Tumor necrosis treatment of ME-180 human cervical carcinoma model with 131I-labeled TNT-1 monoclonal antibody

In contrast to normal tissues, many malignant tumors contain a high proportion of dead and dying cells. The loss of membrane integrity that accompanies cellular degeneration permits macromolecules, including antibodies, to freely enter the cell cytoplasm. Based upon these observations, it was hypothesized that monoclonal antibodies to intracellular antigens, which are integral structural components and are retained by degenerating cells, may be used to target a wide range of human malignancies. Previous studies by our laboratory utilizing these principles have demonstrated the feasibility of imaging four different histological types of human cancer in a nude mouse model, using monoclonal antibodies directed against insoluble intranuclear antigens. The present study describes the application of this approach, designated tumor necrosis treatment, for the radioimmunotherapy of transplantable ME-180 human cervical carcinomas in the nude mouse. Groups of tumor-bearing nude mice received three weekly treatments of 150 or 300 microCi of 131I-labeled experimental (TNT-1) or control (Lym-1) monoclonal antibodies. Detailed biodistribution data, dosimetric evaluations, and therapeutic results are presented to demonstrate the effective and preferential targeting of 131I-labeled TNT-1 monoclonal antibody within the tumor. In the experimental groups, the dose delivered to the tumor was sufficient to induce clinical regressions in 88% of treated animals, without evidence of toxicity to normal tissues. Complete regressions were obtained in 25% of the mice treated with high dose TNT-1. Microscopic examination of the implantation sites of these mice demonstrated the presence of acute radiation damage and residual keratin-positive tumor cells showing marked evidence of degeneration. Dosimetric data obtained over the 3-week treatment period showed that, unlike control treated mice, which received approximately 500 cGy each week, the experimental animals received increasing doses of radiolabeled antibody with each treatment (averages for weeks 1, 2, and 3: 1066, 2046, and 2476 cGy, respectively). In accordance with these data, enhanced imaging and therapeutic responses were observed with each therapeutic dose in the TNT-1-treated groups, compared with controls. These results indicate that TNT-1 therapy produces an ever expanding population of TNT-1-positive targets in the tumor as a result of the centrifugal killing of adjacent viable tumor cells. To help illustrate these results, a four-compartment model of the dose distribution kinetics of TNT-1 is presented for discussion with respect to the possible application of this method for the imaging and treatment of cancer in     

In vitro toxicity of Pt-labeled cisplatin to a human cervical carcinoma cell line (ME-180)

Purpose: The aim of the present work was to examine the effect of ¹⁹¹Pt-cisplatin, and to study the manner in which radiation and cisplatin interact, in a human cervical carcinoma cell line (ME-180).

Methods and Materials: The cells were incubated for 1 hour with nonradioactive cisplatin or ¹⁹¹Pt-cisplatin with specific activities in the range 48–167 MBq/mg. The surviving fraction of the cells after 7 days’ growth was determined with a nonclonogenic tetrazolium-based (MTT) assay. The uptake of platinum into the cell and the amount of platinum bound to DNA was measured.

Results: The 50% inhibition concentration (IC₅₀) decreased with increasing specific activity of the ¹⁹¹Pt-cisplatin. For the specific activities 0 (nonradioactive), 48, 89, 143, 157, and 167 MBq/mg, IC₅₀ was found to be 3.24 ± 0.08, 2.77 ± 0.55, 2.17 ± 0.34, 1.15 ± 0.04, 1.02 ± 0.03, and 0.76 ± 0.13 respectively. Isobologram analysis showed a supra-additive (synergistic) interaction between the radiotoxicity and chemotoxicity for specific activities over 100 MBq/mg.

Conclusion: The cytotoxic effect of cisplatin may be enhanced by labeling the drug with the radionuclide ¹⁹¹Pt.     

Doxorubicin: monoclonal antibody conjugate for therapy of human cervical carcinoma.

