A randomized, triple-masked, active-controlled investigation of the relative effects of dose, concentration, and infusion rate for continuous popliteal-sciatic nerve blocks in volunteers.

BACKGROUND: It remains unknown whether local anaesthetic dose is the only factor influencing continuous popliteal-sciatic nerve block effects, or whether concentration, volume, or both exert an influence as well. METHODS: Bilateral sciatic catheters were inserted in volunteers (n=24). Catheters were randomly assigned to ropivacaine of either 0.1% (8 ml h(-1)) or 0.4% (2 ml h(-1)) for 6 h. The primary endpoint was the tolerance to transcutaneous electrical stimulation within the tibial nerve distribution at hour 6. Secondary endpoints included current tolerance at other time points and plantar flexion maximum voluntary isometric contraction (22 h total). RESULTS: At hour 6, tolerance to cutaneous stimulation for limbs receiving 0.1% ropivacaine was [mean (standard deviation)] 27.0 (20.2) vs26.9 (20.4) mA for limbs receiving 0.4% [estimated mean difference 0.2 mA; 90% confidence interval (CI) -8.2 to 8.5; P=0.02 and 0.03 for lower and upper boundaries, respectively]. Because the 90% CI fell within the prespecified tolerance ±10 mA, we conclude that the effect of the two concentration/volume combinations were equivalent. Similar negative findings were found for the secondary outcomes. CONCLUSIONS: For continuous popliteal-sciatic nerve blocks, we found no evidence that local anaesthetic concentration and volume influence block characteristics, suggesting that local anaesthetic dose (mass) is the primary determinant of perineural infusion effects in this anatomic location. These findings suggest that for ambulatory perineural local anaesthetic infusion-for which there is usually a finite local anaesthetic reservoir-decreasing the basal rate while increasing the local anaesthetic concentration may allow for increased infusion duration without compromising postoperative analgesia. CLINICAL TRIAL REGISTRATION: NCT01898689.

alpha 2-macroglobulin production by cultured human melanoma cells.

alpha 2-Macroglobulin (alpha 2M) was identified in chemically defined spent medium of cultured human melanoma cells. This wide-spectrum protease inhibitor was subsequently shown to be synthesized and was secreted by 10 of 13 cultured melanoma cells but not by neoplastic cells of other histologic origins (0/5 breast, 0/2 colon, 0/2 lung, 0/2 glioblastomas, 0/1 neuroblastoma, 0/4 lymphoid). alpha 2M was also produced by one strain of fetal lung fibroblasts, as was previously shown, but not by two strains of normal fetal uveal melanocytes or two strains of fetal skin fibroblasts. Biochemical characterization of melanoma alpha 2M indicated that it has a similar tetrameric structure but is smaller in size than authentic serum alpha 2M. Preliminary studies indicated that melanoma alpha 2M can bind to protease and has possible functional capability.

Measurement of a monoclonal antibody-defined, melanoma-associated antigen in human sera: correlation of circulating antigen levels with tumor burden.

Quantities of a 100,000-mol wt human melanoma-associated antigen (MAA), defined by both monoclonal and polyclonal xenoantibodies, were measured in sera of 32 patients with malignant melanoma and 15 carefully documented healthy volunteers by use of an amplified sandwich enzyme-linked immunoabsorbent assay. Detectable levels of MAA were found in all normal adult sera. Five of 10 patients with stage III and 9 of 16 patients with stage IV disease had antigen levels that exceeded. the 95th percentile of serum levels in the normal donors (P less than .001). Elevated levels of the 100K MAA were associated with evidence of residual tumor (P less than .008): Only 3 of 13 patients with no evidence of tumor had abnormal values, whereas 9 of 16 patients with evident tumor had increased serum levels. Levels of the 100K MAA were unrelated to the levels of soluble immune complexes as detected by the fluid-phase C1q binding test. Molecular evaluation of the 100K MAA in serum indicated that the molecule associated with albumin in a strong, noncovalent manner. When the complex was dissociated by treatment with sodium dodecyl sulfate, the 100K MAA in serum comigrated with the molecule from spent culture medium of human melanoma cells. Antigen levels in sera were measured under these dissociating conditions. These studies suggest that measurement of the 100K MAA may be useful in monitoring tumor burden in patients with malignant melanoma.

