Micronucleus formation in human melanoma xenografts following exposure to hyperthermia

Summary The formation of micronuclei in two human melanoma xenografts (E. E. and V. N.) following hyperthermic treatment (42.5° C for 60 min) was studied and compared to that following single dose irradiation. The melanomas were grown in the hind leg of athymic mice and heated by immersing the tumour-bearing leg into a water-bath. Histological sections were prepared from tumours removed from the mice at predetermined times after treatment and the fraction of abnormal mitotic figures and the number of micronuclei per nucleus were scored. During the first 24 h after treatment, the fraction of abnormal mitotic figures increased abruptly to 90%–100% followed by a rapid decrease to 40%–50%. It then decreased slowly towards about twice the level in untreated tumours. The number of micronuclei started to increase at about the same time as the fraction of abnormal mitotic figures was highest, reached a maximum at about 2–3 days after treatment, and then decreased slowly. The number of micronuclei seen after the hyperthermic treatment was lower than that seen after radiation treatments causing similar tumour regrowth delays. The same hyperthermic treatment resulted in more micronuclei and larger regrowth delays for E. E. than for V. N. melanoma. The present results indicate that DNA damage is involved in heat-induced cell death in tumours treated in vivo.     

Regression of human melanoma xenografts in nude mice injected with methotrexate linked to monoclonal antibody 225.28 to human high molecular weight-melanoma associated antigen

Summary Intravenous injections into nude mice of 5 mg/kg methotrexate (MTX) linked to the antibody to human high molecular weight-melanoma associated antigen (HMW-MAA), monoclonal antibody (mAb) 225.28, an IgG_2a, on days 1, 4, 7, 10 and 14, starting 24 h after subcutaneous inoculation of 2 × 10⁶ cultured human M21 melanoma cells inhibited mean tumor volume by 90% on day 14 and by 65% on day 50 after the beginning of the treatment. Injections of equimolar amounts of free MTX and MTX linked to normal mouse IgG or to an isotypematched myeloma protein did not inhibit tumor growth significantly. MTX linked to mAb 225.28 did not inhibit the xenograft of a subline of human melanoma cell line M21 without detectable expression of HMW-MAA. In a clonogenic assay, the MTX-225.28 conjugate was three times more potent in inhibiting the growth of M21 melanoma cells than free MTX, but did not inhibit the growth of kidney carcinoma cells Caki-1, which do not express high-M _r MAA. In contrast, MTX linked to the mAb DAL K29, reacting with kidney carcinoma cells Caki-1, inhibited their growth but did not affect that of melanoma cells. M21 melanoma cells isolated from the residual tumor of a mouse treated with the MTX-225.28 conjugate did not differ in their reactivity with mAb 225.28 and in their sensitivity to MTX when compared with M21 cells from an untreated mouse.     

The transition from specific to nonspecific desensitization in human basophils

Human basophils can be desensitized to IgE-mediated stimuli either specifically (to the desensitizing antigen only) or nonspecifically (to all antigens). It has been suggested that the specificity of desensitization depends on the number of membrane-bound, antigen-specific IgE antibody molecules per basophil. We have varied the number of IgE antibody molecules/basophil by passive sensitization of mixed leukocyte preparations with increasing concentrations of purified IgE anti-penicillin (BPO) antibody. The cells were then desensitized with penicillin-human serum albumin (BPO-HSA). Desensitization was specific (lack of response to BPO-HSA only) with 1000 specific antibody molecules/basophil, and increasingly nonspecific (greater than 70% desensitization to rechallenge with anti-IgE and ragweed antigen E as well as lack of response to BPO-HSA) as the number of antibody molecules was increased to 14,000. This formally established that the number of specific IgE antibody molecules/basophil determines the mode of desensitization.     

