Quantitative evaluation of boron neutron capture therapy (BNCT) drugs for boron delivery and retention at subcellular-scale resolution in human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS).

Boron neutron capture therapy (BNCT) of cancer depends on the selective delivery of a sufficient number of boron-10 ((10)B) atoms to individual tumour cells. Cell killing results from the (10)B (n, α)(7) Li neutron capture and fission reactions that occur if a sufficient number of (10)B atoms are localized in the tumour cells. Intranuclear (10)B localization enhances the efficiency of cell killing via damage to the DNA. The net cellular content of (10)B atoms reflects both bound and free pools of boron in individual tumour cells. The assessment of these pools, delivered by a boron delivery agent, currently cannot be made at subcellular-scale resolution by clinically applicable techniques such as positron emission tomography and magnetic resonance imaging. In this study, a secondary ion mass spectrometry based imaging instrument, a CAMECA IMS 3f ion microscope, capable of 500 nm spatial resolution was employed. Cryogenically prepared cultured human T98G glioblastoma cells were evaluated for boron uptake and retention of two delivery agents. The first, L-p-boronophenylalanine (BPA), has been used clinically for BNCT of high-grade gliomas, recurrent tumours of the head and neck region and melanomas. The second, a boron analogue of an unnatural amino acid, 1-amino-3-borono-cyclopentanecarboxylic acid (cis-ABCPC), has been studied in rodent glioma and melanoma models by quantification of boron in the nucleus and cytoplasm of individual tumour cells. The bound and free pools of boron were assessed by exposure of cells to boron-free nutrient medium. Both BPA and cis-ABCPC delivered almost 70% of the pool of boron in the free or loosely bound form to the nucleus and cytoplasm of human glioblastoma cells. This free pool of boron could be easily mobilized out of the cell and was in some sort of equilibrium with extracellular boron. In the case of BPA, the intracellular free pool of boron also was affected by the presence of phenylalanine in the nutrient medium. This suggests that it might be advantageous if patients were placed on a low phenylalanine diet prior to the initiation of BNCT. Since BPA currently is used clinically for BNCT, our observations may have direct relevance to future clinical studies utilizing this agent and provides support for individualized treatment planning regimens rather than the use of fixed BPA infusion protocols.

Agglutination of leukemic and 2,4-dinitrophenyl-tagged normal human lymphocytes by wheat germ agglutinin.

The present study was undertaken to determine if leukemic and 2,4-dinitrophenyl (DNP)-tagged normal human lymphocytes shared common receptors for Wheat Germ Agglutinin (WGA), a lectin capable of reacting with malignant cells. Normal peripheral blood lymphocytes were tagged with 2,4-dinitrofluorobenzene (DNFB) at a ratio of 10-11 molecules/cell. Various concentrations of WGA were added to leukemic or tagged or untagged normal lymphocytes and incubated for 20 minutes at 37 degrees C, after which agglutination was scored visually. A readily discernible quantitative difference in the agglutinability of leukemic and DNP-tagged versus untagged normal cells was seen at all concentrations of WGA in the range of 50-800 mug/ml. The reaction was maximal when a ratio of 10-11 molecules of DNFB/cell was used for tagging and decreased progressively with 10-8, 10-6, 10-4, 0r 10-2 molecules. The agglutination of leukemic and DNP-tagged normal lymphocytes by 200 mug/ml of WGA was completely blocked by 0.1 M N-acetylglucosamine (GlcNAc) while as low a concentration as 0.001 M GlcNAc inhibited the reactivity of untagged cells. Since the agglutination of leukemic and DNP-tagged normal lymphocytes was equally inhibited by GlcNAc, this suggests that the same or similar receptor sites were involved in the two reactions. On the basis of our observations we propose that the initial step in the agglutination of leukemic and DNP-tagged normal lymphocytes by WGA IS THE BINDING OF THE LECTIN TO SPECIFIC RECEPTORS RATHER Than to DNP residues on the cell surface, since leukemic cells tagged with DNFB did not show increased agglutinability.

Agglutination of 2,4-dinitrophenyl-tagged normal human leukocytes by concanavalin A: possible relationship to their ability to evoke production of leukemia-associated antibodies.