Human melanoma cell lines showing striking inherent differences in sensitivity to immunotoxins containing holotoxins.

The in vitro sensitivity of human melanoma cell lines to conjugates of whole abrin or ricin linked through disulfide bonds to the monoclonal antimelanoma antibody 9.2.27 was studied. After passage of the conjugates through a Sepharose 4B column to remove molecular species with exposed binding sites on their B-chains, toxicity of the conjugates to different melanoma cell lines and nonmelanoma tumor lines was assessed by measuring their ability to inhibit cellular protein synthesis. The abrin conjugate was far more toxic to the target cells than the corresponding ricin conjugate. The 8 melanoma cell lines studied differed widely in their sensitivities to the abrin conjugate. The differences were associated with concomitant large differences in the sensitivities of the cells to the native toxins, and the significance of the level of the antigen expression became apparent only when the sensitivities of the different cell lines were normalized with respect to their sensitivity to native abrin. The observed relationship could not be accounted for by unspecific binding via the B-chain binding site of the immunotoxin. The differential sensitivity of the melanoma cell lines to the immunotoxin seems to be related to inherent differences between the cells in their ability to internalize and to process immunotoxins and toxins. The findings may have considerable practical implications.

Immunotoxins to a human melanoma-associated antigen: resistance to pokeweed antiviral protein conjugates in vitro.

Pokeweed antiviral protein (PAP) from the summer leaves of Phytolacca americana was purified and conjugated via N-succinimidyl-3-(2-pyridyldithio)propionate to 9.2.27 anti-melanoma antibody to a glycoprotein-proteoglycan complex. The conjugate was highly potent (50% inhibition dose of 5 X 10(-11)-10(-13) M on antigen-positive melanoma) and highly selective (5 X 10(-8) on antigen-negative melanoma). Human melanoma cells were selected for resistance to in vitro killing of the PAP conjugate by cycling through a killing and recovery sequence. Resultant cultures were shown to be more than 2 logs less sensitive to the killing of the PAP conjugate than untreated cultures. Isolation of clones by limiting dilution and reanalysis indicated that the resistant polyclonal culture contained clones with a range of sensitivities. Resistant cultures were also resistant to other A-chain conjugates of 9.2.27, but not to intact toxins like ricin and abrin. Resistant cultures showed no change in antigen expression after selection with the PAP conjugate of 9.2.27. Thus, just as with many other chemotherapeutic agents, tumor cells can become resistant to agents inhibiting protein synthesis even when targeted with monoclonal antibody. The mechanisms of this resistance and modalities to minimize resistance are currently being explored.

Immunohistochemical detection of the Ca antigen in normal and tumor tissues of humans by use of Ca1 monoclonal antibody.

The Ca1 monoclonal antibody was used to detect Ca antigen in paraffin sections with the use of a two-stage indirect immunoperoxidase technique. A range of normal and malignant human tissues was examined. The antibody reacted with squamous cell carcinomas, small cell carcinomas, adenocarcinomas, ductal carcinomas, and anaplastic carcinomas from gastrointestinal, lung, and breast tissues. Melanomas were negative. Reactivity was also observed against normal tissues: urinary transitional epithelium, fallopian tube epithelium, breast ducts, sweat glands, sebaceous glands, alveolar epithelium, bile duct, gallbladder, kidney tubules, and some digestive tract epithelial cells. Although the study confirms earlier reports that Ca1 is a marker for some normal, nonciliated epithelial cells and for epithelial and some other cancers, a more widespread distribution for normal tissues was found.

Dynamic interaction of 111indium-labeled monoclonal antibodies with surface antigens of solid tumors visualized in vivo by external scintigraphy.

