Monoclonal antibodies to endogenous galactose-specific tumor cell lectins.

A monoclonal antibody, 5D7, was obtained after immunization of syngeneic mice with B16 melanoma cell extracts enriched for endogenous lectin activity and screening for inhibition of lectin-mediated hemagglutination. Binding of this antibody to affinity-purified B16 melanoma galactoside-specific lectin was revealed by solid-phase radioimmunoassay and binding to the surface of viable B16 cells was demonstrated by indirect immunofluorescence. Inhibition of lectin activity and cell surface labeling by 5D7 antibody were also found with several types of cultured human and murine cells including melanoma, sarcoma and carcinoma. This monoclonal antibody should be useful for evaluating the role of tumor cell surface lectins in intercellular interactions and metastasis.     

Synthesis of doxorubicin-peptide conjugate with multidrug resistant tumor cell killing activity

Cell penetrating peptide TAT was introduced into doxorubicin structure. Synthesized doxorubicin-TAT conjugate showed different intracellular distribution pattern and cell killing activity from those of free doxorubicin. Unlike free doxorubicin, doxorubicin-TAT conjugate was highly permeable to drug-resistant cells and was able to kill drug-resistant tumor cells efficiently.     

Inhibition of tumor necrosis factor-induced cell killing by tryptophan and indole

Cells sensitive to the cytocidal effect of tumor necrosis factor (TNF) were protected against this effect when growth in the presence of elevated concentrations of tryptophan. Several other indole derivatives also provided protection against TNF cytotoxicity. Most effective were indole itself and its monomethyl derivatives, providing a degree of protection greatly exceeding that observed with tryptophan. Protection was also observed against the cytocidal effect of TNF applied in the presence of a protein synthesis inhibitor. The protective effect of tryptophan was largely dependent on preexposure of the cells, for several hours, to a high concentration of this amino acid. On the other hand, indole was protective also when applied to cells together with TNF, or even two hours after TNF application. The inhibition of the cytotoxicity of TNF by tryptophan and other indole derivatives may serve as a useful experimental tool in exploring the mechanisms and the physiological implications of TNF cytotoxicity.     

A dose-response model for estimating lifetime tumor risks when cell killing occurs

A model is described in which damage to a single intracellular locus can lead to a tumorigenic transformation. Assuming a large number of independent intracellular loci to be at risk and assuming that damage to a locus sufficient to cause a tumorigenic transformation occurs with probability greater than zero for all doses greater than zero, leads to the use of the Weibull distribution to characterize the probability of a nonspontaneous tumorigenic cellular transformation occurring after exposure to a given dose of carcinogen. The excess lifetime tumor incidence (i.e., the proportion of tumor bearers) above the spontaneous incidence is used as an estimate of the non-spontaneous incidence and is characterized by a tumor incidence function that represents the probability of occurrence of one or more non-spontaneous tumorigenic cellular transformations amongN(D) independent surviving cells per individual, after exposure to a doseD of carcinogen. The tumor incidence function is fitted to published data for the excess tumor incidence after exposure of animals or humans to ionizing radiation and after exposure of animals to chemical carcinogens.     

Engineered drug-resistant immunocompetent cells enhance tumor cell killing during a chemotherapy challenge

Establishment of immunocompetent cell mediated anti-tumor immunity is often mitigated by the myelosuppressive effects during administration of chemotherapy. We hypothesized that protecting these immune cells from drug induced toxicities may allow for the combined administration of immunotherapy and chemotherapy. Using a SIV-based lentiviral gene transfer system we delivered the drug-resistant variant P140KMGMT into the immunocompetent cell lines NK-92 and TALL-104, and the myelogenous leukemia cell line, K562, which is a target for both NK-92 and TALL-104 cells. Genetically engineered immunocompetent cells developed significant resistance to temozolomide compared to non-modified cells, and genetic modification of these cells did not affect their ability to kill K562 cells. We then evaluated the effectiveness of drug-resistant immunocompetent cell mediated killing of tumor cells in the presence and absence of chemotherapy. During a chemotherapy challenge the cytotoxic activity of non-modified immunocompetent cells was dramatically impaired. However, when combined with chemotherapy, genetically-modified immune cells retained their cytotoxic activities and efficiently killed non-modified target cells. These results show that engineering immunocompetent cells to withstand chemotherapy challenges can enhance tumor cell killing when chemotherapy is applied in conjunction with cell-based immunotherapy.     