Normal peripheral blood leukocytes were tagged with 2, 4-dinitrofluorobenzene at a ratio of 10-11 molecules/cell. One-tenth ml of various concentrations of concanavalin A (Con A) was added to 0.2 ml of either tagged or untagged cells (5 times 10-6/ml) and incubated for 20 minutes at ambient temperature, after which agglutination was scored visually. A readily discernible quantitative difference in the agglutinability of 2, 4-dinitrophenyl (DNP)-tagged versus untagged cells was seen at all concentrations of Con A in the range of 12.5 800 mug/ml. The reaction was maximal at 24 degrees C, somewhat diminished at 37 degrees C, and minimal at 4 degrees C. The agglutination of DNP-tagged leukocytes by 50 mug Con A/ml was completely blocked with 0.1 M methyl-alpha-D-glucopyranoside (alpha-MG), but as low a concentration as 0.001 M alpha-MG inhibited agglutination of untagged cells. The ability of Con A to agglutinate DNP-tagged normal leukocutes may be attributed to a lowering of the zeta potential, a topographic rearrangement of receptor sites, or the formation of new antigenic determinants similar to those found on malignant cells. The last alternative would be consistent with the observation that DNP-tagged normal leukocytes could evoke the production of antibodies that reacted with leukemic granulocytes.

Quantitative assay for lectin-induced cytoagglutination by means of an electronic particle-counting technique.

A method employing an electronic particle-counting technique was used to quantify lectin-induced agglutination of human granulocytes and lymphocytes with either concanavalin A or wheat germ agglutinin. The number and mean volume of single cells and aggregates in the presence of increasing concentrations of lectin were computed from 95% confidence intervals. Agglutination depended on both the number of free cells and the number and size of the cell clusters. Changes in these two variables were mutually independent of one another, and both were simultaneously determined. An index of agglutination that takes the effect of these two variables into account was defined as (formula: see text) VA equals mean volume of cell aggregates, NS equals number of single cells, NA equals number of cell aggregates, VS equals mean volume of single cells, rb equals r at a given lectin concentration, and ra equals r in the absence of lectin. For any combination of lectin and cell type, the agglutination curve, as described by zeta, consisted of two components: a) a flat region in which zeta remained constant with increasing lectin concentrations and b) a region in which zeta increased linearly as a function of the logarithm of lectin concentration. The shapes of these curves offered two parameters for quantitative comparison of agglutinability: 1)threshold concentration, defined as the minimum concentration of lectin (microgram/ml) required to bring about a measurable rise in zeta and 2) the concentration gradient, defined as the change in zeta for an increase of one log unit in the concentration of the lectin in the range beyond the threshold concentration. This method offers a high degree of quantification and provides reliable information that can be meaningfully correlated with cell surface characteristics.

Relationship between concanavalin A-induced agglutinability of murine leukemia cells and their propensity to form heterotypic aggregates with syngeneic lymphoid cells.

The agglutinabilities of the murine leukemia cell lines L1210, P388, and C1498 were determined in the presence of concanavalin A (Con A) by a quantitative cytoagglutination assay. The propensity of these cells to form heterotypic aggregates with normal syngeneic spleen cells, those obtained from mice carrying the respective leukemias in ascitic form, and syngeneic lung cells also were determined. Con A caused agglutination of all five types of leukemia cells and the resulting agglutination patterns had certain characteristics. Threshold concentrations of Con A, below which no significant cytoagglutination occurred, were very low. A steady increase in zeta, a previously defined index of agglutination that simultaneously takes into account the number of free cells and the number and size of the aggregates, was observed with increasing concentrations of Con A until a plateau was reached at 25-50 microgram/ml and this extended over a wide range of lectin concentrations (50-1,000 micrograms/ml). Self-aggregation of leukemia cells was not observed, and their propensity to form heterotypic aggregates with syngeneic lung cells was negligible. However, all leukemia cell lines formed measurable aggregates with spleen cells from both normal and leukemia-bearing mice; these aggregates usually reached a maximum plateau between 30 and 35 minutes of incubation and remained constant thereafter. Aggregation of leukemia cells with spleen cells from leukemic mice always was greater than that with spleen cells from normal mice. Con A agglutinability of leukemia cells was correlated with their propensity to form heterotypic aggregates, which suggests that Con A agglutinability of leukemia cells was correlated with their propensity to form heterotypic aggregates, which suggests that Con A receptor carbohydrate moieties may be involved in the intercellular adhesion leading to heterotypic aggregate formation.

Phenotype and functional activity of tumor-infiltrating lymphocytes isolated from immunogenic and nonimmunogenic rat brain tumors.