Doxorubicin (Dox) was conjugated via a dextran linker to the F(ab')2 fragment of monoclonal antibody (MAb) 1H10 which recognizes an antigen expressed on the surface of human cervical carcinoma cells and tissues. Drug-antibody conjugates (1H10-Dox) with a molar ratio of Dox to MAb ranging from 40:1 to 60:1 retained antigen-binding and pharmacological activities. Anti-tumor activity of the conjugate in vitro was evaluated by measuring inhibition of [5-3H]-uridine incorporation into cellular RNA. 1H10-Dox was found to be 30 times more toxic to cervical tumor cells than a control MAb-Dox conjugate and 150 times more potent than Dox coupled to dextran. In addition, 1H10-Dox was less toxic to antigen-negative cells in vitro, suggesting that 1H10-Dox killing of cervical carcinoma cells was antibody-mediated. 125I-labeled 1H10-Dox preferentially localized in solid human cervical carcinoma xenografts in athymic mice with tumor-to-blood ratios of 1H10-Dox reaching 17.9 after 24 hr and 32.8 after 48 hr. Treatment of athymic mice bearing human cervical tumors with 1H10-Dox resulted in a dose-dependent inhibition of tumor growth. Multiple administrations of 1H10-Dox at a dose corresponding to 20 micrograms doxorubicin significantly suppressed the growth of human cervical tumors in nude mice without significant side effects (weight loss), and this suppression was antibody specific. Both i.p. and i.v. administration of 1H10-Dox were found to be equally effective. Our results suggest that 1H10-Dox may be useful for the treatment of human cervical carcinoma.     

Augmentation of antibody dependent cell mediated cytotoxicity following in vivo therapy with recombinant interleukin 2

Monoclonal antibodies (mAB) with tumor specificity are able to enhance the immunological specificity of interleukin 2 (IL-2)-activated lymphokine activated killer (LAK) cells. Antibodies may also be used to broaden the range of tumor types susceptible to immune mediated cytotoxicity by the activated LAK cells. In these studies, mAB with relative tumor specificity were used to target immunologically activated effector cells in an in vitro antibody dependent cell mediated cytotoxicity (ADCC) assay. The mAB included: 3F8 and 14.G2a, which are both specific for neuroblastoma and melanoma and recognize ganglioside GD2, and mAB ING-1, a mouse-human chimeric antibody with constant regions from human IgG1 and kappa chains and variable regions from a mouse mAB that binds to a broad range of human adenocarcinomas. Each of these mAB was able to mediate ADCC with fresh effector cells and antibody binding targets. When peripheral blood mononuclear cells were obtained from cancer patients prior to and following in vivo therapy with interleukin 2, a significant increase was noted in ADCC activity by peripheral blood mononuclear cells obtained following IL-2 therapy. Inclusion of IL-2 in the medium during the cytotoxic assay with mAB further boosted ADCC. The total activity seen was often greater than the sum of the independent LAK activity and standard ADCC activity. The cells responsible for this ADCC had the CD16+ Fc receptor. Combining IL-2 with mAB in clinical tumor therapy may lead to a wider range of tumor types being responsive to immunotherapy and may also enhance the efficacy of therapy by specifically targeting activated effector cells to tumor cells recognized by mAB. Our results provide strong support for the testing of these hypotheses in clinical trials by combining in vivo treatment with IL-2 and mAB able to mediate ADCC.     

Cellular cytotoxicity mediated by isotype-switch variants of a monoclonal antibody to human neuroblastoma

The biological property of an antibody is determined by its antigen binding characteristics and its isotype-related effector functions. We have established monoclonal antibodies of different isotypes by stepwise selection and cloning of the hybridoma CE7. The original CE7 secretes an IgG1/kappa (CE7 gamma 1) antibody that recognises a 185 kD cell surface glycoprotein expressed on all human sympatho-adrenomedullary cells. Isotype-switch variants were isolated in the following sequence: from the original CE7 gamma 1, CE7 gamma 2b variants were isolated, and from a CE7 gamma 2b variant CE7 gamma 2a variants were isolated. The antibodies of three different isotype variant cell lines possess identical antigen binding characteristics, but display distinct effector functions as demonstrated by antibody dependent cell-mediated cytotoxicity (ADCC). ADCC was performed with the neuroblastoma line IMR-32 as the target cells, and different FcR gamma positive cells were either freshly isolated from human peripheral blood leukocytes or cultured for 6-10 days and tested as potential effector cells. Tumour lysis mediated by monocyte-derived macrophages depended on the presence of CE7 gamma 2a antibodies; antibodies from the CE7 hybridomas of gamma 2b and gamma 1 isotypes were virtually inactive in ADCC assay. Pre-exposure of macrophages to rIFN-gamma enhanced their ADCC activity, a result that is compatible with the notion that the high affinity Fc IgG receptor (FcR gamma I/CD64) is involved in the triggering of ADCC in macrophages. In contrast to macrophages, mononuclear cells, nonadherent cells and monocytes displayed considerable non-specific lytic activity, which was little influenced by the presence of antibody regardless of the isotype added.     

Antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC) of breast cancer cells mediated by bispecific antibody, MDX-210

Background: MDX-210 is a bispecific antibody (BsAb) with specificity for both the proto-oncogene product of HER-2/neu (c-erbB-2) and FcRI (CD64). HER-2/neu is overexpressed in malignant tissue of approximately 30% of patients with breast cancer, and FcRI is expressed on human monocytes, macrophages, and IFN- activated granulocytes. We investigated phagocytosis and cytolysis of cultured human breast cancer cells by human monocyte-derived macrophages (MDM) mediated by BsAb MDX-210, its partially humanized derivative (MDX-H210), and its parent MoAb 520C9 (anti-HER-2/neu) under various conditions.Materials and Methods: Purified monocytes were cultured with GM-CSF, M-CSF, or no cytokine for five or six days. Antibody dependent cellular phagocytosis (ADCP) and cytolysis (ADCC) assays were performed with the MDM and HER-2/neu positive target cells (SK-BR-3). ADCP was measured by two-color fluorescence flow cytometry using PKH2 (green fluorescent dye) and phycoerythrin-conjugated (red) monoclonal antibodies (MoAb) against human CD14 and CD11b. ADCC was measured with a non-radioactive LDH detection kit.Results: Both BsAb MDX-210 (via FcRI) and MoAb 520C9 (mouse IgG1, via FcRII) mediated similar levels of ADCP and ADCC. ADCP mediated by BsAb MDX-H210 was identical to that mediated by BsAb MDX-210. Confocal microscopy demonstrated that dual-labeled cells represented true phagocytosis. Both ADCP and ADCC were higher when MDM were pre-incubated with GM-CSF than when incubated with M-CSF.Conclusions: BsAb MDX-210 is as active in vitro as the parent MoAb 520C9 in inducing both phagocytosis and cytolysis of MDM. MDX-210 and its partially humanized derivative, MDX-H210, mediated similar levels of ADCP. GM-CSF appears to superior to M-CSF in inducing MDM-mediated ADCC and ADCP. These studies support the ongoing clinical investigations of BsAb MDX-210 and its partially humanized derivative.     

Antitumour reactions of monoclonal antibody against a human osteogenic-sarcoma cell line.

Monoclonal antibody against an osteogenic-sarcoma cell line (791T) was prepared by production and cloning of a somatic-cell hybrid between the mouse myeloma P3-NS1 and spleen cells from 791T-immunized mice. Three clones of hybridoma producing antibody against 791T, as detected by 125I-labelled Protein A binding, were tested against a range of normal and tumour cell targets to determine the pattern of expression of the antigen detected. The 3 clones had identical activity. They reacted strongly against 791T cells and another osteogenic sarcoma, 788T, and more weakly against a further 2 from a total panel of 10 osteogenic-sarcoma lines. The antibody was negative for fibroblasts from the donor of 791T, and for other fibroblasts, human red blood cells, human peripheral mononuclear cells and sheep red blood cells. When tested against a panel of unrelated tumours, they reacted against individual cell lines derived from carcinomas of colon, lung, bladder and cervix. These cross-reactions were not observed with other colon or lung carcinomas, and it is suggested that the antibody was reacting with a tumour-associated antigen expressed randomly on different tumour types, rather than specifically on osteogenic sarcomas.     

