Monoclonal antibodies to CD44 epitopes on mouse endothelium.

CD44 is a widely expressed, plasma membrane protein. Many molecular forms of CD44 are possible as it is encoded by a gene with multiple exons that can be alternatively spliced and its deduced protein sequence contains numerous glycosylation sites. Through its role as an adhesion molecule, CD44 is involved in many and diverse biological processes, including angiogenesis, lymphogenesis, wound healing, inflammation, and cancer metastasis. We have developed a new panel of rat monoclonal antibodies (MAbs) to murine CD44 by immunization with mouse lung endothelial cells (LEII cells). The antibodies were characterized using immunoprecipitation, mass spectrometry, competition binding, and cross Western blot experiments with MAb 133-13A, which recognizes CD44 expressed on tumor cells. The new MAbs recognize three distinct epitope groups. MAbs 531-2A and 531-32A compete for binding with the MAb 133-13A that was described previously. MAb 531-30A identifies a CD44 epitope found on low molecular weight forms expressed in vivo, while MAb 531-22A appears to recognize an epitope specific for endothelial cells. This novel panel of anti-CD44 antibodies has potential for investigating the role of CD44 in disease pathogenesis models in the mouse. They may be particularly useful for examining the role of endothelial cells in these models.

Therapy of rat tracheal carcinoma IC-12 in SCID mice: vascular targeting with [213Bi]-MAb TES-23.

In previous work, we have demonstrated that vascular targeting of [213Bi], an alpha-emitter, to lung blood vessels could efficiently destroy tumour colonies growing in the lung. In order to expand this approach to treatment of tumours growing in other sites, we employed the monoclonal antibody (MAb) TES-23, which reacts with CD44H, preferentially expressed on new blood vessels in tumours. Biodistribution studies of N-succinimidyl [125I] 3-iodobenzoate (SIB)-radiolabelled MAb TES-23 in ICR-severe combined immunodeficient (SCID) mice bearing subcutaneous (s.c.) and intramuscular (i.m.) IC-12 tumours, demonstrated efficient tumour uptake. At 24 h, accumulation in small tumours was 45%ID/g for s.c. tumours, and 58%ID/g for i.m. tumours and in large tumours it was 25%ID/g for s.c. tumours and 17%ID/g for i.m. tumours. Micro-autoradiography data confirmed that radiolabel accumulated in or near tumour blood vessels. Normal tissues had very low levels of radioactivity. Treatment of mice bearing small IC-12 tumours with [213Bi] MAb TES-23 retarded tumour growth relative to animals treated with cold MAb TES-23. Biodistribution and therapy experiments were also performed in BALB/c mice bearing both s.c. and i.m. syngeneic, lung carcinoma (line 498) tumours. [I(125)] SIB MAb TES-23 accumulated efficiently in both s.c. and i.m. tumours (14%ID/g and 15%ID/g, respectively, at 4 h); however, no therapeutic effect of [213Bi] MAb TES-23 treatment could be demonstrated in this model system. The data demonstrate that the timing of vascularisation of the tumours and the delivery kinetics of MAb relative to the half-life of the therapeutic radionuclide are critical for effective therapy.

Vascular-targeted radioimmunotherapy with the alpha-particle emitter 211At.