Quantitative autoradiographic evaluation of the influence of protein dose on monoclonal antibody distribution in human ovarian adenocarcinoma xenografts

Summary We studied the effect of monoclonal antibody protein dose on the uniformity of radioiodinated antibody distribution within tumor masses using quantitative autoradiography. Groups (n = 11–13/group) of athymic nude mice with subcutaneous HTB77 human ovarian carcinoma xenografts were injected intraperitoneally with an¹²⁵I-labeled anticarcinoma-associated antigen murine monoclonal antibody, 5G6.4, using a high or a low protein dose (500 µg or 5 µg). At 6 days post-injection the macroscopic and microscopic intratumoral biodistribution of radiolabeled antibody was determined. The degree of heterogeneity of the labeled antibody distribution within each tumor was quantified and expressed as thecoefficient of variation (CV) of the activity levels in serial histological sections. Tumors from mice given the 500-µg protein doses had substantially lower CV values, 0.327±0.027, than did tumors from animals given 5-µg protein doses, 0.458±0.041, (P = 0.0078), indicating that the higher protein dose resulted in more homogeneous distribution of radioactivity in tumors than did the lower dose. While the percentage of the injected dose reaching the tumor was comparable between groups, injecting the higher dose of protein resulted in significantly lower tumor to non-tumor uptake ratios than those obtained for the lower protein dose. These data indicate, in this system, that to achieve more uniform intratumoral antibody (and radiation for radioimmunotherapy) delivery, a relatively high protein dose must be administered. However, to obtain this increased uniformity, a substantial drop in tumor/background uptake ratios was seen. Quantitative autoradiographic evaluation of human tumor xenografts is a useful method to assess the intratumoral distribution of antibodies.     

Tropomyosin antibody: the specific localization of tropomyosin in nonmuscle cells

An antibody against purified chicken skeletal muscle tropomyosin is used in indirect immunofluorescence to visualize the localization of tropomyosin in a variety of nonmuscle cells. The antibody produces a fluorescent pattern which is very similar to that obtained with an actin-specific antibody. This pattern is composed of fluorescent fibers which are shown to be coincident with the fibers seen with phase- contrast optics. High resolution epifluorescent microscopy reveals that fibers stained with the actin antibody show a continuous fluorescence, while fibers reacted with the tropomyosin antibody show a periodic fluorescence. Measurements indicate that the lengths of the fluorescent segments are variable with an average of 1.2 mum while the spacing between segments is approximately 0.4 mum.     

Development of specific adsorbents for human tumor necrosis factor-alpha: influence of antibody immobilization on performance and biocompatibility

To develop adsorbents for the specific removal of tumor necrosis factor-alpha (TNF) in extracorporeal blood purification, cellulose microparticles were functionalized either with a monoclonal anti-TNF antibody (mAb) or with recombinant human antibody fragments (Fab). The TNF binding capacity of the adsorbents was determined with in vitro batch experiments using spiked human plasma (spike: 1200 pg TNF/mL; 1 mg particles in 250 muL plasma). Random immobilization of the full-sized monoclonal antibody to periodate-activated cellulose yielded particles with excellent adsorption capacity (258.1 +/- 48.6 pg TNF per mg adsorbent wet weight). No leaching of antibody was detectable, and the adsorbents retained their activity for at least 12 months at 4 degrees C. We found that the conditions used during immobilization of the antibody (pH, nature of the reducing agent) profoundly influenced the biocompatibility of the resulting adsorbents, especially with respect to activation of the complement system. Particles obtained by random immobilization of the monovalent Fab fragments on periodate-activated cellulose using the same conditions as for immobilization of the mAb exhibited only low adsorption capacity (44 +/- 7 pg/mg adsorbent wet weight). Oriented coupling of the Fab fragments on chelate-epoxy cellulose via a C-terminal histidine tag, however, increased the adsorption capacity to 178.3 +/- 8.6 pg TNF/mg adsorbent wet weight. Thus, in the case of small, monovalent ligands, the orientation on the carrier is critical to retain full binding activity.     