INDUCTION OF APOPTOSIS IN HUMAN GASTRIC CARCINOMA CELL LINE MGC-803 BY MONOCLONAL ANTIBODY PD4

Ｔｕｍｏｒｓａｒｉｓｅｎｏｔｏｎｌｙｆｒｏｍａｂｎｏｒｍａｌｃｅｌｕｌａｒｐｒｏｌｉｆｅｒａｔｉｏｎｂｕｔａｌｓｏｆｒｏｍａｄｅｃｒｅａｓｅｉｎａｐｏｐｔｏｓｉｓ，ｗｈｉｃｈｉｓａｐｈｙｓｉｏｌｏｇｉｃａｌｆｏｒｍｏｆｃｅｌｄｅａｔｈｆｉｒｓｔｄｅｓ...     

单克隆抗体PD4诱导人胃癌细胞系MGC-803的凋亡

目的凋亡（Ａｐｏｐｔｏｓｉｓ）是一种区别于坏死的细胞生理性死亡方式。肿瘤发生与凋亡的异常有十分密切的关系。单克隆抗体ＰＤ４能识别胃癌细胞表面的分子量为４００００的分子（Ｐ４０）。本研究是为了观察单抗ＰＤ４是否具有诱导胃癌细胞凋亡的作用。方法我们应用流式细胞术和末端脱氧核苷酸标记及ＤＮＡ电泳法观察单抗ＰＤ４对胃癌细胞ＭＧＣ－８０３增殖周期的影响以及对细胞杀伤作用的方式,并检测了ＭＧＣ－８０３细胞表面Ｆａｓ抗原的表达情况。结果ＰＤ４有阻滞细胞周期、通过诱发凋亡而抑制肿瘤细胞生长的作用,且ＭＧＣ－８０３细胞Ｆａｓ抗原表达为阴性。结论Ｐ４０是一个与细胞凋亡或增殖有关、且不同于Ｆａｓ的肿瘤相关抗原,他的分子克隆有助于抗体诱导细胞凋亡机制的阐明。     

Herpes-related polypeptides from a human cervical carcinoma cell line

Using antiserum against herpes simplex virus type 2 (HSV-2) infected cells, eight polypeptides with similar molecular weights could be immunoprecipitated from the nearly diploid, human cervical carcinoma cell line C4II and from HSV-2 transformed hamster and mouse cells. Only two of these polypeptides corresponded to those from HSV-2 infected cells, including the putative HSV-2 transformation-related 35K protein. Partial proteolytic cleavage products of the immunoprecipitated 35K polypeptides from C4II and HSV transformed hamster and infected cells were indistinguishable; however, viral DNA and mRNA corresponding to the 35K polypeptide could not be detected in C4II or in transformed hamster or mouse cells by dot blot hybridization. A similar mechanism of transformation for the human cervical carcinoma cell line and HSV-2 transformed cells is proposed.     

Therapy of human cervical carcinoma with monoclonal antibody-Pseudomonas exotoxin conjugates.

Pseudomonas exotoxin A (PE) linked to the F(ab')2 fragment of 1H10, a murine monoclonal antibody recognizing a carbohydrate epitope of a glycoconjugate expressed on the surface of human cervical carcinoma tumor cells, was evaluated for in vitro and in vivo activity. PE can kill cells by ADP-ribosylating elongation factor 2 thus inhibiting protein synthesis. Disulfide- as well as thioether-linked immunotoxins (1H10-PE) killed cervical carcinoma cells in vitro and were 20-160 times more inhibitory to target than to control cells. Cell killing was antibody mediated as demonstrated by the reduction of 1H10-PE growth inhibition to target CaSki cells by free 1H10 F(ab')2. In addition, a control antibody immunotoxin was nontoxic to CaSki cells. Thioether-linked 1H10-PE administered either i.v. or i.p. suppressed the growth of established solid s.c. cervical carcinoma tumors xenografted in nude mice for over 30 days. Treatment with antibody alone or a control immunotoxin had no significant effect on tumor growth. Administration of immunotoxin i.p. was associated with less toxicity than administration i.v., but i.v. injections were more effective at suppressing the growth of established solid tumors.     

A novel monoclonal antibody against squamous cell carcinoma.

