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.

A skeptical look at viral immune evasion.

In the past several years, many viral gene products have been found to encode proteins which interfere with immune defense mechanisms. Whether these interactions between virus and immune system components are actually evasion mechanisms used during viral infections in their natural hosts remains to be proven. In vitro studies do, however, reveal several tactics which may aid viral replication and dissemination by interfering with components of both the innate and adaptive immune systems. In this manuscript, we discuss the more intensively studied of these putative in vitro evasion tactics and ponder their relevance in in vivo situations.

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.

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.

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.

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.

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.

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.

Splenectomy in cattle via transthoracic approach.

Sixty-eight cattle under general anesthesia were splenectomized. The transthoracic approach was used to provide better access to the spleen and to facilitate ligature of the major splenic vessels. The procedure was easier and less time-consuming, compared with other surgical approaches, and is considered to be less stressful to the animals. Post-operative recovery was complete in 67 of 68 cattle. After surgery, 1 animal developed respiratory tract disease that was thought to have been unrelated to the surgery.

Identification of select lymphocyte homing molecules and vascular addressins in lymphotoxin-alpha deficient mice.

The transmigration of lymphocytes across vascular endothelium is a critical step for the localization of lymphocytes to lymph nodes in both naive and immune reactive states. Mice deficient in lymphotoxin-alpha (LT-alpha) lack peripheral and gut associated lymph nodes. Lymphocyte function and homing ability are reported to be normal in these mice yet information regarding cell adhesion molecules and counterpart vascular addressins is lacking. The phenotype of peripheral lymphocytes from LT-alpha deficient mice was investigated by the use of fluorescent activated cell sorting and immunohistochemistry. No difference was detected in the splenocyte and tissue expression of L-selectin, alpha4beta7 or its individual integrin components, mucosal addressin cell adhesion molecule (MAdCAM-1), intracellular adhesion molecule (ICAM-1), peripheral node addressin (PNAd), or platelet/endothelial cell adhesion molecule (PECAM-1) between wild-type and LT-alpha deficient mice. Therefore, impaired expression of these lymphocyte homing and vascular addressin molecules is apparently not included in the phenotype of the LT-alpha deficient mouse.

Immune responsiveness of lymphotoxin-alpha-deficient mice: two reconstitution models.

The effects of lymphotoxin-alpha (LT-alpha) deficiency on mucosal immune status has not been defined. We utilized severe combined immunodeficiency (scid) mice as recipients of both mutant and wild-type whole splenocytes to determine whether lymphocytes from mutant mice had impaired homing ability. We also utilized irradiated mutant mice as recipients of wild-type whole splenocytes to determine whether lymphoid tissue anlages had, indeed, failed to develop as a consequence of LT-alpha deficiency. Subsequently, all mice were immunized orally with an attenuated strain of Salmonella typhimurium and mucosal IgA responses were monitored. The data presented here demonstrate that scid recipients generate mucosal responses equally well when reconstituted with mutant or wild-type lymphocytes. In contrast, reconstitution of mutant mice with wild-type cells failed to affect the efficiency of their mucosal immunity. The mutant phenotype, therefore, appears to involve neither impaired lymphocyte homing nor function in the generation of mucosal immunity. However, the mutant phenotype and immune responsiveness cannot be transformed merely by the provision of LT-alpha-expressing donor cell populations. The consequence of LT-alpha deficiency on mucosal immune responsiveness appears to be due to the lack of gut-associated lymphoid tissues, which may include the spleen, in mutant mice.

The spleen and organized lymph nodes are not essential for the development of gut-induced mucosal immune responses in lymphotoxin-alpha deficient mice.

Lymphotoxin knock-out (KO) mice generate specific immune responses to orally administered immunogens despite having neither gut-associated nor peripheral lymphoid tissues. The spleen, therefore, was expected to play a role in the generation of immune responses in these KO mice. KO and wild-type (wt) mice were splenectomized and orally immunized with Salmonella typhimurium. Splenectomy produced the most profound effects on serum and fecal IgA levels in KO mice. Total and antigen-specific serum and fecal IgA were increased in splenectomized wt mice but decreased in splenectomized KO mice. Antigen-specific serum IgG was decreased in both KO and wt splenectomized mice while total IgG increased in splenectomized wt mice. Both splenectomized wt and KO mice demonstrated a compensatory expansion of the lamina propria compartment characterized by a significant increase in the number of IgA spot-forming cells. KO mice demonstrated further compensation for the loss of the spleen in the accelerated development of ectopic lymphoid tissues. We conclude that the spleen plays a prominent role as a lymphoid organ in KO mice but its removal does not abolish immune responsiveness. Residual immune responsiveness in splenectomized KO mice following oral immunization appears to be due to expansion and/or development of alternate effector compartments.

Lymphotoxin alpha-/- mice develop functionally impaired CD8+ T cell responses and fail to contain virus infection of the central nervous system.

Recent observations have indicated that viral persistence and tumor spreading could occur because of effector function-defective CD8(+) T cells. Although chronic exposure to Ag, lack of CD4 help, and epitope dominance are suggested to interfere with CTL differentiation, mechanisms underlying the defective effector function remain obscure. We demonstrate in this report that lymphotoxin alpha-deficient mice develop CD8(+) T cells at normal frequencies when infected with HSV or immunized with OVA Ag but show impaired cytotoxic and cytokine-mediated effector functions resulting in enhanced susceptibility to HSV-induced encephalitis. Although these cells display near normal levels of perforin and Fas ligand, they remain largely at a naive state as judged by high expression of CD62 ligand and failure to up-regulate activation or memory markers. In particular, these CD8(+) T cells revealed inadequate expression of the IL-12 receptor, thus establishing a link between CTL differentiation and LTalpha possibly through regulation of IL-12 receptor. Viruses and tumors could evade immunity by targeting the same pathway.