In vitro tumor cell killing by peritoneal macrophages from mitomycin C-treated rats

Summary The local cellular response induced by intraperitoneal injection of mitomycin C was examined in terms of cell-mediated cytotoxicity for tumor cells. An in vitro cytolysis assay involving ¹²⁵I-iododeoxyuridine-labeled tumor target cells revealed that treatment of normal ACI/N rats (200 g) with a single intraperitoneal injection of mitomycin C (50, 100, or 200 g) induced tumoricidal macrophages in the peritoneal cavity. The tumoricidal activity was dependent on the dose of mitomycin C injected and it was detectable as early as 1 day after the intraperitoneal injection of mitomycin C. In addition to the increased tumoricidal activity, the functional activities of the peritoneal macrophages were found to be increased with respect both to uptake of 2-deoxy-d-glucose and to phagocytosis of latex beads. Additional experiments excluded the possibility that the tumor cell cytolysis was the result of direct cytotoxicity by mitomycin C that might have been incorporated in the peritoneal macrophages or of nutrient depletion in the medium during the cytolysis assay. Furthermore, endotoxin contamination of the mitomycin C, which might have produced the activated macrophages, was not detected. The mechanism by which mitomycin C injected intraperitoneally induced the tumoricidal macrophages locally remains uncertain; however, it is possible also in clinical situations.     

Monoclonal antibody against K562 cell line accelerates killing of the target cells by large granular lymphocytes

A monoclonal antibody (MoAb 11-4) was raised against K562, a human erythroleukemia cell line sensitive to natural killer cell-mediated cytotoxicity (NK-CMC). Immunological analysis revealed MoAb to be IgG_2b. Alone, the MoAb was not cytotoxic for K562 and did not bind to the effector cells, but the addition of this antibody to macrophage-depleted human peripheral blood lymphocytes increased killing of K562 in a 4-hr NK-CMC assay. The maximum increase in NK-CMC was observed when MoAb 11-4 was added to target cells prior to the formation of effector/target cell conjugates. This effect was dose dependent, was specific for K562, and, contrary to conventional antisera, occurred at very low concentrations of MoAb. When MoAb was added either to Percoll-purified large granular lymphocytes (LGL) or to LGL-depleted lymphocytes, only the latter demonstrated a significant increase in the killing of K562 in a 4-hr chromium release assay. Kinetics studies revealed that although the overall LGL-mediated lysis was only slightly increased at 4 hr, the maximum lytic activity was reached within 2 hr. These studies suggest that (1) human LGL and LGL-depleted cell populations bear Fc receptors for mouse IgG_2b and (2) although the cytotoxic activities of both cell populations are increased by treatment with MoAb 11-4, the kinetics of this increase are different.     

Interferon production and tumor cell killing by human lymphocytes stimulated in mixed-lymphocyte culture

The in vitro synthesis of interferon (IFN) by human lymphocytes stimulated in mixed-lymphocyte culture (MLC) was examined. The production of IFN in MLC was restricted to T lymphocytes and maximum levels of IFN were detected in supernatants from cells incubated for 5 to 7 days. The IFN produced was identified as IFN-gamma by antibody neutralization. To identify the T cell responsible for IFN production, purified T lymphocytes were separated into subpopulations after incubation in 5 mM theophylline. Theophylline-resistant (T-res) T cells retain the ability to form sheep erythrocyte (SRBC) rosettes and are depleted in IgG Fc receptor-positive T cells (T gamma cells). Theophylline-sensitive (T-sens) T cells fail to form rosettes after theophylline treatment and are enriched in T gamma cells. In addition, analyses using monoclonal antibodies showed that T-sens cells were enriched in OKM1-, HNK-1-, and 7.2-positive cells and T-res cells contained increased numbers of 9.6- and OKT4-positive cells. Following MLC stimulation, equivalent levels of IFN-gamma were produced by T-res and T-sens cells and both subpopulations maintained natural killer (NK)-like cytotoxicity against K562 target cells. Addition of partially purified IFN-gamma to unstimulated T-res and T-sens cells resulted in the maintenance of NK-like cytotoxicity in a manner analogous to that observed after MLC. Additional experiments indicated that peripheral blood lymphocytes depleted of 9.6- or OKM1-positive cells by complement-mediated lysis were devoid of cytotoxicity against K562 cells. Furthermore, MLC stimulation of 9.6- or OKM1-depleted cells failed to restore cytotoxic activity. In summary, these experiments demonstrate that the maintenance of NK-like cytotoxicity by MLC-stimulated T cells is associated with the synthesis of IFN-gamma, that MLC stimulated T-res and T-sens T-cell subsets produce equivalent amounts of IFN, and that 9.6- or OKM1-positive cells are required for the maintenance of NK-like cytotoxicity in MLC.     

