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Development of distant metastases and acquired multidrug resistance (MDR) are major problems in therapy for human small cell lung cancer (SCLC). MS-209 is a novel quinoline compound, which reverses P-glycoprotein (P-gp)-mediated MDR. We previously reported that MS-209 reversed in vitro MDR of human SCLC (SBC-3 / ADM and H69 / VP) cells expressing P-gp. In the present study, we determined the therapeutic effect of MS-209 in combination with chemotherapy against multiorgan metastases of MDR SCLC cells. SBC-3 / ADM cells expressing P-gp were highly resistant to etoposide (VP-16), adriamycin (ADM), and vincristine (VCR) in vitro, compared with parental SBC-3 cells lacking P-gp expression. MS-209 restored chemosensitivity of SBC-3 / ADM cells to VP-16, ADM, and VCR in a dose-dependent manner in vitro. Intravenous injection with SBC-3 or SBC-3 / ADM cells produced metastatic colonies in the liver, kidneys and lymph nodes in natural killer (NK) cell-depleted severe combined immunodeficiency (SCID) mice, though SBC-3 / ADM cells more rapidly produced metastases than did SBC-3 cells. Treatment with VP-16 and ADM reduced metastasis formation by SBC-3 cells, whereas the same treatment did not affect metastasis by SBC-3 / ADM cells. Although MS-209 alone had no effect on metastasis by SBC-3 or SBC-3 / ADM cells, combined use of MS-209 with VP-16 or ADM resulted in marked inhibition of metastasis formation by SBC-3 / ADM cells to multiple organs. These findings suggest that MS-209 reversed the MDR of SBC-3 / ADM cells, but not SBC-3 cells, growing in the various organs, and inhibited metastasis formation in vivo. Therefore, this chemosensitizing agent, MS-209, may be useful for treatment of refractory SCLC patients with multiorgan metastases.

In the present study, we examined the effect of interleukin-2 (IL-2) gene transfer into multidrug resistance (MDR) cancer cells on the therapeutic efficacy of MRK16. Human MDR ovarian cancer cells, AD10, were transduced with a bicistronic IL-2 retrovirus, Ha-IL2-IRES-Neo. The G418-resistant population, IL2-AD10, secreted IL-2 into the culture supernatant and did not form a tumor mass in nude mice. The IL2-AD10 cells were more susceptible to the cytotoxicity of murine spleen cells than AD10 cells in vitro. For examination of the effect of IL-2 gene transfer on the antitumor activity of MRK16 against P-glycoprotein-positive tumors, IL2-AD10 cells were co-transplanted s.c. with AD10 cells into nude mice in a ratio of 1:3, and the mice were treated with MRK16 on days 2 and 7. MRK16 markedly inhibited the growth of AD10 cells mixed with IL2-AD10 cells under conditions (0.3-1 microgram/body) where it showed only marginal effects on the growth of AD10 tumors. These findings suggest that IL-2 gene transfer potentiates the antitumor activity of MRK16 against MDR tumors.

To develop a therapeutic modality for overcoming multidrug‐resistant (MDR) cancer with anti‐MDR1 antibody, we examined the effect of macrophage colony‐stimulating factor (M‐CSF) gene transfection into MDR AD10 cells on therapy of MDR cancer with anti‐MDR1 antihody (MRK17) in nude mice. MDR human ovarian cancer (AD10) cells were transduced with the human M‐CSF gene inserted into an expression vector to establish gene‐modified cells capable of producing low (ML‐AD10), intermediate (MM‐AD10) and high (MH‐AD10) amounts of M‐CSF. Systemic administration of MRK17 resulted in significant dose‐dependent inhibition of subcutaneous growth of ML‐AD10 tumors. In contrast, systemic administration of recombinant M‐CSF in combination with MRK17 did not augment the therapeutic efficacy of MRK17 alone, but rather promoted the growth of the parent AD10 cells. To test the efficacy of in vivo M‐CSF gene therapy combined with antibody, we mixed the parent AD10 cells with MH‐AD10 cells producing a large amount of M‐CSF, and inoculated the mixed cells subcutaneously. Treatment with MRK17 inhibited growth of the mixed cells more than that of the parent cells alone. Thus, combined therapy with anti‐MDR1 mAb and M‐CSF gene modification of MDR cancer cells may provide a new immunotherapeutic modality for overcoming MDR in humans.