The purpose of the present study was to define the immunogenicity of two transplantable rat gliomas, designated F98 and D74, and to relate this to the phenotype and functional activity of tumor-infiltrating lymphocytes (TIL). Fischer rats, immunized with irradiated F98 tumor cells and challenged with intracerebral implants of ten F98 cells, had a median survival time of 49 days compared to 36 days for nonimmunized controls. In contrast, no statistically significant increases in survival times were noted in animals similarly immunized and challenged with the D74 tumors. No in vivo protection could be demonstrated in animals immunized and cross-challenged with either F98 or D74 glioma cells. Lymph node lymphocytes and TIL, isolated from animals immunized and challenged with F98 cells, were more cytolytically active than effector cells obtained from D74-immunized animals. Phenotypes of TIL isolated from intracerebral F98 gliomas of immunized rats were 52% OX-8+ and 21% W3/25+ compared to 31% OX-8+ and 19% W3/25+ for D74-immunized animals. Cytolytic activity against glioma targets was mediated by OX-8+ TIL, as determined by cell depletion experiments. Limiting dilution analysis showed that cytolytic T-lymphocyte precursors were present in TIL of F98 gliomas of immunized rats at a frequency of 1/3547 and were specific for F98 targets, while natural killer cell-like activity was low. Our data indicate that the F98 glioma was more immunogenic than the D74 glioma, as evidenced by increased numbers and activity of cytolytic effector cells and their precursors among TIL. This may explain in part the longer survival times observed in immunized animals challenged intracerebrally with the F98 gliomas compared to D74-immunized and -challenged hosts.

Intratumoral delivery of boronated epidermal growth factor for neutron capture therapy of brain tumors.

The gene for epidermal growth factor receptor (EGFR) is amplified or overexpressed in high-grade gliomas but is low or undetectable in normal brain. Recently, there has been increasing interest in using epidermal growth factor (EGF)-based bioconjugates as targeting agents for brain tumors. In the present study, we have investigated the potential use of boronated EGF as a delivery agent for boron neutron capture therapy, which is based on the capture reaction that occurs when 10B, a stable isotope, is irradiated with low-energy thermal neutrons. A fourth generation starburst dendrimer was boronated and linked to EGF using heterobifunctional reagents. Either wild-type or EGFR gene transduced C6 glioma cells (C6EGFR), which expressed 10(5)-10(6) receptor sites/cell, were stereotactically implanted into the right cerebral hemisphere of Fischer rats. Four weeks later, the rats received either i.v. or intratumoral (i.t.) injection of 131I-labeled boronated starburst dendrimer (BSD) or BSD-EGF. The biodistribution of 131I-BSD-EGF and 131I-BSD was studied by means of whole-body scintigraphy, autoradiography, and gamma scintillation counting. Following i.t. injection of 131I-BSD-EGF, 21.8% of the injected dose per gram tissue (% ID/g) was localized in C6EGFR tumors at 24 h and 16.3% at 48 h compared to 5 and 1.3% ID/g in C6 wild-type tumors, respectively, and 0.01 and 0.006% ID/g, respectively, for i.v. injected animals at the corresponding times. In contrast, following i.t. injection of BSD-EGF, only 0.01-0.1% ID/g was localized in the liver and spleen at 24 and 48 h compared to 5-12% ID/g following i.v. injection. Our data indicate that direct i.t. injection can selectively deliver BSD-EGF to EGFR-positive gliomas and suggest that intracerebral administration may be the most effective way for delivering EGF-based bioconjugates to EGFR-positive brain tumors.

Adoptive immunotherapy of a rat glioma using lymphokine-activated killer cells and interleukin 2.