Potent and conditional redirected T cell killing of tumor cells using Half DVD-Ig

Novel biologics that redirect cytotoxic T lymphocytes (CTLs) to kill tumor cells bearing a tumor associated antigen hold great promise in the clinic. However, the ability to safely and potently target CD3 on CTL toward tumor associated antigens (TAA) expressed on tumor cells remains a challenge of both technology and biology. Herein we describe the use of a Half DVD-Ig format that can redirect CTL to kill tumor cells. Notably, Half DVD-Ig molecules that are monovalent for each specificity demonstrated reduced non-specific CTL activation and conditional CTL activation upon binding to TAA compared to intact tetravalent DVD-Ig molecules that are bivalent for each specificity, while maintaining good drug like properties and appropriate PK properties.     

Enhancement of murine ascites tumor cell killing with x-irradiation by thiol binding agents

Female mice bearing the Ehrlich carcinoma or P388 lymphocytic leukemia tumors in ascites form were given sublethal doses of whole-body x-irradiation and the thiol binding agents N-ethylmaleimide, hydroxy-mercuribenzoate, or iodoacetamide, injected intraperitoneally prior to irradiation, as a single treatment. These compounds were found previously to sensitize mice to radiation lethality. Enhanced tumor cell killing was observed as measured by tumor cell count, along with slightly longer survival times of the host animal. Increasing the dose of either radiation or drug alone also caused an increase in tumor cell killing, but at the expense of earlier mortality of the host animal. At the doses employed the sensitizers examined appeared more effective on these two ascites tumors han on the host. The mechanism of enhancement of radiation killing of tumor cells by these drugs is not clear, although it appears not to be due to additive toxicity effects. Similar experiments with several cancer chemotherapy agents showed that those compounds did not act as radiosensitizers.     

Immature neutrophils mediate tumor cell killing via IgA but not IgG Fc receptors

Antitumor Abs are promising therapeutics for cancer. Currently, most Ab-based therapies focus on IgG Ab, which interact with IgG FcR (FcgammaR) on effector cells. In this study, we examined human and mouse neutrophil-mediated tumor cell lysis via targeting the IgA FcR, FcalphaRI (CD89), in more detail. FcalphaRI was the most effective FcR in triggering tumor cell killing, and initiated enhanced migration of neutrophils into tumor colonies. Importantly, immature neutrophils that are mobilized from the bone marrow upon G-CSF treatment efficiently triggered tumor cell lysis via FcalphaRI, but proved incapable of initiating tumor cell killing via FcgammaR. This may provide a rationale for the disappointing results observed in some earlier clinical trials in which patients were treated with G-CSF and antitumor Ab-targeting FcgammaR.     

Death receptor-induced cell killing

Apoptosis pathways activated by death receptors of the tumour necrosis factor (TNF) family such as Fas, TNFR1, or the TRAIL receptors DR4 and DR5 are implicated in diverse diseases. These are also the best-understood apoptosis pathways and many of our ideas about apoptosis regulation come from studying these pathways. Cell killing from such receptors occurs because of recruitment to the receptor of the adaptor protein FADD, which in turn recruits the pro form of caspase-8. Aggregation of pro-caspase-8 leads to its auto-activation and subsequent activation of effector caspases such as caspase-3. The apoptotic signal can be amplified through the mitochondria and inhibited through the action of competing molecules such as the inhibitor c-FLIP, which binds to the receptor complex in place of caspase-8. This simple mechanism explains much of the cell death that is induced by death receptors. However, recent studies indicate that we must incorporate new information into this model. Some examples that add new layers of complexity will be discussed in this review.     

Efficient synthesis of trisaccharide saponins and their tumor cell killing effects through oncotic necrosis

To investigate the relationship of cytotoxicity and saponins with varied aglycones, based on the structure of indioside E 1, a natural derived anti-tumor active ingredient from Chinese medicinal plant Solanum indicum L., five novel saponins 2-6 bearing the same trisaccharide moiety together with 1 were efficiently synthesized via a transglycosylation strategy. MTT assay revealed the killing effects to tumor cells of the synthesized saponins are varied with the change of aglycones. Furthermore, time-lapse microscopy, LDH release, PI staining, and immunocytochemical investigations demonstrated that the cell death caused by neosaponin 2 was through oncotic necrosis involving plasma membrane perturbation and destruction of cytoskeleton.     