Two 111indium-labeled murine monoclonal antibodies (MoAb), D3 and 9.2.27, directed to tumor antigens of L-10 hepatocarcinoma and human melanoma, respectively, selectively localized antigen-positive target cells in guinea pigs and nude mice. The fate of MoAb differed in the two antigen-antibody systems after reacting with their corresponding tumor antigens in vivo as reflected by patterns of distribution and turnover in vivo. The 9.2.27 localized in melanoma xenograft in nude mice after intravenous administration with slow loss from tumor but more rapid loss from normal tissues and thus demonstrated optimal imaging of small tumors (approximately equal to 5 mm) between 3 and 6 days after injection of the radiolabeled antibody. In contrast, D3 demonstrated a biphasic localization in guinea pig L-10 hepatocarcinoma with a maximal activity on the 2d day after administration and showed rapid loss from both tumor and normal tissues. Nonspecific localization of antibodies in liver and in kidney was found both in syngeneic (nude mice) and xenogeneic (guinea pig) hosts but was more pronounced in the xenogeneic species. These results indicate that the nature of the antigen-antibody interaction may be of importance in selecting MoAb for both diagnosis and therapy of malignant diseases.

Intratumor localization of monoclonal antibody in patients with melanoma treated with antibody to a 250,000-dalton melanoma-associated antigen.

Antibody localization at the tumor site was assessed in melanoma patients who received the murine monoclonal antibody 9.2.27. Antibody was administered twice weekly in escalating doses from 1 to 500 mg. Localization was assessed by biopsies of cutaneous and lymph node lesions obtained 24-96 hours following therapy. The percentage of tumor cells that bound the antibody in vivo was dose dependent, with similar findings obtained by either flow cytometry or immunoperoxidase staining techniques. Little or no in vivo binding of the 9.2.27 antibody to tumor cells was found following 1- and 10-mg doses, whereas all specimens demonstrated in vivo binding of the antibody following 200- and 500-mg doses. Fluorescence staining intensity, as quantitated by flow cytometry, was employed to determine the degree of in vivo saturation of antibody binding sites following therapy. The degree of saturation was found to vary substantially among patients: Some patients demonstrated nearly 100% saturation after 200-mg doses of 9.2.27 antibody, whereas others demonstrated only half maximal saturation after doses of 500 mg. Although immunoperoxidase staining provided important qualitative information regarding the distribution of antigen and antibody within the tumor, these studies demonstrated the usefulness of immunofluorescent flow cytometry for quantitative assessment of antibody localization in solid tumors and provided information necessary for the design of further trials of monoclonal antibodies and immunoconjugates.

Human neoplastic and normal cells in tissue culture. I. Cell lines derived from malignant melanomas and normal melanocytes.

Forty-six cell lines derived from 31 human melanomas obtained from 28 patients were cultured. Fourteen of 16 lines have produced malignant tumors when injected into nude (thymus-deficient) mice. Tumors in 5 of the nude mice metastasized to distant lymph nodes and/or to the lungs of the mouse host. Extreme variability from line to line was observed for doubling time (34 to 106 hr), plating efficiency (0-86%), and melanin production. All tested lines had type B glucose-6-phosphate dehydrogenase, thereby excluding HeLa cell contamination. HeLa cells have been grown for some time in our laboratory. Our results clearly demonstrated that HeLa cell contamination does not occur invariably in heteroploid lines growing in a laboratory simultaneously with Hela cells, provided that proper care is taken to avoid such occurrence. Multiple cell lines derived from the same tumor had identical phosphoglucomutase enzyme phenotype, which suggested a lack of significant cross-contamination between the lines. Four long-term cultures of normal human uveal embryo melanocytes have also been established and characterized. Although all produced melanin after reaching saturation density, they differed from the melanoma cells morphologically; they were flat, not refringent, and lacked piling up and plating ability. When melanoma cells were exposed to bromodeoxyuridine (BUDR) for long periods, a phenotypic change toward non-neoplastic characteristics was observed. Cells became flat and not refringent and, when injected into nude mice, tumors appeared after a long latent period. These changes were completely reversible in vitro and in vivo. The BUDR-treated cultures were undistinguishable from the untreated mother cultures after 2 to 3 passages. Lines derived from tumors in nude mice (obtained by injection of BUDR-treated cells) were again indistinguishable from the untreated mother line. Normal melanocytes were mostly euploid; all the melanoma cells were aneuploid. All 29 cell lines derived from 14 patients had an average chromosome number higher than 46. Detailed group-by-group chromosome analysis always showed an excess of C chromosomes, which suggested that hyperreduplication of one or more C chromosomes is a specific characteristic of human melanomas.