The aim of the present study was to develop an animal model to test the therapeutic potential of purified adherent lymphokine-activated killer (A-LAK) cells against an intracerebrally implanted rat glioma, designated F98. Highly purified A-LAK cells demonstrated greater activity against F98 tumor cells than conventional lymphokine-activated killer cells, as determined by means of 51Cr-release and clonogenic assays. Therapeutic efficacy was evaluated by means of a Winn neutralization assay, in which F98 targets and A-LAK cells or control nonadherent mononuclear cells were incubated for 18 h in vitro and then implanted stereotactically into the right caudate nuclei of Fischer rats. Animals given injections of 4000 F98 cells alone or control nonadherent mononuclear cells had a mean survival time of 22.3 days, compared to 46.1 days (P less than 0.001) for rats treated with A-LAK cells. Increasing the tumor inoculum to 12,500 cells reduced the survival time of A-LAK-treated animals to 27.8 days, compared to 20.8 days for untreated controls. Systemic administration of 50,000 units/kg of interleukin 2 every 12 h for 5 days failed to improve survival. The mean survival time of rats implanted with the F98 tumor ranged from 16 days for 10(5) cells to 29 days for 10(2) cells. Extrapolating from these survival data, treatment with A-LAK cells may have decreased the number of F98 cells to less than 10, but even this small number was still lethal. Supernatants from F98 cells had immunoinhibitory activity that, further, may have modulated the antitumor effects of A-LAK cells. Our results indicate that curative, adoptive immunotherapy of the F98 glioma by means of A-LAK/interleukin 2 is impossible to achieve and provide some explanation for the clinical failures that have been observed in the adoptive immunotherapy of malignant gliomas.

Phenotypic diversity of murine B16 melanoma detected by anti-B16 monoclonal antibodies.

A panel of monoclonal antibodies (MoAbs), produced against the murine B16 melanoma, has been used to characterize its phenotypic diversity. Six MoAbs that did not bind to primary cultures of kidney, brain or liver, spleen cells, thymocytes, 3T3 fibroblasts, melanin, or transferrin receptors were selected for further evaluation. Five MoAbs, which recognized surface antigens expressed on parental B16 cells and the B16-F1, B16-F10, B16-F10 FLR, and B16-BL6 sublines, did not appear to cross-react with each other, suggesting that they identified antigenically distinct epitopes. Four MoAbs, designated as IB16-2, IB16-4, IB16-8, and IB16-10, recognized B16 surface antigens that were variably expressed over short periods of time. This variable expression was independent of the cell cycle and was characteristic of four B16 sublines. Two of these MoAbs, both of the IgG2b isotype, fixed rabbit and guinea pig complement and were cytolytic in the presence of rabbit complement. One MoAb, designated IB16-6, recognized a surface antigen consistently expressed on greater than 90% of cells of both the parental tumor and the sublines. This MoAb bound to several murine and one human melanoma cell line, but not to other histopathological types of tumors or normal tissues. The cellular antigen that this antibody recognized was not detected in the cytoplasm, did not modulate in the presence of IB16-6, and was sensitive to trypsin, pronase, alcohols, acetone, and detergents, thereby suggesting that it was a protein. Our data are among the first that directly show the extent of phenotypic diversity of the B16 melanoma and sublines that have been derived from it.

Quantitative imaging and microlocalization of boron-10 in brain tumors and infiltrating tumor cells by SIMS ion microscopy: relevance to neutron capture therapy.

Boron neutron capture therapy (BNCT) is dependent on the selective accumulation of boron-10 in tumor cells relative to the contiguous normal cells. Ion microscopy was used to evaluate the microdistribution of boron-10 from p-boronophenylalanine (BPA) in the 9L rat gliosarcoma and the F98 rat glioma brain tumor models. Four routes of BPA administration were used: i.p. injection, intracarotid (i.c.) injection [with and without blood-brain barrier disruption (BBB-D)], and continuous timed i.v. infusions. i.p. injection of BPA in the 9L gliosarcoma resulted in a tumor-to-brain (T:Br) boron-10 concentration ratio of 3.7:1 when measured at the tumor-normal brain interface. In the F98 glioma, i.c injection of BPA resulted in a T:Br ratio of 2.9:1, and this increased to 5.4:1 when BBB-D was performed. The increased tumor boron uptake would potentially enhance the therapeutic ratio of BNCT by >25%. At present, ion microscopy is the only technique to provide a direct measurement of the T:Br boron-10 concentration ratio for tumor cells infiltrating normal brain. In the 9L gliosarcoma, this ratio was 2.9:1 after i.p. administration. In the F98 glioma, i.c injection resulted in a ratio of 2.2:1, and this increased to 3.0:1 after BBB-D. Ion microscopy revealed a consistent pattern of boron-10 microdistribution for both rat brain tumor models. The boron-10 concentration in the main tumor mass (MTM) was approximately twice that of the infiltrating tumor cells. One hour after a 2-h i.v. infusion of BPA in rats with the 9L gliosarcoma, tumor boron-10 concentrations were 2.7 times higher than that of infiltrating tumor cells [83 +/- 23 microg/g tissue versus 31 +/- 12 microg/g tissue (mean +/- SD)]. Continuous 3- and 6-h i.v. infusions of BPA in the 9L gliosarcoma resulted in similar high boron-10 concentrations in the MTM. The boron-10 concentration in infiltrating tumor cells was two times lower than the MTM after a 3-h infusion. After 6 h, the boron-10 concentration in infiltrating tumor cells had increased nearly 90% relative to the 2- and 3-h infusions. A 24-h i.v. infusion resulted in similar boron-10 levels between the MTM and the infiltrating tumor cells. Boron concentrations in the normal brain were similar for all four infusion times (approximately 20 microg/g tissue). These results are important for BNCT, because clinical protocols using a 2-h infusion have been performed with the assumption that infiltrating tumor cells contain equivalent amounts of boron-10 as the MTM. The results reported here suggest that this is not the case and that a 6-h or longer infusion of BPA may be necessary to raise boron-10 levels in infiltrating tumor cells to that in the MTM.

