxins may have a therapeutic role to play in the treatment of SCLC.     

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.     

Biodistribution and tumour localisation of 131I SWA11 recognising the cluster w4 antigen in patients with small cell lung cancer.

The biodistribution of radiolabelled SWA11, a mouse monoclonal antibody recognising the cluster w4 group antigen associated with small cell lung cancer (SCLC) was studied in patients with SCLC. Five patients were injected intravenously with approximately 5 mCi of 131I conjugated to 1 mg of SWA11. The half-life of the radiolabel in blood was short but there was a prolonged second phase of clearance with a half-life of about 40 h. Tumour was detected by gamma camera imaging two patients. However, most of the whole body radioactivity was located in the bone marrow. At least 35% of the radioactivity in blood 18 h after injection was bound to circulating granulocytes and this probably accounted for the unusual biodistribution of the radiolabel in man. This study shows that the biodistribution of radiolabelled SWA11 in man differs from human tumour xenograft models and that the antibody in unsuitable for targeting therapy to SCLC in man.     

Pharmacokinetics in the rat of a panel of immunotoxins made with abrin A chain, ricin A chain, gelonin, and momordin

A panel of immunotoxins was constructed by chemically attaching the ribosome-inactivating proteins abrin A chain, ricin A chain, gelonin, and momordin to the monoclonal mouse IgG2a antibody Fib75 by means of a disulfide linkage. All the immunotoxins were toxic in tissue culture to the EJ human bladder carcinoma cell line expressing the antigen recognized by Fib75, inhibiting the incorporation of [3H]leucine by 50% at concentrations between 1 x 10(-10) M and 8 x 10(-10) M. The pharmacokinetics of the immunotoxins in the normal Wistar rat was determined following i.v. administration. The concentrations of intact immunotoxin in serum samples taken at various intervals after injection for up to 24 h were measured by solid-phase enzyme-linked immunosorbent assays specific for each of the four different ribosome-inactivating proteins. The Fib75 immunotoxins were cleared from the circulation with comparable, but not identical, biphasic kinetics best described by a two compartment open pharmacokinetic model. The alpha-phase half-lives of the panel, between 0.35 and 0.71 h, were similar. The beta-phase half-life of Fib75-abrin A chain, 13.3 h, was significantly longer than the beta-phase half-lives of Fib75-ricin A chain, Fib75-gelonin, and Fib75-momordin, between 7.5 and 8.6 h. Fib75-abrin A chain was found to be about 3- to 4-fold more resistant than the other immunotoxins to breakdown by reduction of the disulfide linkage between the A chain and the antibody with glutathione in vitro. This suggests that the longer serum half-life of Fib75-abrin A chain may have been due to greater stability against reduction in vivo. Analysis of serum samples obtained up to 24 h after injection of Fib75-abrin A chain revealed that the chemically intact immunotoxin present in the circulation retained full cytotoxic activity. An abrin A chain immunotoxin made with a different monoclonal mouse IgG2a antibody was also found to be more stable against reduction by glutathione in vitro than an analogous ricin A chain immunotoxin. Thus, abrin A chain may posses unique molecular properties that endow immunotoxins made with this A chain with greater stability in vivo than immunotoxins made with ricin A chain or other ribosome-inactivating proteins.     

Immunolocalisation and imaging of small cell cancer xenografts by the IgG2a monoclonal antibody SWA11

We describe here a murine monoclonal antibody of the IgG2a isotype which was generated against the SW2 human small cell carcinoma cell line. The antibody, SWA11, was shown to bind to a partially defined antigen preferentially expressed on cell lines of small cell carcinoma origin. In vitro binding studies revealed 6.1 x 10(5) antigenic sites on the SW2 cell line and the Ka to be 1.2 x 10(9)M-1. Following injection into mice bearing 1-2 cm3 SW2 xenografts, SWA11 showed strong selective accumulation in the small cell heterotransplants with a tumour to blood ratio of 7.5:1 at day 4. Other tumour to organ ratios were similarly high at 19:1, 22:1 and 12:1 for liver, kidney and spleen respectively. The absolute amount of SWA11 which localised was 10.5% of total injected material per gram tumour at day 2 and this level did not markedly decrease by day 4. The high ability of SWA11 to localise to SCC xenografts was confirmed by external gamma scintigraphy. The potential application of SWA11 as a model system for in vivo radioimmunotherapy is discussed.     

Selective immunotherapy of small cell cancer xenografts using 131I-labelled SWA11 antibody.

