Murine natural anti-tumor antibodies. I. Rapid in vivo binding of natural antibody by tumor cells in syngeneic mice.

A competitive radioimmunoassay (RIA) for the detection of cell-bound antibody was used to study the in vivo acquisition of immunoglobulin (Ig) by tumor cells. Several tumor lines acquired Ig rapidly between 3 and 18 h after intraperitoneal implantation into normal syngeneic mice and this Ig was recovered by elution with basic or acid buffers. The Ig eluted from the L5178Y lymphoma showed higher binding to the L5178Y than to thymocytes, bone-marrow cells, 1509a sarcoma and P-815-X2 mastocytoma. In addition, binding of the eluates to the L5178Y was specifically inhibited by L5178Y cells or by solubilized membrane antigens of the L5178Y. The in vivo acquisition of Ig by the L5178Y could also be blocked by the IV and IP injections of tumor antigen although both L5178Y and 1509a solubilized membrane antigens were effective. Some of the Ig acquired by the tumor cells was found to be complement-fixing antibody since normal rabbit complement lysed 80% of L5178Y cells obtained from the peritoneal cavity of syngeneic mice 18 h after implantation, but did not lyse in vitro L5178Y cells. The in vivo binding of the complement-fixing antibodies was also inhibited by tumor antigens in the same way as the acquisition of Ig detected by RIA. It was shown that the acquisition of Ig during the first 18h of IP growth was a T-independent phenomenon because tumor cells acquire as much Ig in AT X BM mice as in sham-thymectomized controls. In a study with 11 different clones derived from the L5178Y lymphoma, a high correlation (r = 0.75, p less than 0.005) was found between the amount of Ig acquired after in vivo implantation and the amount of Ig bound to the cells after in vitro incubation with normal syngeneic serum. It is suggested that the rapid in vivo acquisition of Ig was due to the in vivo binding of natural antibodies to tumor cells.     

Murine natural antitumor antibodies. III. Interferon treatment of a natural killer-resistant lymphoma: augmentation of natural antibody reactivity and susceptibility to in vivo natural resistance

Treatment of a natural killer cell-resistant (NKR) DBA/2 lymphoma with L-cell interferon (IFN) enhanced its reactivity to serum natural antibody in vitro in cytolysis and absorption studies and increased the in vivo acquisition of natural and antitumor antibody in the peritoneal cavity. The IFN effects were both time- and dose-dependent. In vitro IFN-treated, [131I]5-iodo-2'-deoxyuridine-labeled tumor cells, when injected ip into normal syngeneic mice, were more rapidly eliminated than were untreated control cells. IFN treatment of the NKR tumor decreased "cold-target" inhibition of NK lysis and did not alter binding or lysis by macrophages. These findings indicated that the enhancement of natural resistance to the IFN-treated tumor did not involve NK cells or macrophages and suggested that IFN may enhance host antitumor resistance by increasing tumor reactivity to antibody.     

The contribution of antibodies to targeted cancer therapy

The fight against cancer is the focus of many research groups, as it is classified as one of the leading causes of death. The treatment of cancer is mainly composed of surgery, chemotherapy, radiation therapy, immunotherapy, and nowadays through targeted therapy as well. Targeted therapy targets the molecules whose expression correlates to carcinogenesis. It can be performed either with small molecule drugs or with monoclonal antibodies. Based on the capacity of the monoclonal antibodies to recognize a certain epitope of an antigen, and the fact that cancer cells express many antigens compared with normal (noncancer) cells abnormally in their surface, cytotoxicity may be induced directly in these cells. The cytotoxicity can be caused either via antibody-dependent cellular interaction or via the complement system. The possibility to create monoclonal antibodies against any antigen led to production of new medicines, many of which are already in clinical use. The ability to conjugate several molecules to antibodies also made the delivery of drugs/substances directly to cancer cells possible, thereby reducing the side effects of cancer treatment. This review presents the implementation of antibodies to targeted therapy, emphasizing the antibody-dependent cytotoxicity, as well as the monoclonal antibodies that are used in cancer treatment.     