Astatine-211, an alpha-particle emitter, was employed in a model system for vascular-targeted radioimmunotherapy of small tumors in mouse lung to compare its performance relative to other radioisotopes in the same system. Astatine-211 was coupled to the lung blood vessel-targeting monoclonal antibody 201B with N-succinimidyl N-(4-[211At]astatophenethyl) succinamate linker. Biodistribution data showed that the conjugate delivered 211At to the lung (260-418% ID/g), where it remained with a biological half-time of about 30 h. BALB/c mice bearing about 100 lung tumor colonies of EMT-6 cells, each about 2000 cells in size, were treated with 211At-labeled monoclonal antibody 201B. The administered activity of 185 kBq per animal extended the life span of treated mice over untreated controls. Injections of 370 kBq, corresponding to an absorbed dose of 25-40 Gy, were necessary to eradicate all of the lung tumors. Mice receiving 740 kBq of 211At-labeled monoclonal antibody 201B developed pulmonary fibrosis 3-4 months after treatment, as did mice treated with 3700 kBq of the alpha-particle emitter 213Bi-labeled monoclonal antibody 201B in previous work. Animals that were injected with 211At bound to untargeted IgG or to glycine, as control agents, also demonstrated therapeutic effects relative to untreated controls. Control groups that received untargeted 211At required about twice as much administered activity for effective therapy as did groups with lung-targeted radioisotope. These results were not consistent with radioisotope biodistribution and dosimetry calculations that indicated that lung-targeted 211At should be at least 10-fold more efficient for lung colony therapy than 211At bound to nontargeting controls. The data showed that 211At is useful for vascular-targeted radioimmunotherapy because lung tumor colonies were eradicated in the mice. Work in this model system demonstrates that vascular targeting of alpha-particle emitters is an efficient therapy for small perivascular tumors and may be applicable to human disease when specific targeting agents are identified.

Evaluation of 225Ac for vascular targeted radioimmunotherapy of lung tumors.

Several alpha particle emitting radioisotopes have been studied for use in radioimmunotherapy. Ac-225 has the potential advantages of a relatively long half life of 10 days, and a yield of 4 alpha emissions in its decay chain with a total energy release of approximately 28 MeV. A new, 12 coordination site chelating ligand, HEHA, has been chemically modified for coupling to targeting proteins without loss of chelating ability. HEHA was coupled with MAb 201B which binds to thrombomodulin and accumulates efficiently in murine lung. Ac-225 was bound to the HEHA-MAb 201B conjugate and injected into BALB/c mice bearing lung tumor colonies of EMT-6 mammary carcinoma. Biodistribution data at 1 and 4 h postinjection indicated that, as expected, 225Ac was delivered to lung efficiently (> 300% ID/g). The 225Ac was slowly released from the lung with an initial t1/2 = 49 h, and the released 225Ac accumulated in the liver. Injection of free HEHA was only partially successful in scavenging free 225Ac. In addition to the slow release of 225Ac from the chelate, data indicated that decay daughters of 225Ac were also released from the lung. Immediately after organ harvest, the level of 213Bi, the third alpha-decay daughter, was found to be deficient in the lungs and to be in excess in the kidney, relative to equilibrium values. Injected doses of 225Ac MAb 201B of 1.0 microCi, delivering a minimum calculated absorbed dose of about 6 Gy to the lungs, was effective in killing lung tumors, but also proved acutely radiotoxic. Animals treated with 1.0 microCi or more of the 225Ac radioconjugate died of a wasting syndrome within days with a dose dependent relationship. We conclude that the potential for 225Ac as a radioimmunotherapeutic agent is compromised not only by the slow release of 225Ac from the HEHA chelator, but most importantly by the radiotoxicity associated with decay daughter radioisotopes released from the target organ.

High resolution X-ray computed tomography: an emerging tool for small animal cancer research.

Dedicated high-resolution small animal imaging systems have recently emerged as important new tools for cancer research. These new imaging systems permit researchers to noninvasively screen animals for mutations or pathologies and to monitor disease progression and response to therapy. One imaging modality, X-ray microcomputed tomography (microCT) shows promise as a cost-effective means for detecting and characterizing soft-tissue structures, skeletal abnormalities, and tumors in live animals. MicroCT systems provide high-resolution images (typically 50 microns or less), rapid data acquisition (typically 5 to 30 minutes), excellent sensitivity to skeletal tissue and good sensitivity to soft tissue, particularly when contrast-enhancing media are employed. The development of microCT technology for small animal imaging is reviewed, and key considerations for designing small animal microCT imaging protocols are summarized. Recent studies on mouse prostate, lung and bone tumor models are overviewed.

High resolution computed tomography and MRI for monitoring lung tumor growth in mice undergoing radioimmunotherapy: correlation with histology.