Intact SWA11 antibody was evaluated as a potential radioimmunotherapeutic agent against small cell lung cancer xenografts in a nude mouse model system. 131I-labelled SWA11 was given either in a single injection regimen (1 x 300 microCi) or as a multiple injection regimen achieving a total of 900 microCi (3 x 300 microCi with a 2 week interval between injections). Treatment of both small (mean volume approximately 0.2 cm3) and large (mean volume approximately 1.0 cm3) established xenografts resulted in significant anti-tumour effects and, in the case of the fractionated protocol, the failure of the xenografts to regrow within the period of observation (84 days). Xenograft histology following radioimmunotherapy showed large areas of necrosis, fibrosis and very few residual cells of SCLC origin.     

The selection of antibodies for targeted therapy of small-cell lung cancer (SCLC) using a human tumour spheroid model to compare the uptake of cluster 1 and cluster w4 antibodies.

Spheroids of a small-cell lung cancer (SCLC) cell line POC were used to evaluate the uptake and penetration of two antibodies recognising different SCLC antigens. Spheroids approximately 300-400 microns in diameter were incubated with 1 microgram ml-1 125I-labelled NY.3D11, an antibody which reacts with the cluster 1 group antigen (neural cell adhesion molecule; NCAM) and [125I]SWA11, which binds to the cluster w4 antigen. The rate of uptake of both antibodies was similar; an initially rapid phase was seen during the first 8 h and maximum uptake occurred by 24 h. The mean uptake per spheroid at 24 h was 0.97 ng for [125I]NY.3D11 and 0.45 ng for [125I]SWA11. An objective measurement of antibody penetration into spheroids was developed using a computerised image analysis of immunostained sections of spheroids. The concentration of antibody and incubation times were varied. Both antibodies penetrated the spheroids to a depth of 50 microns after 30 min. This increased to about 100 microns after 4 h incubation with 1 or 100 micrograms ml-1 SWA11. The results with 1 microgram ml-1 NY.3D11 were similar, but in the presence of 100 micrograms ml-1 NY.3D11 penetration into the spheroid was deep and diffuse. These results demonstrate a major concentration-dependent difference in the uptake and penetration of cluster 1 and cluster w4 antibodies in this spheroid model and they have implications for the selection of antibodies for targeted therapy of SCLC.     

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.     

Action of a cd24-specific deglycosylated ricin-a-chain immunotoxin in conventional and novel models of small-cell-lung-cancer xenograft

The therapeutic efficacy of an immunotoxin, SWA II-SPDB-dg.ricin A chain, recognizing the leukocyte-differentiation antigen CD24, was evaluated against SCLC cell lines in tissue culture and in 2 nude-mouse models. The first model used conventional S.C. solid-tumor xenografts. The second used small tumor-cell deposits established in S.C. implanted sponge matrices and allowed us to directly estimate the killing efficiency of the immunotoxin under experimentally defined conditions in vivo. It also mimics the clinical setting of disseminated tumor cells which form the basis of residual disease in SCLC. The cytotoxic potency of SWAII-SPDB-dg.ricin A chain was demonstrated in tissue culture by the inhibition of ³H-leucine incorporation and by the selective elimination of CD24-positive tumor cells in clonogenic assays. In nude mice, SWAII-SPDB-dg.ricin A chain was cleared from the blood circulation with biphasic kinetics: an initial (α phase of 1 hr and a second β phase of 20.5 hr. Following i.v. injection of a dose equivalent to 30% of the LD₅₀, the immunotoxin delayed the growth of SW2 solid-tumor xenografts by 16 days. The therapeutic efficacy of SWAII-SPDB- dg.ricin A chain was further demonstrated by the selective elimination of clonogenic SW2 cells from small tumor-cell deposits established in sponge matrices. Regrowth of the solid tumors after the initial response and the clonogenic activity in the sponge-derived cell population were mediated by CD24-positive cells, excluding the selection of CD24-negative mutants during immunotoxin therapy.     