Murine monoclonal IgG3 antibodies to human colorectal tumor-associated antigens: production and characterization of antibodies active in both antibody-dependent cellular cytotoxicity and complement-mediated cytolysis.

Murine monoclonal antibodies have been developed against human colorectal tumors using immunogens consisting of extracts from xenografted human colon carcinoma bound to lectin-conjugated agarose beads. Antibodies of the IgG3 isotype were selected from the resulting fusions, since we and other investigators have reported that this class of antibody can mediate antibody-dependent cellular cytotoxicity (ADCC). They were characterized with respect to specificity, ADCC activity, and complement-mediated cytolytic activity. One antibody, NR-Co-01, was produced using wheat germ agglutinin-agarose, whereas NR-Co-03, NR-Co-04, and NR-Co-05 were independently derived after immunization with a Dolichos biflorus lectin-agarose carrier. All four antibodies reacted intensely with the cytoplasm and cell surface of colonic adenocarcinomas and showed a restricted normal tissue reactivity. Recognition of epithelial cells in some normal adult tissues was revealed by immunoperoxidase staining of frozen tissues. No binding was found to normal lymphocytes, monocytes, granulocytes, and erythrocytes by immunofluorescence flow cytometry. All four of the antibodies were reactive with neutral glycolipids partitioned by Folch extraction of human colon carcinoma cell lines and also with a glycoprotein extracted from ovarian cyst mucin, suggesting that they may recognize one or more carbohydrate structures. The NR-Co-01, NR-Co-03, NR-Co-04, and NR-Co-05 are potent inducers of ADCC with human peripheral blood mononuclear effector cells and of complement-mediated cytolysis with human complement. However, they exhibited no ADCC activity with nude or normal mouse peripheral blood mononuclear cells at target:effector ratios ranging from 40:1 to 160:1. Their tumor reactivity, restricted normal tissue distribution, and ability to direct ADCC and complement-mediated cytolysis make these antibodies promising candidates for cancer therapy.

Monoclonal antibodies to human melanoma-associated antigens: an amplified enzyme-linked immunosorbent assay for the detection of antigen, antibody, and immune complexes.

An amplified, indirect biotin-avidin micro-enzyme-linked immunosorbent assay was developed for the measurement of human melanoma-associated antigens, either free or circulating with associated immunoglobulin in patient sera. Parameters and specificity of detection were assessed using monoclonal antibody to human melanoma-associated antigens. The main advantages of the assay are its flexibility, through the use of indirect detection and a variety of formats, and its sensitivity, with a lower limit of antibody detection at 100 pg/well and a lower limit of soluble antigen detection at 10 pg/well. The assay was applied to cell surface antigen detection with monoclonal antibody 9.2.27 to a melanoma-associated antigen against a panel of glutaraldehyde-fixed cells, and gave similar binding specificity as assessed by a previous 125I-Protein A assay. Utilizing a unique "sandwich" format, aMr 100,000 melanoma-carcinoma-associated antigen was quantitated in melanoma patient sera and found highly elevated in Stage IV disease. The same sandwich format was also used to detect and determine the class of human immunoglobulin associated with circulating Mr 100,000 human melanoma-associated antigens in normal donor sera. Thus, the sensitivity and flexibility of this enzyme-linked immunosorbent assay system make it particularly suitable for numerous applications in the study of monoclonal antibody-defined tumor-associated antigens.

Selective lethal effect of supranormal temperatures on human neoplastic cells.

The effects of supranormal temperatures upon normal human cells (derived from normal adult and embryonal tissues) and neoplastic human cells (derived from biopsies of malignant tumors) have been quantitatively studied in tissue culture. Melanoma cells have been compared with melanocytes derived from fetal uveas. Colon carcinoma cells have been compared with embryonal intestinal epithelial cells. Malignant neuroepithelial cells derived from a teratocarcinoma of the ovary have been compared with neuroepithelial cells derived from fetal brain. Fibrosarcoma cells have been compared with normal adult fibroblasts. All cells defined as neoplastic have produced malignant tumors when injected into nude thymus-deficient mice at doses of 1 X 10(7) cells or less. Exposure to temperatures of 42.5-43.0 degrees for 4 to 8 hr has been shown to have significantly greater lethal effect on the tumor cells than on the nonneoplastic cells.