Monoclonal antibodies directed against preneoplastic and neoplastic murine mammary lesions.

We have produced a panel of monoclonal antibodies directed against a dimethylbenzanthracene-induced murine mammary tumor. Five rat-mouse hybridomas produced antibodies that bound to some murine mammary tumors, but not to normal renal adherent cells, lymphocytes, 3T3 fibroblasts, red blood cells, or mammary gland. One of these antibodies, designated AMT8, was selected for further evaluation based on its relatively strong reactivity, as determined by immunofluorescence. Indirect immunofluorescent studies on frozen histological tissue sections and quantitative immunofluorescent binding studies on cultured normal and tumor cells revealed that AMT8 was bound to certain murine mammary tumors and their preneoplastic hyperplastic nodules, but not to normal murine organs including normal mammary glands. Two tumors and their hyperplastic alveolar nodule counterparts that contained the antigen recognized by AMT8 did not express functional estrogen and progesterone receptors, indicating that antigen expression was not dependent on functional receptors. The antigen recognized did not cap, was found to modulate slowly, and was reexpressed in the presence of excess AMT8. From these findings, we conclude that AMT8 may prove to be a valuable tool for the study of early mammary tumorigenesis.

Boron neutron capture therapy of intracerebral melanoma using boronophenylalanine as a capture agent.

A rat brain tumor model has been developed utilizing nude rats and the human melanoma cell line MRA 27. For pharmacokinetic and tissue distribution studies, 2 10(5) MRA 27 cells were implanted intracerebrally (i.c.), and 30 days later, 120 mg of 10B-enriched L-boronophenylalanine were injected i.p. into nude rats. 10B concentrations in the tumor, blood, and normal brain were 23.7, 9.4, and 8.4 micrograms/g, respectively, 6 h following administration. For therapy experiments, tumor bearing rats were irradiated at the Brookhaven Medical Research Reactor 30 days following implantation. The median survival time was 44 days for untreated rats, 76 days for those receiving a physical dose of 2.7 Gy, and 93 days for those receiving 3.6 Gy. Animals receiving both 10B-L-boronophenylalanine and physical doses of 1.8, 2.7, or 3.6 Gy (total tumor physical doses of 5.0, 7.5, or 10.1 Gy) had median survival times of 170, 182, and 262 days, respectively. Forty % of rats that received the highest tumor dose (10.1 Gy) survived > 300 days. In a replicate experiment 21% of animals that had received L-boronophenylalanine and irradiation (total tumor physical dose of 10.1 Gy) were alive 220 days after therapy. In a parallel study, animals that were irradiated with gamma photons from a 137Cs source with 12 Gy or 2.0 Gy 9 delivered to the head had median survival times of 86 and 79 days, respectively, compared to 47 days for untreated animals. Our results indicate that boron neutron capture therapy is effective against i.c. melanoma in a rodent model and suggest that large animal studies are warranted to further assess its efficacy.

Boron neutron capture therapy of brain tumors: enhanced survival following intracarotid injection of either sodium borocaptate or boronophenylalanine with or without blood-brain barrier disruption.