A model lung tumor system has been developed in mice for the evaluation of vascular targeted radioimmunotherapy. In this model, EMT-6 mammary carcinoma tumors growing in the lung are treated with 213Bi, an alpha particle emitter, which is targeted to lung blood vessels using a monoclonal antibody. Smaller tumors (< 100 microm in diameter) are cured, but larger tumors undergo a period of regression and then regrow and ultimately prove lethal. The goal of this work was to determine if external imaging with MRI or CT could be used routinely to monitor the growth/ regression of lung tumors in live mice. To attempt to evaluate individual tumors in vivo, animals were initially imaged with magnetic resonance imaging (MRI). High resolution MRI images could be obtained only after sacrifice when lungs were not moving. In contrast, high resolution computed tomography (CT) produced evaluable images from anesthetized animals. Serial CT images (up to 5/animal) were collected over a 17 day period of tumor growth and treatment. When tumored animals became moribund, animals were sacrificed and lungs were inflated with fixative, embedded in paraffin, and then sectioned serially to compare the detection of tumors by high resolution CT with detection by histology. CT proved most useful in detecting lung tumors located in the hilar area and least useful in detecting serosal surface and anterior lobe tumor foci. Overall, CT images of live animals revealed tumors in approximately 2/3 of cases detected in histologic serial sections when relatively few tumors were present per lung. Detection of lesions and their resolution post therapy were complicated due to residual hemorrhagic, regressing tumor nodules and the development of lung edema both of which appeared as high density areas in the CT scans. We conclude that the microCT method used could identify some lung tumors as small as 100 microm in diameter; however, no concrete evaluation of therapy induced regression of the tumors could be made with CT analyses alone.

Labeling and distribution of linear peptides identified using in vivo phage display selection for tumors.

To develop targeting molecules to be used for vascular targeting of short half-lived alpha-emitters for radioimmunotherapy, linear peptide phage display libraries were selected in vivo for binding to IC-12 rat tracheal tumors growing in severe combined immune deficient mice. After three rounds of selection, 15 phage clones were analyzed for DNA sequence, and the deduced translation products of cDNA inserts were compared. Three consensus sequences were chosen from three separate experimental selection series and peptides of these sequences with added -gly-gly-tyr were obtained. Peptides were radiolabeled on tyrosine with (125)I and the biodistribution in tumor-bearing mice was determined. The radioiodinated peptides were stable in vitro and when injected in tumor-bearing mice approximately 3.0 %ID/g accumulated in the tumor; however, much of the (125)I was found in the gastrointestinal tract and thyroid, indicative of dehalogenation of the labeled peptide. Radiolabeling peptide 2 with N-succinimidyl-3-(125)I-iodobenzoate resulted in faster excretion, which in turn resulted in lower levels in tumor and other organs, especially thyroid and gastrointestinal tract. Peptide 2 was derivatized with the bifunctional isothiocyanates of cyclohexyl-B diethylenetriaminepentaacetic acid (DTPA) or CHX-A" DTPA by direct conjugation or with a hydroxylamine derivative of 1B4M-DTPA (2-(p-[O-(carboxamylmethyl)hydroxylamine]benzyl)-6-methyl-diethylenetriamine-N,N,N',N",N"-pentaacetic acid ) coupled at the N-terminus. The primary molecular species in the conjugated products were shown by mass spectrometry to have one DTPA per peptide. Peptide chelate conjugates were radiolabeled with (213)Bi and the products tested for biodistribution in tumor-bearing mice. The data show that chelation of (213)Bi to peptides was accomplished by both the direct method of DTPA attachment and by the method using the linker at the N-terminus. Only small amounts of peptide accumulated at tumor sites. We conclude that phage display is a powerful tool to select peptides with restricted binding specificity; however, the peptides isolated to date do not bind with high retention to tumor sites in vivo.

Mutation analyses of a series of TNT-related compounds using the CHO-hprt assay.