Radiolocalization of xenografted human malignant melanoma by a monoclonal antibody (9.2.27) to a melanoma-associated antigen in nude mice.

A murine monoclonal antibody (9.2.27), directed to a Mr 250,000 glycoprotein-chondroitan sulfate proteoglycan complex, was radiolabeled with 125I and assessed for radiolocalization in tumor and normal tissues of normal and tumor-bearing nude mice. The 125I-9.2.27 localized in vivo preferentially in Mr 250,000 antigen-expressing human melanomas (FMX-Met, SESX) but not in low antigen-expressing tumors (LOX-L) xenografted in nude mice. The imaging index of tumor cells was positively correlated with the antigen density of the various melanoma cell lines as measured by flow cytometry. The nonspecific immunoglobulin RPC-5 of the same IgG2a subclass as 9.2.27 did not specifically localize to xenografts of melanoma. The total amount of 125I-9.2.27 accumulated in the tumor was directly correlated with tumor size. However, the specific radioactivity (cpm/g) in smaller tumors was higher than that in larger tumors. Nonspecific uptake and circulating antibody levels differed between normals and tumor-bearers. The organs of the reticuloendothelial system of normal mice accumulated more labeled antibody than did those of tumor bearers, and conversely, tumor bearers had higher levels of circulating labeled antibody in the blood than normals. The circulating labeled antibody in tumor bearers was still monomeric but had no detectable antigen-binding capacity.

Murine monoclonal IgG3 to human colorectal tumor-associated antigens: enhancement of antibody-dependent cell-mediated cytotoxicity by interleukin 2.

Murine monoclonal antibodies (MAB) of the IgG3 subclass generated to colorectal tumor-associated glycolipids were assessed for enhancement of antibody-dependent cell-mediated cytotoxicity (ADCC) by interleukin 2 (IL-2). Mononuclear cell preparations containing large granular lymphocyte effectors required only low doses of IL-2 (0.1-10 units/ml) and short exposure (3 h) for maximal enhancement of ADCC. Exposure of mononuclear cells for longer periods (1-6 days) to higher levels of IL-2 (100-1000 units/ml) resulted in the generation of lymphokine-activated killer N cells which were lytically active without antibody. A non-ADCC MAB of the IgG1 subclass showed no ADCC even after stimulation of effector cells with IL-2. Effector cells pretreated with IL-2 showed an enhanced rate of cytolysis of target cells in the presence of antibody. Pretreatment of effector cells with IgG3 MAB resulted in lower ADCC, but pretreatment of target cells or simultaneous addition of MAB and effectors to target cells gave higher levels. These results indicated that ADCC with murine IgG3 is enhanced by levels of IL-2 achievable in current patient trials. The combination of antibody and IL-2-boosted effector cells gives comparable levels of killing to lymphokine-activated killer cells but should not suffer from similar toxicity. The NR-Co-04 antibody in combination with lymphokine may prove effective in treating colon cancer, a disease for which both chemotherapeutic and current biological therapies suggest a need for improved forms of therapies.

Immunotoxins to a human melanoma-associated antigen: comparison of gelonin with ricin and other A chain conjugates.

Gelonin, a ribosome-inactivating protein from the seeds of Gelonium multiflorum, has been conjugated to antibodies. Previous reports have indicated variable potency of such immunotoxins. The lack of toxicity of gelonin, however, makes it attractive for immunoconjugate production. The ribosome-inactivating protein was covalently linked (using N-succinimidyl-3-(2-pyridyldithio)propionate) to monoclonal antibody, 9.2.27, directed to a human melanoma-associated glycoprotein/proteoglycan. The immunoconjugate showed high selectivity with dose-dependent cytotoxic activity to cultured human melanoma cells (50% inhibitory dose; 1-3 X 10(-11) M versus antigen-positive cells; 1-3 X 10(-7) M versus antigen-negative cells). Specificity and immunoreactivity of the conjugate were similar to those of unconjugated antibody. Biodistribution studies with iodine trace-labeled conjugate in nude mice indicated that tumor localization of the gelonin conjugate was decreased compared to unconjugated antibody. However, a significant therapeutic effect of the conjugate was found with multiple but not single dose i.v. treatment in nude mice bearing established palpable melanoma. These in vivo experiments showed that gelonin conjugates are not toxic up to 2 mg total dose/mouse and significantly retarded the growth of established s.c. tumor. Comparison of gelonin conjugates in vitro and in vivo with other A-chain conjugates of 9.2.27 (abrin and ricin) indicated that gelonin had similar potency, better selectivity, better tumor localization, and more significant therapeutic effects.