Specific localization of In-111-labeled monoclonal antibody versus 67-Ga-labeled immunoglobulin in mice bearing human breast carcinoma xenografts

Summary A murine monoclonal antibody reacting with more than 95% of all breast cancers was radiolabeled with In-111 and injected IP into nude mice bearing human breast carcinoma xenografts, together with Ga-67-labeled normal mouse immunoglobulin. Images were produced with a gamma camera in dual isotope mode. Tumors could be localized clearly with In-111-labeled specific monoclonal antibody, but improved visualization was obtained after computer-assisted subtraction of the image with Ga-67-labeled nonspecific immunoglobulin. The tumor-to-tissue contrast was improved from 2.3 to 5.9 after subtraction. Imaging with In-111-radiolabeled monoclonal antibody was superior to imaging with iodinated antibody. For the first time it was shown that images of two chemically related isotopes, Ga-67 and In-111, coupled to nonspecific and specific antibody, respectively, and simultaneously injected, can be subtracted to show the preferential uptake of the specific antibody in the tumor. As these isotopes are routinely used in clinical practice this technique may prove to be more practical for immunodetection of tumors in patients than existing imaging techniques.     

IFN-beta pretreatment sensitizes human melanoma cells to TRAIL/Apo2 ligand-induced apoptosis

All human melanoma cell lines (assessed by annexin V and TUNEL assays) were resistant to apoptosis induction by TRAIL/Apo2L protein. TRAIL/Apo2L activated caspase-8 and caspase-3, but subsequent apoptotic events such as poly(ADP-ribose) polymerase cleavage and DNA fragmentation were not observed. To probe the molecular mechanisms of cellular resistance to apoptosis, melanoma cell lines were analyzed for expression of apoptosis regulators (apoptotic protease-associated factor-1, FLIP, caspase-8, caspase-9, caspase-3, cellular inhibitor of apoptosis, Bcl-2, or Bax); no correlation was observed. TRAIL/Apo2L was induced in melanoma cell lines by IFN-beta and had been correlated with apoptosis induction. Because IFN-beta induced other gene products that have been associated with apoptosis, it was postulated that one or more IFN-stimulated genes might sensitize cells to TRAIL/Apo2L. Melanoma cell lines were treated with IFN-beta for 16-24 h before treatment with TRAIL/Apo2L. Regardless of their sensitivity to either cytokine alone, >30% of cells underwent apoptosis in response to the combined treatment. Induction of apoptosis by IFN-beta and TRAIL/Apo2L in combination correlated with synergistic activation of caspase-9, a decrease in mitochondrial potential, and cleavage of poly(ADP-ribose) polymerase. Cleavage of X-linked inhibitor of apoptosis following IFN-beta and TRAIL/Apo2L treatment was observed in sensitive WM9, A375, or WM3211 cells but not in resistant WM35 or WM164 cells. Thus, in vitro IFN-beta and TRAIL/Apo2L combination treatment had more potent apoptotic and anti-growth effects when compared with either cytokine alone in melanoma cells lines.     

Altered biodistribution of indium-111-labeled monoclonal antibody 96.5 to tumors and normal tissues of nude mice bearing human melanoma xenografts in visceral organs