Tumor necrosis factor is an important mediator of tumor cell killing by human monocytes

The mechanism of human peripheral blood monocyte-mediated cytotoxicity for tumor cells was investigated, using the A673 human rhabdomyosarcoma and HT-29 human colon adenocarcinoma lines as target cells. A673 cells were shown to be susceptible to the cytotoxic action of purified recombinant human tumor necrosis factor (TNF). A673 cells were also highly sensitive to the cytotoxic action of peripheral blood monocytes. Clones of A673 cells sensitive and resistant to TNF were isolated and characterized for their sensitivity to monocyte killing. A good correlation was found between the sensitivity of these clones to the cytotoxicity of TNF and their susceptibility to killing by monocytes. A TNF-specific neutralizing monoclonal antibody (MAb) reduced monocyte killing of parental A673 cells and of a TNF-sensitive clone of A673 cells. Inhibition of monocyte killing by this MAb was particularly pronounced at a low effector to target cell ratio. HT-29 cells were relatively resistant to the cytotoxic action of recombinant TNF and to monocyte killing. Treatment of HT-29 cells with recombinant human IFN-gamma increased their susceptibility to both TNF cytotoxicity and monocyte killing. In addition, MAb to TNF inhibited monocyte killing in HT-29 cells sensitized by incubation with IFN-gamma. Our data show that TNF is an important mediator of the cytotoxicity of human monocytes for tumor cells and that IFN-gamma can increase monocyte cytotoxicity by sensitizing target cells to the lytic action of TNF.     

Levels of specific peptide-HLA class I complex predicts tumor cell susceptibility to CTL killing

Recognition of tumor-associated Ags (TAAs) on tumor cells by CTLs and the subsequent tumor cell death are assumed to be dependent on TAA protein expression and to correlate directly with the level of peptide displayed in the binding site of the HLA class I molecule. In this study we evaluated whether the levels of Her-2/neu protein expression on human tumor cell lines directly correlate with HLA-A*0201/Her2/neu peptide presentation and CTL recognition. We developed a TCR mimic (TCRm) mAb designated 1B8 that specifically recognizes the HLA-A2.1/Her2/neu peptide (369-377) (Her2(369)-A2) complex. TCRm mAb staining intensity varied for the five human tumor cell lines analyzed, suggesting quantitative differences in levels of the Her2(369)-A2 complex on these cells. Analysis of tumor cell lines pretreated with IFN-gamma and TNF-alpha for Her2/neu protein and HLA-A2 molecule expression did not reveal a direct correlation between the levels of Her2/neu Ag, HLA-A2 molecule, and Her2(369)-A2 complex expression. However, compared with untreated cells, cytokine-treated cell lines showed an increase in Her2(369)-A2 epitope density that directly correlated with enhanced tumor cell death (p = 0.05). Although a trend was observed between tumor cell lysis and the level of the Her2(369)-A2 complex for untreated cells, the association was not significant. These findings suggest that tumor cell susceptibility to CTL-mediated lysis may be predicted based on the level of specific peptide-MHC class I expression rather than on the total level of TAA expression. Further, these studies demonstrate the potential of the TCRm mAb for validation of endogenous HLA-peptide epitopes on tumor cells.     

Protein changes associated with stages of activation of mouse macrophages for tumor cell killing

Bone marrow-derived mouse macrophages become activated for tumor cell killing by traversing a series of stages. The stages studied here were as follows: unstimulated (exposed to nothing but medium), primed (prepared to become cytolytic), fully activated (primed macrophages exposed to a triggering agent), and postcytolytic (previously activated macrophages that had gradually lost cytolytic activity after the removal of stimuli). Macrophages were labeled with [35S]methionine, lysed, and subjected to 2-D gel electrophoresis and fluorography. The priming agent used was recombinant mouse IFN-gamma, 10 to 20 U/ml. Bacterial lipopolysaccharide (LPS), 0.4 to 1 ng/ml, was used as the triggering agent. A total of 40 major changes was identified in macrophages treated with both agents. Twenty-six of these were seen in macrophages treated with IFN-gamma, and 35 were found in LPS-treated macrophages. Twenty-two of the 40 changes were found in both IFN-gamma- and LPS-treated macrophages. The major reason for this overlap was the autocrine action of IFN-alpha/beta secreted from LPS-treated macrophages. Changes in expression of specific proteins, designated p47b and p71/73, were found to correlate closely with the development and loss of the activated state. With the use of these proteins as markers, phenotypes could be constructed that distinguished unstimulated, LPS-treated, primed, and fully activated macrophages. Postcytolytic macrophages had a phenotype similar to unstimulated macrophages and could be reactivated by reexposure to inducing agents. They also reexpressed the protein markers that were characteristic of fully activated macrophages.     