Enhanced therapeutic efficacy of an immunotoxin in combination with chemotherapy against an intraperitoneal human tumor xenograft in athymic mice.

A mouse IgG2b anti-pan carcinoma monoclonal antibody, NR-LU-10, was shown to bind homogeneously to ascites xenografts of both ovarian and colon carcinoma. Following linkage to a highly potent holotoxin, Pseudomonas exotoxin A (PE), NR-LU-10 demonstrated high potency and selectivity in vitro (ID50 = 100 pg/ml; elimination of greater than or equal to 4.5 logs of cells). The conjugate was evaluated for therapeutic efficacy against a human colon tumor (HT-29) transplantable in the peritoneal cavity of nude mice. Beginning 3 days after HT-29 injection, mice received either three or six i.p. injections of 0.5 micrograms of unconjugated NR-LU-10 or immunotoxin conjugate (NR-LU-10/PE) every other day. Mice that received three or six treatments of NR-LU-10 alone had median survival times (MSTs) of 39 and 40 days, respectively, which did not differ significantly from the MST observed for the untreated control groups (MST = 35 days). In contrast, treatment with three or six injections of 0.5 micrograms NR-LU-10/PE exhibited significantly increased MSTs (P = 0.002) of 50 and 60 days, respectively. Coinjection of unconjugated NR-LU-10 (20 micrograms) and 0.5 micrograms of NR-LU-10/PE blocked the therapeutic effect of the immunotoxin (MST = 33 days). The therapeutic efficacy of NR-LU-10/PE was further enhanced against HT-29 when administered i.p. during and after cytoreductive chemotherapy. The i.p. administration of 300 mg/lg of cyclophosphamide plus 100 mg/kg of the chemoprotective drug, WR-2721, 10 and 17 days posttumor cell inoculation induced a significant increase in MST from 36 days to 59 days (P = 0.002). Interestingly, groups of mice that received either two, four, or seven treatments of NR-LU-10/PE following cytoreductive therapy exhibited a further significant increase (P = 0.001) in MSTs of 89, 97, and 105 days, respectively. Therefore, the use of immunotoxin therapy following cytoreductive chemotherapy significantly prolonged survival time of mice bearing the HT-29 colon tumor over that observed with chemotherapy or NR-LU-10/PE alone.

Human anti-murine immunoglobulin responses in patients receiving monoclonal antibody therapy.

Human anti-murine immunoglobulin responses were assessed in serum from three groups of patients receiving murine monoclonal antibody therapy. Each of the three patient groups responded differently. Chronic lymphocytic leukemia patients demonstrated little or no preexisting murine immunoglobulin G-reactive antiglobulin prior to treatment, while the cutaneous T-cell lymphoma and melanoma patients demonstrated preexisting antiglobulin levels in the same range as those demonstrated in healthy controls. None of 11 chronic lymphocytic leukemia patients receiving the T101 monoclonal antibody demonstrated an antiglobulin response, whereas all four of the cutaneous T-cell lymphoma patients receiving the same antibody developed increased levels of antiglobulins. Three of nine malignant melanoma patients receiving the 9.2.27 monoclonal antibody showed an increase in antiglobulin titers. In patients developing antiglobulin responses, the response was rapid, typically being detectable within 2 weeks. The antiglobulins were primarily immunoglobulin G and, with the exception of a single melanoma patient in whom the response appeared to have a substantial 9.2.27-specific component (i.e., antiidiotype), were cross-reactive with most murine immunoglobulin G preparations tested. This pattern of results suggested that the antiglobulin was a secondary immune reaction with elevation of the levels of preexisting antiglobulin which was cross-reactive with the mouse antibody administered. While the presence of serum antiglobulin would be expected to present major complications to monoclonal antibody therapy, no clinical toxicity related to antiglobulin responses was observed in these patients, and no inhibition of antibody localization on tumor cells was seen.