The monoclonal antibody, INS-2, was raised against rat fibroblasts transformed by open reading frames E6 and E7 of human papillomavirus (HPV) DNA. In immunoperoxidase testing of frozen sections, the INS-2 antibody was reactive with all squamous cell carcinomas of the uterine cervix and esophagus tested. In contrast, no antibody binding was detected with adenocarcinomas of various origins. Similarly, normal tissues, lymphoid cells and erythrocytes from multiple donors were negative, except that binding localized at basal cells in normal squamous epithelium was observed. Interestingly, strong staining was observed in dysplastic cells of cervical intraepithelial neoplasia and at the growing edge of squamous cell carcinomas. The antigen for the INS-2 antibody is a non-sialyl glycoprotein with Mw. 40,000 and appears to be a squamous cell-specific cell differentiation marker, although it is not related to HPV-DNA-derived protein.     

THE DIFFERENTIATION INDUCING EFFECT OF TANSHINONE AND RETINOIC ACID ON HUMAN CERVICAL CARCINOMA CELL LINE（ME180） IN VITRO

ＴＨＥＤＩＦＦＥＲＥＮＴＩＡＴＩＯＮＩＮＤＵＣＩＮＧＥＦＦＥＣＴＯＦＴＡＮＳＨＩＮＯＮＥＡＮＤＲＥＴＩＮＯＩＣＡＣＩＤＯＮＨＵＭＡＮＣＥＲＶＩＣＡＬＣＡＲＣＩＮＯＭＡＣＥＬＬＬＩＮＥ（ＭＥ１８０）ＩＮＶＩＴＲＯＹｕａｎＳｈｕｌａｎ袁淑兰，ＨｕａｎｇＧ...     

多柔比星联合顺铂对宫颈癌Caski细胞株的凋亡诱导作用

目的:体外观察多柔比星（Doxorubicin,ADM）联合顺铂（Cisplatin,CDDP）对宫颈癌Caski细胞株的增殖抑制作用及可能的协同机制。方法:MTT法检测浓度分别为9、4.5、2.25、1.125、0.562 5μg/ml ADM、CDDP以及两种药物联合对Caski细胞的增殖抑制作用。RT-PCR法检测ADM、CDDP及两种药物联合对Caski细胞Bax和Bcl-2基因表达的影响。结果:MTT法检测显示ADM浓度为0.562 5μg/ml联合相同浓度的CDDP时,两药联合能明显增强其对Caski细胞的增殖抑制作用,具有协同抑制作用;RT-PCR法检测显示ADM、CDDP联合处理Caski细胞后Bax基因表达明显增高,同时下调Bcl-2基因的表达。结论:合适浓度的ADM和CDDP联合可以增强化疗效果,而且两药物在联合作用下可通过诱导Bcl-2/Bax 基因表达量的改变,协同发挥其促凋亡作用,从而增强肿瘤细胞对化疗药物的敏感性。     

抗人stathmin单克隆抗体与紫杉醇联用对人肝癌细胞增殖的抑制作用

目的: 探讨抗人stathmin单克隆抗体和紫杉醇单用或联用对肝癌细胞系HepG2增殖的抑制作用。方法: 以不同浓度的抗人stathmin单克隆抗体、紫杉醇分别组成单药组和联合用药组, 另设不加药的空白对照组,分别作用于HepG2细胞24、48、72和96 h,观察细胞数量和形态的变化,MTT法检测各用药组对HepG2细胞增殖的抑制作用,AnnexinV/PI双染法检测各组细胞凋亡率的改变。结果: 不同浓度的各组药物作用后细胞数量明显减少,形态不规则,部分细胞变圆、细胞核固缩和胞质减少,而对照组细胞生长状态良好。抗人stathmin单克隆抗体、紫杉醇单药与联用均能抑制HepG2细胞增殖, 呈剂量时间依赖效应, 联用组细胞增殖抑制率较单药组明显增高(P<0.05),两药联用有交互效应( P<0.05)。抗人stathmin单克隆抗体、紫杉醇单用与联用均能诱导HepG2细胞凋亡, 联合组作用更为明显(P<0.05)。结论: 抗人stathmin单克隆抗体、紫杉醇单药与联用均能抑制HepG2细胞增殖和诱导其凋亡,两药联合使用具有协同作用。     

Lymphokine-activated killer cell cytotoxicity against human colon carcinomas enhanced by monoclonal antibody D612