Comparative biochemical, cytotoxic and pharmacokinetic properties of immunotoxins made with native ricin A chain, ricin A1 chain and recombinant ricin A chain

Immunotoxins were constructed by attaching native ricin A chain, ricin A1 chain and recombinant ricin A chain to the mouse monoclonal IgG2a antibody Fib75 by means of a disulphide linkage using the hetero-bifunctional cross-linker SPDP. The Fib75 immunotoxins were of similar composition and exerted identical cytotoxic effects against the EJ human bladder carcinoma cell line in tissue culture. All 3 immunotoxins broke down to the same extent upon incubation with glutathione in vitro. The clearance of the immunotoxins from the circulation of normal Wistar rats was determined following i.v. administration. The concentration of intact immunotoxin in serum samples taken at various intervals up to 48hr after injection was measured by a ricin A chain-specific ELISA. The Fib75 immunotoxin made with native ricin A chain was removed from the circulation most rapidly. Fib75-recombinant ricin A chain persisted in the circulation at a higher level than Fib75-ricin A1 chain. A higher proportion of the ricin A1 chain immunotoxin was lost from the bloodstream during the alpha-phase. The beta-phase half-lives of Fib75-recombinant ricin A chain and Fib75-ricin A1 chain were similar, consistent with the identical susceptibility of the immunotoxins to cleavage by glutathione. The presence of the complex-type oligosaccharide side-chain on the A1 chain may have accelerated the clearance of the A1 chain immunotoxin in relation to that of the immunotoxin made with the aglycosyl recombinant A chain.     

Application of anti-sialyl Lea monoclonal antibody, KM231, for immunotherapy of cancer.

A mouse monoclonal antibody (MoAb), KM231 raised against human gastric cancer was found to recognize sialyl Lea -epitope expressed on glycoprotein and glycolipid with high affinity. KM231 reacted with many human gastrointestinal cancer tissues and could detect the antigen shed in sera of cancer patients. The present study was designed to evaluate competence of KM231 for immunotherapy of cancer. We first confirmed that KM231 could probe the cancer cells in vivo by injecting biotinylated KM231 into nude mice bearing human colorectal carcinoma cell, SW1116. Light- and electron-microscopic examination showed that the MoAb was localized in the tumor tissues and bound to the plasma membrane and cytoplasmic endosomes. Imaging studies with 125I-labeled KM231 revealed specific localization of the antibody in SW1116 tumors transplanted into nude mice. From Scatchard analysis of KM231 binding, the number of KM231 molecules bound to per SW1116 cell was calculated approximately 1.9 x 10(6) and the association constant was 1.3 x 10(8) liter/mol. We made KM231-ricin A chain immunotoxin for evaluating the tumoricidal effect of KM231. The immunotoxin exerted strong cytotoxicity toward sialyl Lea-expressing tumor cells specifically in vitro, but not toward sialyl Lea non-expressing cells. The in vivo tumoricidal effect of the immunotoxin was examined on ascites and subcutaneous xenograft tumors in nude mice. Three intraperitoneal injections of the immunotoxin (1.6 x 10(-6) mol) into nude mice bearing SW1116 ascites tumor resulted in extension of survival by 204% compared with controls. Further, repeated intraperitoneal administration of the immunotoxin (1.4 - 2 x 10(-6) mol) significantly inhibited the growth of established subcutaneous tumor (ratio of tumor inhibition = 0.7 - 0.54). These results indicated that KM231 has the ability to probe sialyl Lea-expressing tumor cells in vivo with high efficiency and to become tumoricidal drug when it conjugated with cytotoxic reagents like ricin A chain.     

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) SEN3I, 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 clono-genic 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.     

Antitumor activity of intraperitoneal immunotoxins in a nude mouse model of human malignant mesothelioma

Immunotoxins directed against human transferrin receptor have been evaluated in a nude mouse model of human malignant mesothelioma. Immunotoxins were constructed by linking ricin A chain to murine monoclonal antibodies reactive with the human transferrin receptor. A chain was obtained either by isolation from the parent toxin or by recombinant DNA techniques. These immunotoxins acted as potent in vitro cytotoxins against human malignant mesothelioma cells (H-MESO-1) (ID50, 2 X 10(-9) M). Cytotoxic potency and kinetics of cell kill were potentiated in vitro by the carboxylic ionophore monensin. For in vivo trials, nude mice were injected i.p. with 6-9 X 10(6) human malignant mesothelioma cells 24 h prior to the start of i.p. immunotoxin treatments. The survival of tumor-bearing mice was extended by 149-404%, representing a probable cell kill of 2-4 logs. Specificity of this antitransferrin receptor immunotoxin response was confirmed by the ineffectiveness of irrelevant control immunotoxins and blockade of specific immunotoxin action by excess free antibody. Monensin showed limited in vivo potentiation of immunotoxin effect, but a derivative formed by esterification of monensin with linoleic acid gave improved survival times over treatment with immunotoxin alone. Immunotoxins constructed with ricin A chain have significant tumoricidal activity in this model of regional antitumor therapy. These results may have direct relevance for treatment of i.p. malignancy in clinical settings.     