Trinitrotoluene (TNT) and related compounds were tested for induction of mutation in the CHO-hprt mutation assay. The parent compound, TNT, was consistently found to be mutagenic at concentrations above 40 microg ml(-1), whether or not S9 activating enzymes were added. Five TNT metabolites gave statistically significant but small increases in mutation frequency over solvent controls: 4-amino-2,6 dinitrotoluene, 2,4',6,6'-tetranitro-2',4-azoxytoluene, 2,2',6,6'-tetranitro-4,4'-azoxytoluene, 2',4,6,6'-tetranitro-2,4'-azoxytoluene and triaminotoluene. Clear dose-response relationships could not be established for the mutagenic response of these compounds. They are considered as very weak mutagens in this mammalian test system. Five compounds did not produce statistically significant mutation frequencies at the levels tested: 2-amino-4,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene, 1,3,5-trinitrobenzene, 2,6-diamino-4-nitrotoluene and 4,4',6,6'-tetranitro-2,2'-azoxytoluene. The results indicate that none of the TNT metabolites tested pose a significant mutational health risk, at least as judged by the CHO-hprt assay.

Radioimmunotherapy using vascular targeted 213Bi: the role of tumor necrosis factor alpha in the development of pulmonary fibrosis.

A monoclonal antibody (201B) specific to murine thrombomodulin, covalently linked to cyclohexyl diethylenetriaminepentaacetic acid, successfully delivers chelated 213Bi, an alpha-particle emitter, (213Bi-201B) rapidly to lung vascular endothelium. When injected at doses of 1 MBq/mouse, 213Bi-201B destroyed most of the 100 colonies of EMT-6 mammary carcinomas growing as lung tumors of up to 2000 cells/colony. Some mice were cured of lung tumors, and others had extended life spans compared to untreated control animals but eventually succumbed to tumor recurrence. At injected doses of 4-6 MBq/mouse, 100% of lung tumor colonies were eliminated; however, 3-4 months later, these mice developed pulmonary fibrosis and died. The mechanisms leading to the fibrotic response in other pulmonary irradiation models strongly implicate tumor necrosis factor alpha (TNF-alpha), released from damaged tissues, as the pivotal inflammatory cytokine in a cascade of events that culminate in fibrosis. Attempts to prevent the development of pulmonary fibrosis, by using antibodies or soluble receptor (rhuTNFR:Fc) as inhibitors of TNF-alpha, were unsuccessful. Additionally, mice genetically deficient for TNF-alpha production developed pulmonary fibrosis following 213Bi-201B treatment. Interestingly, non-tumor-bearing BALB/c mice receiving rhuTNFR:Fc or mice genetically deficient in TNF-alpha production and treated with 213Bi-201B, had significantly reduced life spans compared to mice receiving no treatment or 213Bi-201B alone. We speculate that in normal mice, although TNF-alpha may induce an inflammatory response following alpha-particle radiation mediated tumor clearance and pulmonary damage, its effects in the post-tumor clearance time period may actually retard the development of fibrosis.

Analysis for TNF-alpha using solid-phase affinity capture with radiolabel and MALDI-MS detection.

Screening of mutant mice for subtle phenotypes requires sensitive, high-throughput analyses of sentinel proteins in functional pathways. The cytokine TNF-alpha is upregulated during inflammatory reactions associated with autoimmune diseases. We have developed a method to monitor the concentration of TNF-alpha under physiological conditions. TNF-alpha is captured, purified, and concentrated using monoclonal antibody-coated microbeads. The capture is efficient (> 80%) and can be used in the concentration range < 100 pg/mL to > 50 ng/mL, as determined by detection of 125I-labeled TNF-alpha. The bead capture of TNF-alpha can be combined with direct detection by MALDI-MS for sample concentrations of > 10 ng/mL. TNF-alpha can be captured and detected from diluted mouse serum, with minimal interferences observed in the MALDI spectrum. This method is adaptable to high-throughput sample handling with microfluidic devices and automated mass spectrometric analysis.

Comparison of 225actinium chelates: tissue distribution and radiotoxicity.