The purpose of the present study was to determine whether the efficacy of boron neutron capture therapy could be enhanced by means of intracarotid (i.c.) injection of sodium borocaptate (BSH) or boronophenylalanine (BPA) with or without blood-brain barrier disruption (BBB-D). For biodistribution studies, F98 glioma-bearing rats were injected i.v. or i.c. with either BSH (30 mg of boron/kg of body weight) or BPA (24 mg of boron/kg of body weight) with or without mannitol-induced, hyperosmotic BBB-D and killed 2.5 h later. The highest tumor boron concentrations for BSH and BPA were attained following i.c. injection with BBB-D (48.6 and 94.0 microg/g, respectively) compared to i.c. (30.8 and 42.7 microg/g) and i.v. injection (12.9 and 20.8 microg). Using the same doses of BSH and BPA, therapy experiments were initiated 14 days after intracerebral implantation of F98 glioma cells. Animals were irradiated 2.5 h after i.v. or i.c. administration of the capture agent with or without BBB-D using a collimated beam of thermal neutrons at the Brookhaven Medical Research Reactor. The median survival times of rats given BSH or BPA i.c. were 52 and 69 days, respectively, for rats with BBB-D; 39 and 48 days for rats without BBB-D; 33 and 37 days for i.v. injected rats; 29 days for irradiated controls; and 24 days for untreated controls. i.c. injection of either BSH or BPA resulted in highly significant enhancement (P = 0.01 and P = 0.0002, respectively) of survival times compared to i.v. injection, and this was further augmented by BBB-D (P = 0.02 and P = 0.04, respectively) compared to i.c. injection. Normal brain tissue tolerance studies were carried out with non-tumor-bearing rats, which were treated in the same way as tumor-bearing animals. One year after irradiation, the brains of these animals showed only minimal radiation-induced changes in the choroid plexus, but no differences were discernible between irradiated controls and those that had BBB-D followed by i.c. injection of either BSH or BPA. Our data clearly show that the route of administration, as well as BBB-D, can enhance the uptake of BSH and BPA, and, subsequently, the efficacy of boron neutron capture therapy.

Comparative studies of the preparation of immunoliposomes with the use of two bifunctional coupling agents and investigation of in vitro immunoliposome-target cell binding by cytofluorometry and electron microscopy.

The two coupling agents SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate) and SATA (N-succinimidyl-S-acetylthioacetate) were compared in their efficiency and feasibility to couple monoclonal antibodies (Abs) via thioether linkage to liposomes functionalized by various lipophilic maleimide compounds like N-(3-maleimidopropionyl)-N2-palmitoyl-L-lysine methyl ester (MP-PL), N-(3-maleimidopropionyl)phosphatidylethanolamide (MP-PE), N6-(6-maleimidocaproyl)-N2-palmitoyl-L-lysine methyl ester (EMC-PL), and N-(6-maleimidocaproyl)phosphatidylethanolamine (EMC-PE). The composition of the liposomes was soy phosphatidylcholine (SPC), cholesterol, maleimide compounds and alpha-tocopherol (1:0.2:0.02:0.01, mol parts), plus N4-oleylcytosine arabinoside (NOAC) as cytostatic prodrug (0.2 mol parts) and a new, lipophilic and highly fluorescent dye N,N'-bis(1-hexylhfetyl)-3,4:9,10-perylenebis(dicarboximid ) (BHPD, 0.006 mol parts). From the maleimide derivatives MP-PL was the most effective in terms of preservation of the coupling activity in dependence of liposome storage. The coupling of the monoclonal A B8-24.3 (mouse IgG2b, MHC class I, anti H-2kb) and IB16-6 (rat IgG2a, anti B16 mouse melanoma) to the drug carrying liposomes was more effective and easier to accomplish with SATA as compared to SPDP. Coupling rates of 60-65% were obtained with SATA at molar ratios of 12 SATA:1 Ab:40 maleimide spacer groups on the surface of one liposome. The highest coupling rates with SPDP were obtained at the ratio of 24 SPDP:1 Ab:40 liposomal maleimide groups, with an Ab binding efficiency of only 20-25%. The optimal in vitro binding conditions to specific target cells (EL4 for B8-24.3-liposomes and B16-F10 for IB16-6-liposomes) were determined by cytofluorometric measurement of the liposomal BHPD fluorescence with SATA linked Abs. Optimal immunoliposome binding to specific epitopes on the target cells was achieved with 1-2 Ab molecules coupled to one liposome, with immunoliposome concentrations of 20-130 nM and with a small incubation volume of 0.3-0.4 ml. The specificity of the binding of B8-24.3-liposomes to EL4 target cells was visualized by scanning electron microscopy. Antibody mediated endocytic uptake of immunoliposomes could be demonstrated by transmission electron microscopy.