Murine monoclonal antibodies to colon-specific antigen p

A previous report described the production of monoclonal antibodies Mu-1, -2, -3, and -4 against colon-specific antigen p. We now report the tissue distribution of the reactive epitopes. Each of the monoclonal antibodies was reactive with epitopes shared by the entire length of the intestine, with Mu-2 and Mu-3 determinants being present in the stomach as well. Mu-1 and Mu-4 appeared to have a more restricted distribution than did Mu-2 and Mu-3, which were present in several nongastrointestinal tissues. Unfortunately, these MAbs were not ideal for in vivo use against colorectal carcinoma. Mu-1 was not present in neoplastic colon tissue, Mu-2 and Mu-4 were reactive with granulocytes, and Mu-3 was reactive with kidney tubules and connective tissue. An additional fusion was performed from which Mu-9 was selected for further study. Among normal adult tissues this monoclonal antibody gave reactivity restricted to the intestines. The stomach and all nonintestinal tissues were negative by immunoperoxidase. Among colon tumors 60% were positive for the Mu-9 epitope. The data suggest Mu-9 may be a good candidate for in vivo targeting of diagnostic and therapeutic agents to colon cancer.     

Murine in vitro antigenic modification of tumor cells: effect on susceptibility to natural cell-mediated cytotoxicity

A 3-methylcholanthrene [(MCA) CAS: 56-49-5]-induced fibrosarcoma cell line and its Friend murine leukemia virus-infected counterpart were assessed for their susceptibility to lysis by so-called "natural" effector cells in a series of 51Cr release assays. Detailed functional and phenotypic analysis of lytic effector cell populations from normal C57BL/6 mouse spleens revealed an identity most closely associated with natural cytotoxic cells. Neither tumor cell line was found to be sensitive to natural killer-mediated lysis. Additionally, virus infection of the MCA-induced fibrosarcoma cell line did not affect susceptibility to natural cell-mediated cytotoxicity.     

Murine tumor cell lysis by antibody-dependent macrophage-mediated cytotoxicity using syngeneic monoclonal antibodies

Four monoclonal antibodies to MH134 murine syngeneic hepatoma cells, 3H1, 7C2, 11G2, and 12A2, were produced by hybridomas constructed by fusing P3-X63-Ag8-U1 murine myeloma cells with spleen cells of a C3H/HeN mouse immunized with the syngeneic tumor cells. Immunodiffusion analysis with rabbit anti-mouse immunoglobulin antisera showed that 3H1, 7C2, 11G2, and 12A2 are IgG2a, IgM, IgG1, and IgG2a, respectively. Enzyme-linked immunosorbent assay using cells of five syngeneic tumor lines, MH134, MM102, MM46, MM48, and X5563, and lymph node cells of C3H/HeN and C57BL/6 mice showed that 3H1 specifically bound to MH134 tumor cells, whereas 7C2, 11G2, and 12A2 reacted not only with MH134 but also with MM102 and MM46 tumor cells. None of these monoclonal antibodies bound either to cells of MM48 or X5563 tumor lines or to normal lymph node cells. These results strongly suggest that MH134 tumor cells display at least two kinds of tumor-associated antigens on their cell surfaces: one is expressed uniquely by MH134 tumor cells, which are recognized by 3H1; the other is commonly shared by MH134, MM102, and MM46 tumor cells, which are determined by the other three antibodies. 3H1, 11G2, and 12A2 but not 7C2 were found to be able to induce antibody-dependent cellular cytotoxicity (ADCC) against MH134 tumor cells. Target specificity of ADCC induced by these monoclonal antibodies was identical with that seen in enzyme-linked immunosorbent assay. 3H1, 7C2, and 12A2 but not 11G2 exhibited complement-dependent cytotoxicity, showing the same specificity in target cell lysis as that seen in enzyme-linked immunosorbent assay or ADCC. Pretreatment of MH134 tumor cells with 7C2 inhibited ADCC of both 11G2 and 12A2. Pretreatment of the tumor cells with 11G2 inhibited complement-dependent cytotoxicity of both 7C2 and 12A2. Neither ADCC nor complement-dependent cytotoxicity of 3H1 was inhibited by the pretreatment of the cells with 7C2 or 11G2. These results strongly suggest that tumor-associated antigens recognized by 3H1 are located apart from that recognized by 7C2, 11G2, and 12A2 and that the binding sites of the latter three antibodies are closely associated with, or identical with, each other in the tumor-associated antigen. The ability of 12A2 to induce ADCC against MH134 tumor cells was significantly stronger than that of 3H1 or 11G2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Promotion of tumor invasion by cooperation of granulocytes and macrophages activated by anti-tumor antibodies