Ganglioside GM2 (GM2) frequently appears on the cell surface of human cancers of neuroendocrine origin. A mouse-human chimeric monoclonal antibody (mAb), KM966, against GM2 was previously found to promote the lysis of various cancer cells by human blood mononuclear cells (MNC). In this study, we analyzed the effector cells responsible for the chimeric mAb-dependent cell-mediated cytotoxicity (ADCC) against small cell lung cancer (SCLC) cells and examined the enhancing effect of various cytokines on the ADCC activity. The ADCC activity was assessed by 4-h 51Cr release assay. Highly purified lymphocytes (> 99%) and monocytes (> 90%) were separated by centrifugal elutriation from peripheral blood MNC of the same healthy donor. KM966 induced lysis of SCLC cells mediated by both lymphocytes and monocytes to similar extents, in a dose-dependent manner. Pretreatment of lymphocytes with various cytokines [interleukin (IL)-2, IL-12 and interferon-gamma] and that of monocytes with macrophage-colony-stimulating factor significantly augmented the killer activity against SCLC cells in the presence of KM966 mAb. KM966 was also effective for the lysis of non-small cell lung cancer cells in direct proportion to the GM2 expression levels. These findings suggest that combined treatment of KM966 mAb with cytokines may be therapeutically useful for in vivo killing of lung cancer cells expressing GM2 through the ADCC reaction.

Cyclosporin A (CsA) was previously found to bind to P-glycoprotein expressed on multidrug-resistant (MDR) cancer cells. In the present study, the effect of CsA on anti-P-glycoprotein monoclonal antibody (mAb)-dependent cell-mediated cytotoxicity (ADCC) against human MDR cells was examined. The ADCC reaction was assessed by 4-h 51Cr-release assay. Highly purified lymphocytes (> 99%) and monocytes (> 99%) obtained from blood mononuclear cells (MNC) of healthy donors were used as effector cells. CsA decreased the cytotoxic activity of MNC against MDR cells, but enhanced their ADCC activity in the presence of anti-P-glycoprotein mAb MRK16. Lymphocyte-mediated ADCC and natural killer activity against MDR cells were also suppressed by addition of CsA. CsA induced a significant dose-dependent increase in monocyte-mediated ADCC activity. Interestingly, pretreatment of MDR cancer cells, but not of monocytes, with CsA significantly enhanced ADCC activity mediated by monocytes, but not by lymphocytes. A CsA analog (PSC833) and FK-506, but not verapamil also increased the sensitivity of MDR cells to ADCC by monocytes. CsA did not affect the binding of monocytes to MDR cells in the presence of MRK16 mAb. These results indicate that CsA may directly enhance the susceptibility of MDR cancer cells to the monocyte-mediated ADCC reaction.

Pore-forming protein (PFP) is an important effector molecule for cytotoxicity mediated by cytotoxic T cells and NK cells. In the present study, the effect of monocytes on PFP production by interleukin-2 (IL-2)-stimulated T lymphocytes was examined. Highly purified lymphocytes (> 99%) and monocytes (> 90%) were isolated by centrifugal elutriation from peripheral blood of healthy donors, and, CD4+ and CD8+ cells were isolated from the purified lymphocytes by using antibody-bound magnetic beads. PFP production was quantitated with a universal microspectrophotometer in combination with immunostaining using anti-PFP antibody. Monocytes did not produce any PFP. High levels of PFP production were observed in CD8+ cells, but not CD4+ cells after incubation for 4 days with IL-2. Addition of monocytes to cultures of CD8+ cells resulted in significant augmentation of PFP production after 3 days' stimulation with IL-2. Monokines (TNF alpha and IL-6) caused a significant increase in PFP production by IL-2-stimulated CD8+ cells. Northern blot analysis revealed that the PFP mRNA levels was enhanced by stimulation with IL-2, and that addition of monocytes to cultures of CD8+ cells plus IL-2 augmented their PFP mRNA expression. These observations suggest that monocytes are important in in situ regulation of the CD8+ T cell-mediated cytotoxic response through production of PFP.

The present study was undertaken to examine the potential value of cetylmannoside‐modified multilamellar liposomes (Man‐MLV) as carriers for transfer of macrophage activators to blood monocytes. Highly purified blood monocytes were isolated by centrifugal elutriation from healthy donors under cndotox in‐free conditions. Freshly prepared monocytes phagocytosed Man‐MLV to a lesser extent than monocyte‐derived macrophages, but they took up Man‐MLV much more effectively than control liposomes without cetylmannoside (control MLV). Phagocytosis of Man‐MLV, but not control MLV by monocytes was inhibited by addition of D‐mannose, but not of D‐galactose. Desmethyl‐muramyl dipeptide (norMDP) entrapped in Man‐MLV was far more effective than norMDP entrapped in MLV in activating monocytes to the tumoricidal state. The effect of encapsulation of recombinant human macrophage colony‐stimulating factor (M‐CSF) in Man‐MLV on prolongation of survival of monocytes was examined. Blood monocytes that had been incubated for up to 21 days with Man‐MLV containing 5–20 U of M‐CSF per ml were effective in prolonging monocyte survival, but monocytes that had been incubated in medium with less than 50 If/ml of M‐CSF or with control MLV containing 5–10 U of M‐CSF showed no increase of monocyte survival over that in medium alone. Addition of rabbit anti‐M‐CSF antiserum did not affect survival prolongation of monocytes by M‐CSF encapsulated in Man‐MLV. We conclude that liposomes modified with cetylmannoside are far more effective than unmodified liposomes as a carrier to deliver biological response modifiers to human blood monocytes.