The biodistribution and tissue toxicity of intravenously administered 225-actinium (225Ac) complexed with acetate, ethylene diamine tetraacetic acid (EDTA), 1, 4, 7, 10, 13-pentaazacyclopentadecane-N, N', N", N"', N""-pentaacetic acid (PEPA), or the "a" isomer of cyclohexyl diethylenetriamine pentaacetic acid (CHX-DTPA), were examined. The percent of injected dose per organ and per gram of tissue for each chelate complex was determined. 225Ac-CHX-DTPA was evaluated further for radiotoxic effects. Mice receiving > or =185 kBq 225Ac-CHX-DTPA suffered 100% morbidity by 5 days and 100% mortality by 8 days postinjection, and all animals evaluated had significant organ damage. The in vivo instability of the 225Ac-CHX-DTPA complex likely allowed accumulation of free 225Ac in organs, which resulted in tissue pathology.

Radioimmunotherapy of micrometastases in lung with vascular targeted 213Bi.

A model system has been used to test the efficacy of vascular targeting of alpha-particle emitter 213Bi for therapy of small, 'artificial' metastases in mouse lung. Specific monoclonal antibody (mAb) 201 B was used to deliver greater than 30% of the injected dose to lung where tumours had developed due to intravenous injection of cells. Specific 213Bi-mAb 201B treatment of BALB/c mammary carcinoma EMT-6 tumours in lung resulted in a dose-dependent destruction of tumours and an extended lifespan of treated animals relative to controls. Significant reduction of lung tumour burden was noted in animals treated with 0.93 MBq injected dose or as little as 14 Gy absorbed dose to the lung. Animals treated with higher doses (2.6-6.7 MBq) had nearly complete cure of lung tumours but eventually died of lung fibrosis induced by the treatment. Four other tumour cell types were studied: murine Line 1 lung carcinomas in syngeneic BALB/c mice, rat IC-12 tracheal carcinoma growing in severe combined immune deficient (SCID) mice, and two human tumours--epidermoid carcinoma A431 and lung carcinoma A549--growing in SCID mice. In all cases, the number of lung tumour colonies was reduced in animals treated with specific, labelled mAb relative to those in animals treated with control 213Bi MAb or EDTA complexed 213Bi. Tumours treated in immunodeficient SCID mice were partially destroyed or at least retarded in growth, but ultimately regrew and proved fatal, indicating that an intact immune function is necessary for complete cure. The data show that the short-lived alpha-particle emitter 213Bi can be effectively targeted to lung blood vessels and that tumour cells growing in the lung are killed. The mechanism may involve direct killing of tumour cells from alpha-particle irradiation, killing through destruction of blood supply to the tumour, or a combination of the two.

Improved in vivo stability of actinium-225 macrocyclic complexes.

The favorable nuclear properties of actinium-225 ((225)Ac) have led to proposal of this isotope for use in radioimmunotherapy. In an effort to reduce the toxicity of free (225)Ac, a series of ligands were evaluated for stability in vivo. Loss of (225)Ac from acyclic chelating agents resulted in high liver uptake and poor whole body clearance. The macrocyclic ligands c-DOTA, PEPA, and HEHA were evaluated, and (225)Ac-HEHA showed exceptional stability in vivo. (225)Ac chelated with EDTA, DTPA, DOTA, or PEPA permitted substantial accumulation of the radionuclide to the liver, while the (225)Ac-HEHA complex was essentially excreted within minutes of administration. The preparation of the ligands and radiolabeled complexes and the biodistribution results will be discussed.

In vivo studies of fullerene-based materials using endohedral metallofullerene radiotracers.

Biodistribution studies of a water-soluble radioactive metallofullerene compound have been conducted using BALB/c mice. To this end, a sample containing Hox@C82 (x = 1, 2) was purified and derivatized to prepare the water-soluble metallofullerol, Hox@C82(OH)y. This metallofullerol was then neutron-activated (165Ho[n,gamma]166Ho) to prepare the 166Hox@C82(OH)y analog as a radiotracer, which was monitored, after intravenous administration, for up to 48 hours by using dissection radioanalysis, and its biodistribution was compared with a control compound, Na2[166Ho(DTPA)(H2O)]. Results showed selective localization of the 166Hox@C82(OH)y tracer in the liver but with slow clearance, as well as uptake by bone without clearance. In contrast, excretion of the control compound was nearly quantitative after 1 hour. The fate of 166Ho was also explored by a metabolism study of 166Hox@C82(OH)y in Fischer rats. Results indicated 20% excretion of intact 166Hox@C82(OH)y within 5 days. The present findings demonstrate the feasibility of using water-solubilized metallofullerene radiotracers to monitor the fate of fullerene-based materials in animals, and suggest that water-solubilized fullerene materials, in general, may be useful components in drug design.