We investigated the potential role of anti-tumor antibodies and tumor antigens in the formation of immune complexes which promote matrix degradation and angiogenesis. B-cell deficient or B-cell depleted mice showed a reduction in tumor invasion and metastasis. In vitro invasion assays and in vivo models of metastasis showed that anti-sTn antibodies and sTn tumor antigens form complexes which induce granulocytes and macrophages together to mediate tumor invasion and metastasis by processes including extracellular matrix degradation and angiogenesis. These results suggest the existence of a tumor promoting role of a B-cell immune response induced by shed tumor associated antigens of solid, nonlymphoid tumors.     

苓甲抗癌复方提取物在荷瘤小鼠中抗肿瘤免疫应答机制

目的:初步探究苓甲抗癌复方提取物(LAM)在荷瘤小鼠中抗肿瘤免疫应答机制.方法:LAM治疗荷瘤小鼠,检测小鼠生长、瘤块体积与重量的变化;流式细胞术和ELISA分析小鼠脾脏中Treg细胞、NK细胞和各种细胞因子;Western blot分析LAM对Treg细胞相关蛋白作用.结果:LAM抑制荷瘤小鼠肿瘤的生长,引发肿瘤坏死,抑制肿瘤促增殖蛋白Ki67的表达,降低血液中CA153的含量;降低脾脏中Treg细胞比例和血清中TGF-β与IL-10的含量,提高IL-2、IFN-γ、穿孔素、颗粒酶B的含量和NK细胞的比例.结论:LAM抑制肿瘤的生长,并抑制Treg细胞的数量与功能,提高机体的免疫功能.     

Assessment of reactivities of natural antibodies to endogenous RNA tumor virus envelope antigens and virus-induced cell surface antigens.

The autogenous humoral immune response of mice to their endogenous leukemia virus (MuLV) has been examined with respect to the reactivities of natural antibodies to MuLV envelope antigens and virus-induced cells surface antigens. The natural reactivity of MuLV envelope antigens was evaluated by means of a radioimmune precipitation assay of intact and disrupted virus, as well as by virus neutralization tests. The specificity of natural antibody for MuLV envelope antigens was determined by immunoelectron microscopy and radioimmune precipitation. Antibody reactivity to virus-induced cell-surface antigens was evaluated by immunoelectron microscopy and a complement-dependent cytotoxicity test. The strains of mice seleced for study were C57BL/6, C3H/Anf, and the C57BL/6 X C3H/Anf F1 hybrid. Although there were quantitative differences in the antibody levels among these various strains, the naturally recognized antigenic determinants of the virus were consistent, i.e., gp68, gp-43, and p15. High levels of neutralizing antibody against the xenotropic BALB:virus-2 were detected in these various normal sera with the focus reduction assay; however, only marginal levels of neutralizing activity against Moloney leukemia virus were detected with the XC virus assay. No anticellular antibody could be detected in these normal sera with the complement-dependent cytotoxicity assay.     

Nonimmune and immune surveillance. II. Effect of recipient's age, tumor immunogenicity, and neonatal thymectomy on tumor growth inhibition.

After a suspension of tumor pieces was grafted into newborn and adult (CBA X C57BL/6J)F1 and BALB/c mice, the growth of Lewis lung adenocarcinoma and mammary gland adenocarcinoma was inhibited in newborn mice, whereas sarcoma of the rectum (SR-1-75) grew at the same rate in newborn and adult recipients. Neonatal thymectomy stimulated the growth of hepatoma (H-2-73) in newborns. The degree of tumor growth inhibition was age-dependent: The maximum inhibition was observed in 1- to 6-day-old recipients, but later it gradually decreased. The hepatoma (H-2-73) and ovarian carcinoma (OC-1-75) with inhibited growth in newborns and the tumor (SR-1-75) with uninhibited growth had equally low immunogenicity. The data suggested that newborns possess factors that inhibit tumor growth but these factors disappear with increased age of recipients.     