Summary Human melanoma xenografts were produced in the subcutis, kidney, cecum and liver of different nude mice. An¹¹¹In-labeled anti-(human melanoma) monoclonal antibody (96.5) or an¹¹¹In-labeled nonspecific control monoclonal antibody (ZCE-025) was injected intravenously in separate groups of mice. Radioactive antibody accumulation was measured in tumor, blood, viscera, and carcasses. mAb 96.5 targeted specifically to tumor tissue regardless of site of growth. Tumors in the liver exhibited significantly (P <0.05) higher tumor-to-blood ratios (45±6, mean ±SEM) than xenografts at other visceral organs, the lowest value being found for subcutaneous melanoma (2.6±0.5). The differences in tumor-to-blood ratio were due to significant alterations of antibody biodistribution, since the actual antibody concentration in the different tumor sites was similar. The percentage of recovered anti-melanoma antibody per milliliter of blood in mice with visceral lesions (4.6±1.1%/ml) was significantly lower than that found in mice with subcutaneous tumors (9.5±1.4%/ml,P <0.05). Moreover, significantly higher levels (18.2±3.2%/g, 31.0±5.1%/g, respectively) of the melanoma mAb 96.5 were found in normal liver and spleen tissue recovered from mice with visceral tumors as compared to tissue from mice with subcutaneous tumors (9.2±0.9%/g, 13.5±1.9%/g, respectively;P <0.05). These results demonstrate that the presence of visceral tumor can significantly affect tumor-to-blood ratios, blood levels, and biodistribution of¹¹¹In-labeled mAb 96.5.This work was supported in part by funds from the National Institutes of Health, National Cancer Institute, Grant R35-CA42107 and Core Grant CA16672     

Antitumor effects of a monoclonal antibody to human CCR9 in leukemia cell xenografts

Tumor expression of certain chemokine receptors is associated with resistance to apoptosis, migration, invasiveness and metastasis. Because CCR9 chemokine receptor expression is very restricted in healthy tissue, whereas it is present in tumors of distinct origins including leukemias, melanomas, prostate and ovary carcinomas, it can be considered a suitable candidate for target-directed therapy. Here, we report the generation and characterization of 91R, a mouse anti-human CCR9 IgG2b monoclonal antibody that recognizes an epitope within the CCR9 N-terminal domain. This antibody inhibits the growth of subcutaneous xenografts from human acute T lymphoblastic leukemia MOLT-4 cells in immunodeficient Rag2^−/− mice. Tumor size in 91R-treated mice was reduced by 85% compared with isotype-matched antibody-treated controls. Tumor reduction in 91R-treated mice was concomitant with an increase in the apoptotic cell fraction and tumor necrotic areas, as well as a decrease in the fraction of proliferating cells and in tumor vascularization. In the presence of complement or murine natural killer cells, 91R promoted in vitro lysis of MOLT-4 leukemia cells, indicating that this antibody might eliminate tumor cells via complement- and cell-dependent cytotoxicity. The results show the potential of the 91R monoclonal antibody as a therapeutic agent for treatment of CCR9-expressing tumors.     

Antitumor effects of a monoclonal antibody to human CCR9 in leukemia cell xenografts

Tumor expression of certain chemokine receptors is associated with resistance to apoptosis, migration, invasiveness and metastasis. Because CCR9 chemokine receptor expression is very restricted in healthy tissue, whereas it is present in tumors of distinct origins including leukemias, melanomas, prostate and ovary carcinomas, it can be considered a suitable candidate for target-directed therapy. Here, we report the generation and characterization of 91R, a mouse anti-human CCR9 IgG2b monoclonal antibody that recognizes an epitope within the CCR9 N-terminal domain. This antibody inhibits the growth of subcutaneous xenografts from human acute T lymphoblastic leukemia MOLT-4 cells in immunodeficient Rag2^?/? mice. Tumor size in 91R-treated mice was reduced by 85% compared with isotype-matched antibody-treated controls. Tumor reduction in 91R-treated mice was concomitant with an increase in the apoptotic cell fraction and tumor necrotic areas, as well as a decrease in the fraction of proliferating cells and in tumor vascularization. In the presence of complement or murine natural killer cells, 91R promoted in vitro lysis of MOLT-4 leukemia cells, indicating that this antibody might eliminate tumor cells via complement- and cell-dependent cytotoxicity. The results show the potential of the 91R monoclonal antibody as a therapeutic agent for treatment of CCR9-expressing tumors.     