Evidence for absence of toxicity of T101 immunotoxin on human hematopoietic progenitor cells prior to bone marrow transplantation

T101-ricin A-chain immunotoxin is a hybrid molecule made up of the T101 monoclonal antibody bound to the A-chain of ricin. It specifically destroys cells expressing the cell surface T65 antigen. We have designed a preclinical study to evaluate its possible use for the in vitro treatment of T-cell hematological cancers prior to autologous bone marrow transplantation. The data presented here show that conditions previously defined to produce high tumor cell killing, i.e., a 20-hr incubation at 37 degrees in the presence of T101-ricin A-chain immunotoxin up to 10(-7) M in a 10 mM ammonium chloride solution, do not affect the in vitro proliferative capacity of human hematopoietic stem cells studied by means of semisolid medium cultures (granulocyte-macrophage progenitors, burst-forming units-erythrocyte) and continuous liquid cultures (pre-granulocyte-macrophage progenitors). Therefore, autologous bone marrow transplantation with T101-ricin A-chain immunotoxin-treated graft should be feasible.     

Recombinant ricin A chain conjugated to monoclonal antibodies: improved tumor cell inhibition in the presence of lysosomotropic compounds

Recombinant ricin A chain was chemically linked to monoclonal antibodies directed toward human breast cancer cells, a human T-cell differentiation antigen, and mouse transferrin receptor. Three types of immunotoxins were prepared; in two of them the antibody was linked to recombinant ricin A chain by a disulfide bond and in the third, a nonreducible thioether bond was used. Immunotoxins containing a nonreducible linkage may have some advantage over conjugates containing a reducible linkage because of improved stability in vivo. Conjugation of recombinant ricin A chain through either the endogenous thiol group or through a derivatized amino group produced immunotoxins with comparable cytotoxicity. The thioether conjugate was 1000-fold less cytotoxic to target tumor cells than the respective disulfide-linked immunotoxin. However, addition of monensin, a monocarboxylic ionophore, greatly enhanced the cytotoxicity of the thioether-linked immunotoxin. Monensin increased the immunotoxin activity better than other lysosomotropic reagents that were tested. The increase in activity of recombinant ricin A chain-containing immunotoxins mediated by monensin argues against a role for contaminating ricin B chain in potentiation.     

Radioimmunotherapy of transplanted small cell lung cancer with 131I-labelled monoclonal antibody

Monoclonal antibody TFS-4 has previously been shown to react selectively with human small cell lung cancer (SCLC). We evaluated the use of 131I-labelled TFS-4 for the treatment of established human SCLC transplanted in nude mice. The specific accumulation of the antibody in the transplanted tumour was recorded by both scintigraphic and biodistribution studies. Administration of 200 microCi 131I-labelled TFS-4 inhibited tumour growth when compared with the same radiation dose of the control monoclonal antibody. The therapeutic effect was dose-dependent and complete disappearance of the tumour was observed transiently in one out of the three animals following the administration of 500 microCi 131I-labelled TFS-4.     

Epidermal growth factor receptor expression in human lung cancer cell lines

Non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cell lines were studied for epidermal growth factor (EGF) receptor expression. All NSCLC cell lines tested (eight of eight) had specific EGF binding sites, whereas only five of 11 SCLC cell lines bound EGF. NSCLC and SCLC cell lines expressed the same type of high affinity EGF binding sites with a Kd of 0.5 to 4.5 nM; however, NSCLC cells bound significantly more EGF than SCLC cell lines. The amount of binding sites in NSCLC cells ranged between 71 and 1,000 fmol/mg of protein and in SCLC cells, between 26 and 143 fmol/mg of protein. The two SCLC cell lines with EGF binding values within the range of NSCLC belonged to the variant subtype of SCLC. By means of an anti-erbB serum and indirect radioimmunoprecipitation, a strong Mr approximately 170,000 protein band could be detected in the NSCLC cell lines. This protein corresponds to the EGF receptor molecule. Its identity was proven by competition with excess erbB antigen for the antibody during the radioimmunoprecipitation. Furthermore, this Mr 170,000 protein exhibited protein kinase activity as evidenced by in vitro autophosphorylation. The radioactivity incorporated into the Mr 170,000 band in radioimmunoprecipitation and protein kinase assays was 10 to 100 times lower in these SCLC cell lines which were positive in the EGF binding assay compared to the NSCLC cell lines. We conclude that NSCLC in contrast to SCLC expresses high levels of EGF receptors which may be used to facilitate the differential diagnosis in some cases of lung cancer. These data suggest that EGF may play a role in growth and differentiation of NSCLC.