The fate of antibodies bound to the surface of tumor cells in vitro.

The fate of monoclonal antibodies binding to the surface of human tumor cells in vitro was investigated. Seven antibodies, labeled with 125I, were tested on four cell lines, which included a melanoma and carcinomas of the ovary, kidney, and lung. The antibodies were selected only by the criterion that they not be rapidly internalized via coated pits, so that they would be representative of most antibodies reacting with cell surface antigens. After allowing binding during a 2-h incubation, unbound antibody was removed, and the release of intact or degraded antibody in the supernatant was monitored. The data demonstrate that most bound antibody was gradually degraded and released from the cell over a 2-3-day period, probably via internalization, while only a small fraction, less than 20% for most antibodies, appeared to dissociate intact. One exceptional antibody, MW207, dissociated largely intact. The release of intact antibody was virtually complete within 4 h, and radioactivity released after this time was predominantly in degraded form. These results demonstrate that antibody binding to the surface of viable cells must in general be considered irreversible, and hence the concept of affinity is not applicable. Since an Fab fragment of one of the antibodies dissociated rapidly, such irreversible binding appears to require bivalent attachment. Another conclusion of this study is that most antibodies binding to the cell surface are gradually internalized, which we suggest is due to the normal turnover of cell surface constituents via non-clathrin-dependent endocytosis. Several experimental approaches indicated that a large fraction of antibody retained by the cells, for at least 2 days after binding, was present at the cell surface.     

MHC class I-related chain A conjugated to antitumor antibodies can sensitize tumor cells to specific lysis by natural killer cells.

PURPOSE: As a first step for the development of a new cancer immunotherapy strategy, we evaluated whether antibody-mediated coating by MHC class I-related chain A (MICA) could sensitize tumor cells to lysis by natural killer (NK) cells. EXPERIMENTAL DESIGN: Recombinant MICA (rMICA) was chemically conjugated to Fab' fragments from monoclonal antibodies specific for tumor-associated antigens, such as carcinoembryonic antigen, HER2, or CD20. RESULTS: Flow cytometry analysis showed an efficient coating of MICA-negative human cancer cell lines with the Fab-rMICA conjugates. This was strictly dependent on the expression of the appropriate tumor-associated antigens in the target cells. Importantly, preincubation of the tumor cells with the appropriate Fab-rMICA conjugate resulted in NK cell-mediated tumor cell lysis. Antibody blocking of the NKG2D receptor in NK cells prevented conjugate-mediated tumor cell lysis. CONCLUSIONS: These results open the way to the development of immunotherapy strategies based on antibody-mediated targeting of MICA.     

Murine colon carcinoma cells engineered to produce human interleukin-2 induce tumor-specific anti-tumor response

Murine colon carcinoma cells (colon 26) transduced by a retrovirus vector with the human interleukin-2 (IL-2) cDNA were studied for their tumorigenicity. Although cell growth in vitro was not affected by integration of the IL-2 gene, s.c. tumors of IL-2-producing colon 26 cells (H2) in syngeneic mice regressed spontaneously after producing small masses. Histological examination of the sites of tumor rejection revealed predominant infiltration of macrophages around the tumor necrotic mass. Subsequent challenge with parent colon 26 cells, but not with Meth A cells (fibrosarcoma of the same genetic background), did not result in tumor formation in mice which had been protected against H2 cells. Inoculation of H2 cells into syngeneic nude mice resulted in tumors with a retarded growth rate. Taken together, T cell-dependent, tumor-specific immunity is obtained by local IL-2 secretion around colon tumors, and this experimental animal model gives us a clue(s) for investigating host anti-tumor responses by cytokine production. © 1996 Wiley-Liss, Inc.     