Monoclonal antibody specific for carbodiimide-fixed glutamate: immunocytochemical localization in the rat CNS

Glutamate is widely distributed in the central nervous system (CNS) and is present in greater amounts than any other putative neurotransmitter. To study its distribution in the CNS, a monoclonal antibody was raised against gamma-L-glutamyl-L-glutamic acid (gamma-Glu-Glu) conjugated to keyhole limpet hemocyanin (KLH) using glutaraldehydeborohydride. By use of this antibody, indirect immunoperoxidase staining was observed in CNS tissue fixed with carbodiimide to form gamma-Glu-Glu from glutamate and post-fixed with glutaraldehyde or paraformaldehyde. In contrast, immunoreactivity was quite low in tissues fixed only with glutaraldehyde. Absorption controls indicated that the staining of carbodiimide-fixed tissue could be inhibited by micromolar concentrations of gamma-Glu-Glu but not by other small molecules. Using ELISA, the antibody reacted strongly with the gamma-Glu-Glu/KLH conjugate used to immunize the mouse, but not with other small molecules conjugated to KLH. The reactivity of the antibody with the gamma-Glu-Glu/KLH conjugate on ELISA was inhibited by free gamma-Glu-Glu in micromolar concentrations, but not by similar dipeptides or amino acids. Dense immunocytochemical staining was observed in cortical pyramidal cells, cerebellar granule cells, and the cochlear nuclei. Staining with this monoclonal antibody correlated well with other methods of localizing glutamate in the CNS.     

Neutralizing monoclonal antibody specific for alpha-bungarotoxin: preparation and characterization of the antibody, and localization of antigenic region of alpha-bungarotoxin

We prepared an alpha-bungarotoxin-specific monoclonal antibody that neutralizes the biological activity of the toxin in vivo. The antigenic determinant combining specifically with this antibody was determined on the basis of cross-reaction experiments using three other long neurotoxins and peptide fragments of alpha-bungarotoxin. The antigenic determinant was located on the peptide fragment containing S34-S35-R36-G37-K38, which forms a part of the expected site that binds to the acetylcholine receptor proteins.     

Phenotypic and functional changes of human melanoma xenografts induced by DNA hypomethylation: immunotherapeutic implications

Emerging in vitro evidence points to an immunomodulatory activity of DNA hypomethylating drugs in human malignancies. We investigated the potential of 5-aza-2'-deoxycytidine (5-AZA-CdR) to modulate the expression of cancer testis antigens (CTA) and of HLA class I antigens by melanoma xenografts, and the resulting modifications in immunogenicity of neoplastic cells. Three primary cultures of melanoma cells, selected for immune phenotype and growth rate, were grafted into BALB/c nu/nu mice that were injected intraperitoneally with different dose- and time-schedules of 5-AZA-CdR. Molecular analyses demonstrated a de novo long-lasting expression of the CTA MAGE-1, -2, -3, -4, -10, GAGE 1-6, NY-ESO-1, and the upregulation of MAGE-1, MAGE-3, and NY-ESO-1 levels in melanoma xenografts from 5-AZA-CdR-treated mice. Serological and biochemical analyses identified a de novo expression of NY-ESO-1 protein and a concomitant and persistent upregulation of HLA class I antigens and of HLA-A1 and -A2 alleles. Immunization of BALB/c mice with 5-AZA-CdR-treated melanoma cells generated high titer circulating anti-NY-ESO-1 antibodies. Altogether, the data obtained identify an immunomodulatory activity of 5-AZA-CdR in vivo and strongly suggest for its clinical use to design novel strategies of CTA-based chemo-immunotherapy for melanoma patients.     