"Hidden" cytotoxic antibodies that react with allogeneic cultured fetal and tumor cells contained in soluble immune complexes from normal human sera.

Hidden complement-dependent cytotoxins were demonstrated in normal donor sera after removing anti-immunoglobulins that normally block the ability of these immunoglobulin Gs to react with surface antigens on tumor cells. The blocking antibodies had certain properties of anti-idiotypes. Immunoglobulin G from Cohn Fraction II, after removing these anti-immunoglobulins, was cytotoxic for cultured fetal cells and for malignant melanoma, breast, lung, colon, and other tumor cells maintained either in tissue culture or by serial passage in nude mice. The cytotoxins were not adsorbed by extracts from normal lymphoid, liver, skin, or red blood cells. These results suggest that a heterogeneous group of natural antibodies reactive with antigens expressed on a variety of neoplastic and fetal cells circulate in normal donor sera as part of a soluble immune complex, together with a blocking anti-immunoglobulin.

Selective elimination of breast cancer cells from human bone marrow using an antibody-Pseudomonas exotoxin A conjugate.

A pancarcinoma monoclonal antibody (NR-LU-10), homogeneously reactive with human breast cancer cells, was conjugated to Pseudomonas exotoxin A. The immunotoxin was evaluated for its potential for purging breast cancer cells from human bone marrow. The immunotoxin NR-LU-10 antibody did not react with normal bone marrow preparations yet readily detected 1% contamination of bone marrow by MCF-7 breast cancer cells added to normal bone marrow without significantly inhibiting the colony-forming ability of bone marrow progenitor cells. NR-LU-10-Pseudomonas exotoxin A has potential for purging bone marrow of breast cancer cells without impairing the growth of bone marrow progenitor cells.

Biodistribution, pharmacokinetic, and imaging studies with 186Re-labeled NR-LU-10 whole antibody in LS174T colonic tumor-bearing mice.

Biodistribution, pharmacokinetic, and radioimaging studies were performed with 186Re-labeled NR-LU-10 whole antibody in athymic nude mice bearing the LS174T tumor growing either s.c. or in an experimental hepatic metastasis model. NR-LU-10 is an IgG2b murine monoclonal antibody (MAb) that reacts with virtually all human tumors of epithelial origin. NR-BC-1, a IgG2b murine MAb that reacts with normal human B-cell and B malignancies, was used as an isotype-matched control. These MAbs were radiolabeled with 186Re (3.7-day physical half-life; 1.07-MeV beta particle and 137-keV gamma, 9% abundance) by a preformed chelate approach by using the triamide thiolate ligand system. 186Re-labeled NR-LU-10 (50 microCi) was injected into nude mice bearing LS174T tumors growing s.c. Biodistribution studies revealed that the LS174T tumor retained the highest concentration of 186Re-labeled NR-LU-10 (5.3% injected dose/g) at day 6. The tumor:blood ratio ranged from 0.1:1 to 10.8:1 by day 6, the last day of analysis. In contrast the tumor:blood ratio of 186Re-labeled NR-BC-1, the isotype-matched MAb control, was 1:1 on day 6. Pharmacokinetic analysis indicated that the t1/2 beta of NR-LU-10 for blood and other tissues ranged from 21 to 25 h, while the t1/2 beta for the LS174T tumor averaged 52 h. The area under the curve for tumor compared to blood was 2.8- to 5.7-fold higher than the area under the curve for all other tissues and organs. The mean residence time for NR-LU-10 in blood and all other organs ranged from 23 to 26 h, while the mean residence time for NR-LU-10 in the LS174T tumor was 72 h. Scintigraphic images revealed selective uptake of the 186Re-labeled NR-LU-10, but not of the 186Re-labeled NR-BC-1, at the LS174T tumor site. Studies in an experimental model of hepatic metastasis revealed a similar selective pattern of 186Re-labeled NR-LU-10 accumulation. Scintigraphic images of the LS174T tumor growing within the athymic nude mouse liver were obtained. The biodistribution, pharmacokinetic, and scintigraphic image results suggest that 186Re-labeled NR-LU-10 shows promise as a therapeutic agent for gastrointestinal cancer.