Variability of tumor response to chemotherapy II. Contribution of tumor heterogeneity

Summary The role of tumor-to-tumor variability in response to chemotherapy was investigated in mice bearing mammary adenocarcinoma 16/C treated with melphalan. Lissamine green, a triphenylmethane dye, was given systemically to delineate areas of perfusion in the tumors. The regions of low perfusion ranged from 10% to >90% of the mass of individual tumors. The variation in perfusion was as large between bilateral tumors in a mouse as between tumors in different hosts. The presence of viable cells capable of continued growth in the regions of low perfusion was demonstrated by bioassay. Concentrations of melphalan following i.p. administration varied by as much as tenfold or more between regions of low and high perfusion. Concentrations of melphalan in the well-perfused regions were similar to plasma concentrations at 30 min after administration, but elimination from the plasma was more rapid. The levels of melphalan in the tumor were higher following the initial dose than following succeeding doses in a multiple dose schedule. The results indicate that tumor-to-tumor variations in perfusion and drug distribution are major factors in variable tumor response.     

Enhanced effects of monoclonal antibody carboplatin immunoconjugates uptake and anti-tumor effects with angiotensin II and tumor necrosis factor.

The most important factor influencing the use of monoclonal antibody immunoconjugates for cancer treatment is effectiveness of enhancement for tumor targeting. We call attention to the vasoactive agents angiotensin II (ATII) for selective increase in tumor blood flow and cytokine tumor necrosis factor (TNF) to improve the vascular permeability of the tumor. Tumor localization and biodistribution were investigated by nude mice transplanted human colon adenocarcinoma (LS-180) with radiolabeled carboplatin immunoconjugates. Tumor activity 48 h after administration of ATII and TNF was significantly higher than in the control group. However, nonspecific accumulation in normal organs was not observed. In vivo anti-tumor effects in animals with ATII and TNF was significantly stronger than in those given the same dose drug alone or immunoconjugates alone. These results indicate that ATII and TNF could be useful tools for induction of stronger inhibition of tumor growth without systemic toxicity.     

Determination of specificity in natural cell-mediated cytotoxicity by natural antibodies.

Natural cell-mediated cytotoxicity (NCMC) and antibody-dependent cell-mediated cytotoxicity (ADCC) appeared to involve similar cytotoxic mechanisms; effector N-cells killed target cells with IgG antibodies attached to the Fc receptor determining the specificity of the reaction. In NCMC the wide range of specificities detected by natural antibodies provided an effector system capable of recognizing numerous antigens on cultured target cells. When several target cells were tested concurrently, an apparent nonselective cytotoxicity resulted. The specificity of individual reactions against each of the target cells could be demonstrated by selective inhibition with competitor cells. The inhibition of cytotoxicity by competition and the effect of proteases on the effector cell for NCMC, but not for ADCC, initially suggested an antibody on the surface of the natural cytotoxic effector cell. This suggestion was supported by the loss of activity with treatments that removed immunoglobulins on the effector cell and by the recovery of reactivity with incubation of the cells in normal human serum. Absorption of the reconstituting serum with target cells resulted in loss of activity against that target cell, substantiating the role of natural antibodies.     

Detection of anti-tumor antibody in virally induced tumors and its relationship to tumor growth.

The humoral antibody response to virally induced tumors insyngeneic hosts has been studied. The tumors include an SV40 tumor SVT2, the Friend virus-induced leukemias FBL-3 and FLC; and Moloney sarcoma virus-induced tumors. It was found that antitumor antibodies could be detected by the isotopic antiglobulin technique in these tumor systems at a relatively early stage of tumor growth. The kinetics of the antibody response in relation to the status of tumor growth varied between different tumors. In geneumor growth than in the regressors of tumor-free hosts. Reinoculation of tumor cells or recurrence of tumor growth produced elevation of antibody levels (secondary response). The specificity of the antibody reactions also varied in different tumor systems: some antibodies were truly tumor-specific and thus might produce a biological effect on in vivo tumor immunity, whereas others were not. These studies indicated that a sensitive antibody assay could be used for early detection of tumor growth. However, its usefulness in evaluation of the status of tumor growth should be carefully studied in each tumor system.