The specificity of cross-reactivity: promiscuous antibody binding involves specific hydrogen bonds rather than nonspecific hydrophobic stickiness

Proteins are renowned for their specificity of function. There is, however, accumulating evidence that many proteins, from enzymes to antibodies, are functionally promiscuous. Promiscuity is of considerable physiological importance. In the immune system, cross‐reactive or multispecific antibodies are implicated in autoimmune and allergy conditions. In most cases, however, the mechanism behind promiscuity and the relationship between specific and promiscuous activities are unknown. Are the two contradictory? Or can a protein exhibit several unrelated activities each of which is highly specific? To address these questions, we studied a multispecific IgE antibody (SPE7) elicited against a 2,4‐dinitrophenyl hapten (DNP). SPE7 is able to distinguish between closely related derivatives such as NP (nitrophenol) and DNP, yet it can also bind a number of unrelated ligands. We find that, like DNP, the cross‐reactants are themselves bound specifically—close derivatives of these cross‐reactants show very low or no binding to SPE7. It has been suggested that cross‐reactivity is simply due to “hydrophobic stickiness”, nonspecific interactions between hydrophobic ligands and binding sites. However, partitioning experiments reveal that affinity for SPE7 is unrelated to ligand hydrophobicity. These data, combined with crystal structures of SPE7 in complex with four different ligands, demonstrate that each cross‐reactant is bound specifically, forming different hydrogen bonds dependant upon its particular chemistry and the availability of complementary antibody residues. SPE7 is highly homologous to the germline antinitrophenol (NP) antibody B1–8. By comparing the sequences and binding patterns of SPE7 and B1–8, we address the relationship between affinity maturation, specificity, and cross‐reactivity.     

A human monoclonal antibody specific to placental alkaline phosphatase,a marker of ovarian cancer

Placental alkaline phosphatase (PLAP) is a promising ovarian cancer biomarker. Here, we describe the isolation, affinity-maturation and characterization of two fully human monoclonal antibodies (termed B10 and D9) able to bind to human PLAP with a dissociation constant (K_d) of 10 and 30 nM, respectively. The ability of B10 and D9 antibodies to recognize the native antigen was confirmed by Biacore analysis, FACS and immunofluorescence studies using ovarian cancer cell lines and freshly-frozen human tissues. A quantitative biodistribution study in nude mice revealed that the B10 antibody preferentially localizes to A431 tumors, following intravenous administration. Anti-PLAP antibodies may serve as a modular building blocks for the development of targeted therapeutic products, armed with cytotoxic drugs, radionuclides or cytokines as payloads.     

Growth characteristics of human melanoma multicellular spheroids in liquid-overlay culture: comparisons with the parent tumour xenografts

The growth characteristics of multicellular spheroids, derived from human melanoma xenografts and cultivated in liquid-overlay culture, were studied and compared with those of the parent tumours. Six of the seven melanomas investigated formed spheroids, which grew exponentially up to a volume of 1-2 X 10(7) microns 3 (a diameter of 270-340 microns) before the growth rate tapered off. The morphology of the spheroids varied considerably among the melanomas; some spheroids grew as densely packed, spherical structures of cells whereas others were loosely packed and showed an irregular shape. Central necrosis developed when the spheroids attained a diameter of 150-200 microns. The histological and cytological appearance of the spheroids was remarkably similar to that of the parent xenograft in five of the six cases. The sixth melanoma contained two subpopulations with distinctly different DNA content, one of which was predominant in the spheroids, the other in the tumours. This gave rise to clear histological and cytological differences. The volume-doubling time of the spheroids during the exponential growth phase ranged from 1.7 +/- 0.2 to 2.7 +/- 0.4 days and the fraction of cells in S from 13 +/- 1 to 28 +/- 2%. The volume-doubling time decreased with increasing fraction of cells in S, indicating that the differences in growth rate were due mainly to differences in the growth fraction or to differences in the duration of G1. The spheroid volume-doubling times did not correlate with those of the parent xenografts (Td = 4.2-22.5 days at V = 200 mm3), possibly because the cell loss factors of the xenografts were large and varied among the melanomas. The fractions of cells in G1/G0, S and G2 + M in the spheroids and the xenografts did not correlate either, but were found to be within the same narrow ranges in the spheroids and the xenografts--i.e. 50-80% (G1/G0), 10-30% (S) and 10-20% (G2 + M).