Treatment of lung tumor colonies with 90Y targeted to blood vessels: comparison with the alpha-particle emitter 213Bi.

An in vivo lung tumor model system for radioimmunotherapy of lung metastases was used to test the relative effectiveness of the vascular- targeted beta-particle emitter 90Y, and alpha-particle emitter, 213Bi. Yttrium-90 was shown to be stably bound by CHXa" DTPA-MAb 201B conjugates and delivered efficiently to lung tumor blood vessels. Dosimetry calculations indicated that the lung received 16.2 Gy/MBq from treatment with 90Y MAb 201B, which was a sevenfold greater absorbed dose than any other organ examined. Therapy was optimal for 90Y with 3 MBq injected. Bismuth-213 MAb 201B also delivered a similar absorbed dose (15Gy/MBq) to the lung. Yttrium-90 was found to be slightly more effective against larger tumors than 213Bi, consistent with the larger range of 2 MeV beta particles from 90Y than the 8 MeV alpha particles from 213Bi. Treatment of EMT-6 tumors growing in immunodeficient SCID mice with 90Y or 213Bi MAb 201 resulted in significant destruction of tumor colonies; however, 90Y MAb 201B was toxic for the SCID mice, inflicting acute lung damage. In another tumor model, IC-12 rat tracheal carcinoma growing in SCID mouse lungs, 90Y therapy was more effective than 213Bi at destroying lung tumors. However, 90Y MAb 201B toxicity for the lung limited any therapeutic effect. We conclude that, although vascular-targeted 90Y MAb can be an effective therapeutic agent, particularly for larger tumors, in this model system, acute damage to the lung may limit its application.

Radiotoxicity of bismuth-213 bound to membranes of monolayer and spheroid cultures of tumor cells.

Monoclonal antibody 13A to murine CD44 was used to bind the alpha-particle emitter 213Bi to cell surfaces of cultured EMT-6 or Line 1 tumor cells. Data on kinetics and saturation of binding, cell shape and nuclear size were used to calculate the absorbed dose to the nuclei. Treatment of monolayer cells with [213Bi]MAb 13A produced a classical exponential survival curve with no apparent shoulder. Microdosimetry analyses indicated that 1.4-1.7 Gy produced a 37% surviving fraction (D0). Multicellular spheroids were shown to bind [213Bi]MAb 13A mainly on the outer cell layer. Relatively small amounts of activity added to the spheroids resulted in relatively large absorbed doses. The result was that 3-6-fold less added radioisotope was necessary to kill similar fractions of cells in spheroids than in monolayer cells. These data are consistent with the interpretation that the alpha particles from a single 213Bi atom bound to one cell can penetrate and kill adjacent cells. Flow cytometry was used to sort cells originating from the periphery or from the interior of spheroids. Cells from the outside of the [213Bi]MAb 13A exposed spheroids had a lower surviving fraction per administered activity than cells from the interior. Cells were killed efficiently in spheroids up to 20-30 cells in diameter. The data support the hypothesis that alpha-particle emitters should be very efficient at killing cells in micrometastases of solid tumors.

Combination vascular targeted and tumor targeted radioimmunotherapy.