Monoclonal antibodies against tumor associated antigens

By the use of xeno-immunization, syngeneic immunization, or human lymphocytes, we prepared monoclonal antibodies against human epithelial tumors, especially against bladder tumors. Here, we describe results of antigen analyses and application to the tumor diagnosis.     

Independent control of Ly49g alleles: implications for NK cell repertoire selection and tumor cell killing

A novel murine NK cell-reactive mAb, AT8, was generated. AT8 recognizes Ly49G from 129/J, BALB/c, and related mouse strains, but does not bind to Ly49G(B6). Costaining with AT8 and a Ly49G(B6)-restricted Ab (Cwy-3) provides the first direct evidence that Ly49G protein is expressed from both alleles on a significant proportion of NK cells from four different types of F(1) hybrid mice. The observed level of biallelic Ly49G expression reproducibly followed the product rule in both freshly isolated and cultured NK cells. Surprisingly, the percentage of NK cells expressing both Ly49G alleles could be dramatically increased in vitro and in vivo through IL-2R- and IFN receptor-dependent signaling pathways, respectively. Unexpectedly, Ly49G(B6+) NK cells in an H-2(d), but not H-2(b), background were more likely to lyse D(d+) and Chinese hamster ovary tumor cells than Ly49G(BALB/129+) NK cells. Furthermore, Ly49G(B6+) NK cells also proliferated to a higher degree in response to poly(I:C) than NK cells expressing a non-Ly49G(B6) allele in an H-2(d), but not H-2(b), background. These results suggest that Ly49G(B6) has a lower affinity for H-2D(d) than Ly49G(BALB/129), and the genetic background calibrates the responsiveness of NK cells bearing self-specific Ly49. Other H-2D(d) receptors on the different Ly49G(+) NK cell subsets were unequally coexpressed, possibly explaining the disparate responses of Ly49G(B6+) NK cells in different hybrid mice. These data indicate that the stochastic mono- and biallelic expression of divergent Ly49G alleles increases the range of MHC affinities and the functional potential in the total NK cell population of heterozygous mice.     

Comparative effects of various classes of mouse interferons on macrophage activation for tumor cell killing

The effects of mouse interferon-alpha (MuIFN-alpha), -beta (MuIFN-beta), and -gamma (MuIFN-gamma) on macrophage activation for tumor cell killing were determined by using proteose peptone-elicited peritoneal macrophages from C3H/HeN and C3H/HeJ mice under conditions that either included or were free of detectable endotoxin. Alone, under the conditions used, none of the interferons was able to activate macrophages directly for tumor cell killing. However, with a second signal provided to responsive macrophages by contaminating endotoxin, added bacterial lipopolysaccharide (LPS), or heat-killed Listeria monocytogenes (HKLM), all three types of interferon induced cytolytic activity, with MuIFN-gamma approximately 500 to 1000-fold more active than either MuIFN-alpha or -beta. Thus, all three interferons were able to prime macrophages for killing but required a second signal before cytolytic activity could be expressed. When MuIFN-gamma was mixed with either MuIFN-alpha or -beta and placed on macrophages, little or no killing developed. Mixtures of MuIFN-gamma with either MuIFN-alpha or -beta did increase the sensitivity of macrophages to triggering by LPS, however, compared with macrophages treated with MuIFN-gamma alone. The results are collectively important because they i) confirm that significant quantitative differences exist between the various interferons with regard to their capacity to prime macrophages for tumor cell killing; ii) indicate that to be an efficient activator each type of interferon must be combined with a second stimulus, such as LPS or HKLM; iii) show that neither MuIFN-alpha nor -beta can provide an efficient second triggering signal for macrophages that are primed by MuIFN-gamma; and iv) document that mixtures of MuIFN-gamma with either MuIFN-alpha or -beta are most efficient at inducing priming, compared with any one of the interferons used alone.