The antibody-dependent cellular cytotoxicity (ADCC) properties of a murine monoclonal antibody (MAb), designated D612 (IgG2a), which reacts with human colon carcinomas, was studied using normal human peripheral blood lymphocytes (PBMNC). Although the level of ADCC of PBMNC with D612 varied among different donors, it was 20 to 30 times higher than the lytic activity of control cultures containing isotype-matched control MAb. Incubation of PBMNC with recombinant interleukin-2 (IL-2) resulted in a 2- to 5-fold augmentation in the cytotoxicity of effector cells exposed to MAb. This augmentation was apparent after subtracting nonspecific cellular cytotoxicity from the total cytotoxicity mediated by activated effector cells in the presence of D612. Optimal stimulation of specific ADCC with IL-2 appeared after 24 hr of culture in 500 U/ml of IL-2, resulting in a 3.8 +/- 1.7 fold increase in lytic units. However, stimulation of ADCC was also evident at 10 U/ml of IL-2. Furthermore, antibody dose titrations with untreated and IL-2 activated effectors showed that the threshold dose of MAb needed for efficient ADCC was reduced by 200-fold with IL-2. Depletion of FcR gamma III-positive lymphoid cells markedly reduced D612 ADCC, demonstrating the participation of NK/LAK cells in D612-mediated ADCC. Low levels of ADCC activity were found associated with adherent cells, either untreated or following their activation with gamma-interferon, while D612 was most active with non-adherent effectors. The specificity and ADCC properties of the D612 MAb suggest that it should be considered as a candidate for immunotherapy of colon cancer, particularly when used in combination with IL-2 plus LAK cell treatment.     

The human squamous cervical carcinoma cell line, HOG-1, is responsive to steroid hormones.

Growth of the human squamous cervical carcinoma cell line, HOG-I, was stimulated in response to oestradiol in serum-containing and chemically defined medium. The oestradiol-stimulated growth could be inhibited by 4-OH tamoxifen, progesterone and medroxyprogesterone acetate; the last 2 compounds also inhibited basal cell growth in serum-containing and chemically defined media. The data are consistent with the sensitivity of human squamous cervical cancer to sex-steroid hormones and suggest that endocrine therapies may be of benefit in this disease.     

Augmentation of lymphokine-activated killer cell cytotoxicity by monoclonal antibodies against human small cell lung carcinoma

This study examined the lymphokine-activated killer (LAK) cell cytotoxicity on monoclonal antibody (MoAb)-bound tumor cells from the human small cell lung carcinoma cell lines H69 and H128. LAK cells were generated from normal peripheral blood mononuclear cells by incubation with interleukin 2 for 3 or more days. Cells from the LAK culture were cytotoxic to natural killer-sensitive (K562, 84% cytotoxicity) and natural killer-resistant (Daudi, 85%; H69 and H128, 69% and 97%, respectively) cell lines, and to freshly excised human lung (49%) and breast (57%) tumors. LAK cytotoxicity to H69 or H128 cells was significantly augmented by target cell preincubation with the small cell lung carcinoma-reactive MoAbs 1096 (increases of up to 271%) or 5023 (up to 223%). SCLC 5023 or 1096 did not enhance LAK cytotoxicity to Daudi cells of lymphoblastoid origin. Pretreatment of LAK cells with an anti-Fc receptor antibody blocked MoAb augmentation by 1096 or 5023 (but not LAK cytotoxicity), suggesting that LAK-MoAb interaction may be mediated by Fc binding. LAK activity coincided with emergence of a large cell [interleukin 2-stimulated large mononuclear leukocyte (LML)] subset expressing the CD16 and NKH-1 surface determinants. Serial immunophenotyping of the LAK cell culture harvested at Days 3, 5, and 7 indicated that the level of LAK cytotoxicity, with or without MoAb augmentation, correlated with frequency of NKH-1-reactive LMLs. These observations support the hypothesis that LAK cytotoxicity is mediated by a NKH-1-reactive LML subpopulation. Antitumor cytotoxicity may be augmented by tumor-reactive MoAbs through Fc binding to this LML subset.     

A human squamous cell carcinoma cell line.

An epithelial cell line COLO 16 has been established from a human squamous carcinoma, characterized and maintained for over two years. The cells produce a parathyroid-like hormone and carcinoembryonic antigen. The line is definitely not a "HeLa contaminant." The cell line is available to other investigators.