Rat MAb 201B, which binds to murine thrombomodulin, can deliver up to 50% of the injected dose of attached radioisotopes to the lung vascular endothelium. We have shown previously that intravenous injection of about 30 microCi of 213Bi-MAb 201B, which delivers about 15 Gy of alpha irradiation to the lung, is capable of eradicating small lung colonies (500-1000 cells) of the mammary tumor line, EMT-6. Larger tumors (> 5000 cells) were not completely cured by this vascular targeted radioimmunotherapy (VT-RAIT) approach. We reasoned that VT-RAIT might make the lung vessels serving the tumor cells more permeable, allowing MAb targeted to the tumor cells to extravasate more readily and mediate more efficient standard radioimmunotherapy (RAIT). Distribution experiments with the tumor targeted MAb 13A (RAIT MAb), following VT-RAIT, did not demonstrate a large increase in tumor uptake; however, microautoradiography did indicate that MAb 13A was distributed more evenly throughout the tumor when administered after VT-RAIT. Therapy experiments on lung tumors of approximately 5000 cells each, combining 213Bi-MAb 201B (VT-RAIT) with 213Bi-MAb 13A (RAIT) 24 hours later, resulted in a better outcome (3 cured/10 at risk) than for control groups: RAIT only (0/10), VT-RAIT only (1/10), or no therapy (0/10). RAIT therapy delivered 48 hours after VT-RAIT had no apparent benefit. 213Bi-MAb 201B VT-RAIT followed by 90Y-MAb 13A Fab' RAIT showed only a slight improvement in tumor cures (2/10) over that in control groups: (0/9), (0/10), (0/10), respectively. These results suggest that optimal timing, dosage, and choice of MAb for RAIT should enhance the double MAb therapy approach significantly.

Vascular targeted radioimmunotherapy with 213Bi--an alpha-particle emitter.

To destroy both tumor blood vessels and adjacent tumor cells, an alpha-particle emitter, 213Bi, has been targeted with a monoclonal antibody (MAb) to vessels that feed lung tumors in mice. Animals, bearing approximately 100 EMT-6 carcinomas each of 50-400 cells in size in the lung, that were treated with 120 muCi of 213Bi-MAb 201B were all cured of their disease. Animals treated when tumors were larger (10(3)-10(4) cells) had extended life spans, but a small number of residual tumors eventually killed the animals. Significant extension of life span was also induced with another tumor model-rat tracheal carcinoma growing in the lungs of SCID mice that were then treated with 136 muCi 213Bi-MAb 201B. These studies indicate that attack of both blood vessels and tumor cells simultaneously is an effective mode of cancer treatment.

Monoclonal antibody to rat CD63 detects different molecular forms in rat tissue.

From mice immunized with rat endothelial cell membranes, we isolated several hybridomas secreting monoclonal antibodies (MAbs) to a 45-kDa glycoprotein expressed on the surface of cultured cells. One of these antibodies, 523-14A, was purified and used for immunoaffinity chromatography, Western blotting, and immunohistochemistry. The glycoprotein containing the antigen for MAb 523-14A, gp45, was isolated from rat lung endothelial cell membranes using wheat germ agglutinin and antibody affinity chromatography sequentially. Mass spectrometry of tryptic peptides from gel purified bands identified gp45 as rat CD63, a member of the transmembrane-4 superfamily. Western blot analyses of tissues from F344 rats showed that kidney, spleen, uterus, and ovaries expressed CD63 at high levels. Thymus, salivary gland, testicles, intestines, pancreas, and adrenals expressed lower amounts. Tissue cell types expressing CD63 were also examined and the results showed that, in addition to the expected expression on lymphoid cells, CD63 was expressed on many epithelial and muscle cells as well. The mobility of CD63 on SDS-PAGE varied widely, indicative of molecular masses ranging from 45 kDa in some tissues to nearly 60 kDa in others.

Distinct substrate preference of human and mouse N-methylpurine-DNA glycosylases.

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair protein, removes several N-alkylpurine adducts, hypoxanthine, cyclic ethenoadducts of adenine, guanine and cytosine and 8-oxoguanine from DNA. The recombinant human and mouse MPGs, purified from Escherichia coli, show a significant difference in substrate preference. While both proteins prefer 3-methyladenine over other N-alkylpurines in DNA, the mouse MPG removes 7-methylguanine and 3-methylguanine at an approximately 2- to 3-fold higher rate than the human protein when adjusted for equal activity for the release of 3-methyladenine from DNA. Hybrid recombinant proteins containing N-terminal and C-terminal halves of the human and mouse glycosylases were partially purified from MPG-negative E.coli. Their substrate preferences suggest that the N-terminal half is more critical for the recognition of 3-methylguanine and 7-methylguanine.