The effect of epidermal growth factor receptor (EGFR) expression on in vivo growth of rat C6 glioma cells.

The discovery of EGFR gene amplification in glioblastoma multiforme has prompted interest in experimental therapies to target the receptor on brain tumor cells. To develop an animal model for in vivo study of such strategies, we transfected C6 glioma cells with a plasmid containing the neomycin resistance gene and the human EGFR gene under the control of the glucocorticoid-inducible MMTV promoter. Following selection with G418, individual clones that expressed EGFR at high levels were selected. Kinetics of EGF binding fit a dual site model indicating the presence of both high (KA = 2.5 x 10(9) M-1) and low (KA = 3.3 x 10(7) M-1) affinity receptors. To assess growth in vivo, graded numbers of either wild-type or transfected cells were implanted into the brains of CD Fischer 344 rats. No differences in survival were observed between groups of animals injected with either wild-type or transfected cells at inocula of 10(3) or 10(4) respectively. In addition, one-third of animals (7/21) challenged with 10(5) or 10(6) transfected cells survived > 50 days compared to 0% of animals (0/12) challenged with 10(5) or 10(6) wild-type cells. Such an effect suggests greater immunogenicity of transfected cells, but only at the larger inocula. Since C6 glioma cells will grow in both outbred and inbred strains, our model should have a number of applications including the in vivo study of EGFR targeting for glioma therapy.

Antigenic cross-reactivity between human leukemic cells and dinitrophenylated normal leukocytes.

Dinitrofluorobenzene-modified normal human leukocytes (DNP-LK) have been shown to evoke the production of antibodies, which following absorption with erythrocytes and unmodified leukocytes (UN-LK) had residual leukoagglutinating activity against human leukemic cells (HLC). In the present study we observed that rabbit antisera prepared against cell membrane extracts of DNP-modified granulocytes (DNP-GM) or lymphocytes (DNP-LM) were cytotoxic to HLC. By passive hemagglutination, these antisera were specifically reactive with DNP-GM or DNP-LM conjugated to sheep erythrocytes (SRBC) but not with DNP-modified bovine serum albumin (DNP-BSA) or bovine gamma globulin (DNP-BGG) nor with UN-LK, suggesting that the antisera were devoid of anti-DNP antibodies. Rabbit anti-DNP-BSA or anti-DNP-BGG failed to react with DNP-LK, and anti-DNP antibodies in these sera could not be absorbed by DNP-LK, suggesting that DNP groups either were not expressed on the surface of DNP-LK or were not detectable by these methods. These data, together with our recent finding that dinitrophenylation alters the expression of histocompatibility antigens, suggest that neoantigens cross-reactive with HLC antigens are induced by DNP modification of membrane antigens of normal leukocytes, and that the antibodies produced against these DNP-modified cells are directed mainly against the modified protein and not against the DNP moiety per se.

Increased in vitro and in vivo tumoricidal activity of a macrophage cell line genetically engineered to express IFN-gamma, IL-4, IL-6, or TNF-alpha.

Genetically engineered monocytes and macrophages may have potential as effector cells for the adoptive immunotherapy of cancer. As a first step, we have transfected the genes encoding either mouse interferon (IFN)-gamma, human interleukin (IL)-6, mouse IL-4, or mouse tumor necrosis factor (TNF)-alpha into the mouse macrophage cell line, J774A.1 cells using retroviral vectors. In vitro activation of J774A.1 cells by gene modification was assessed by morphological changes, proliferative activity was determined by [3H]-TdR uptake, and cytolytic activity was assessed using an 18-hour chromium-51 (51Cr) release assay. In vivo tumoricidal activity was studied by means of local adoptive immunotherapy using intratumoral injection of transfected effector cells. IFN-gamma gene-transfected J774A.1 [J7(IFN-gamma)] cells developed filamentous processes, increased doubling times, and enhanced tumoricidal activity against three tumor cell lines: the TNF-sensitive fibrosarcoma line WEHI 164 and the TNF-alpha-resistant cell lines B16 melanoma and C1300 neuroblastoma. IL-6-, TNF-alpha-, and IL-4-gene-transfected J774A.1 cells also had augmented tumoricidal activity but did not display any changes in morphology or growth. Cytolytic activity was markedly reduced after the addition of anti-TNF-alpha antibodies. Cytolytic J7(IFN-gamma) cells showed upregulated expression of TNF-alpha messenger RNA. After intratumoral injection of J7(IL-4) and J7(IFN-gamma) cell mixtures, 50% of established B16 melanomas were rejected by C57BL/6 mice, thereby demonstrating synergistic killing. Further studies on gene-transfected macrophages should better define their potential usefulness in tumor immunotherapy.

Sensitivity of B16 melanoma sublines to lymphokine-activated killer cells as determined by 51Cr-release and clonogenic assays.

The aim of this study was to compare the differential sensitivities of B16 melanoma sublines to LAK cells by means of the standard 51Cr release assay and a clonogenic assay, which measures both cell survival and proliferation. LAK cells, generated after 4 days incubation with 150 international units (IU)/ml of interleukin-2 (IL-2), showed both cytolytic and anti-proliferative activities against B16 targets. Using an 18 h 51Cr release assay, murine LAK cells showed the highest cytolytic activity against B16 parental cells compared to B16-F1, B16-F10, B16-FLR and B16-BL6 sublines at effector/target (E/T) ratios ranging from 6/1 to 100/1. Purified adherent LAK (A-LAK) cells showed greater cytolytic activity against B16 parental cells and other B16 sublines compared to LAK cells, but otherwise the pattern of reactivity was similar. Using a clonogenic assay, the surviving fraction of B16 parental cells co-cultivated with LAK cells decreased to 0 at an E/T ratio of 50/1, while a 400/1 ratio was required to achieve a similar reduction of B16-F1, B16-F10, B16-FLR, and B16-BL6 sublines. No differences in subline sensitivity were seen with the 51Cr release assay, but these were observed using the clonogenic assay. An inverse linear relationship existed between % surviving fraction, as determined by the clonogenic assay, and cytolytic activity, as determined by the 51Cr release assay. Our data indicate that the clonogenic assay can detect differences in target cell sensitivity that otherwise are undetectable by the standard 51Cr release assay. The clonogenic assay may prove useful in delineating the long-term anti-adherent and anti-proliferative properties of effector cells from their cytolytic activity.

Quantitation of glioma-reactive cytolytic T lymphocyte precursors by means of limiting dilution analysis.

The purpose of the present study was to define the usefulness of limiting dilution analysis (LDA) to enumerate glioma-reactive cytolytic T lymphocytes (CTL) as a constituent of tumor infiltrating lymphocytes (TIL) isolated from rat gliomas. Optimum LDA microculture conditions were defined by co-cultivating graded numbers of responder TIL together with 10(5) irradiated syngeneic rat splenocytes, 10(3) irradiated glioma cells, and 10 U/well of recombinant interleukin-2 incubated for 8 days. Antigenic specificity of the anti-tumor response was demonstrated by high levels of [3H]thymidine incorporation by TIL derived from F98 gliomas following stimulation with irradiated F98 glioma cells compared to low levels following stimulation with the antigenically distinct D74 glioma cells. Limiting dilution analysis showed that cytolytic T lymphocyte-precursors were present in TIL of F98 gliomas of immunized rats at an approximate frequency of 300 CTL/10(6) TIL, indicating that less than 1% of the TIL were tumor-reactive CTL. As determined by cell depletion experiments using various MoAbs and complement, the majority of the cytolytic activity detected against glioma targets was mediated by OX-8+ TIL. Split culture experiments revealed that high levels of glioma-reactive CTL activity and low levels of NK activity, which are simultaneously detected among TIL, were mediated by separate cell populations. Our data demonstrate that LDA microcultures can be used as a powerful tool to differentiate tumor-reactive CTL from other effector cell populations.

Giant cell myocarditis.

The present report describes a 45-year-old man with giant cell myocarditis who died of heart failure eight months after the onset of symptoms. On postmortem examination, the heart showed extensive myocardial fibrosis with numerous multinucleated giant cells. The lungs and a series of 20 lymph nodes showed no evidence of granulomatous disease, thereby excluding a diagnosis of sarcoidosis. Circumstantial evidence supports the view that giant cell myocarditis may have an autoimmune origin, and the histopathology suggests that cellular immune mechanisms might have a role in the pathogenesis of this disease. On this basis, it is suggested that cyclosporine, a selective inhibitor of T lymphocyte-mediated immune responses, may be useful for the treatment of this presently fatal disease.