Mouse monoclonal antibodies (MRK16 and MRK17) against human multidrug-resistant cancer cell lines were tested for antibody-dependent cytotoxicity mediated by human blood mononuclear cells, using a 4-h 51Cr release assay. MRK16 (IgG2a isotype) was shown to be more effective than MRK17 (IgG1 isotype). Moreover, when four pairs of drug-resistant and their parent sensitive human cancer cells were tested for antibody-dependent cell-mediated cytolysis (ADCC) using MRK16, only the drug-resistant cell lines were susceptible to ADCC reaction. When highly purified lymphocytes (greater than 99%) and monocytes (greater than 97%) were isolated from blood mononuclear cells by centrifugal elutriation and adherence, MRK16 promoted both lymphocyte- and monocyte-mediated tumor cell killing, whereas MRK17 induced only a lymphocyte-mediated ADCC reaction. These results suggest that MRK16 of IgG2a subtype may be a useful therapeutic agent in eradication of drug-resistant cancer cells expressing P-glycoprotein through ADCC reaction.

The abilities of human alveolar macrophages (AM) obtained from healthy donors and patients with lung cancer to produce tumor necrosis factor (TNF) were compared with those of their blood monocytes after activation with lipopolysaccharide (LPS). TNF activity was assayed by measuring cytotoxicity against actinomycin D-treated L929 cells and TNF was determined quantitatively by sandwich enzyme-linked immunosorbent assay (ELISA) with polyclonal and monoclonal antibodies against TNF-alpha. Unstimulated AM from healthy donors released variable amounts of TNF spontaneously, whereas blood monocytes did not. When treated with LPS for 24 h, AM and monocytes produced TNF dose-dependently, but TNF production by AM was significantly more than that by blood monocytes. This TNF activity was inhibited completely by monoclonal anti-TNF-alpha antibody. Macrophages generated by in vitro maturation of monocytes induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) produced more TNF than freshly isolated monocytes. No difference was found in the abilities of AM from healthy donors and patients with lung cancer to produce TNF after activation stimuli. These observations suggest that human AM may be important in in vivo antitumor defense of the lung through TNF-alpha production.

Killer helper factor (KHF) was previously found to be produced by a human T cell hybridoma, 24A . CA2. We studied the therapeutic effects of interleukin-2 (IL-2) and KHF on the inhibition of pulmonary metastases of syngeneic Lewis lung carcinoma (3LL) in C57BL/6N mice. Multiple subcutaneous (sc) injections of IL-2 plus KHF had significantly more effect than injections of IL-2 alone in inhibiting spontaneous pulmonary metastases and prolonging survival of the mice. The effect of KHF with IL-2 on induction of lymphokine (IL-2)-activated killer (LAK) activity against P-29 cells was examined in the murine system. Spleen cells generated LAK activity after incubation for 4 days with more than 500 U/ml of IL-2. In contrast, KHF alone did not render spleen cells cytotoxic. The combination of these lymphokines at subthreshold concentrations, however, resulted in significant in vitro induction of LAK activity. The LAK activity of splenocytes incubated with IL-2 plus KHF was maximal after 4 days, and persisted for longer than that of cells treated with IL-2 alone. The LAK cells induced by KHF plus IL-2 were also cytotoxic to FBL and YAC-1 cells. Moreover, spleen cells of mice bearing lung metastases could be induced to the cytotoxic state by sc injections of IL-2 plus KHF. These results indicate that combination treatment with IL-2 and the new lymphokine KHF should be useful clinically in inducing LAK activity for inhibition of pulmonary metastases.

Mononuclear cells (MNC) from pleural effusions and peripheral blood of 18 patients with primary lung cancer with malignant pleural effusion were studied. Pleural and blood MNC generated lymphokine-activated killer (LAK) activity similarly when cultured for 4 days with an optimal concentration of interleukin 2 (IL-2). Highly purified lymphocytes (greater than 98%) and monocyte-macrophages (greater than 90%) were isolated by discontinuous Percoll gradient centrifugation from pleural and blood MNC. Pleural macrophages, as well as blood monocytes, showed significant augmenting effects on in vitro LAK cell induction from pleural and blood lymphocytes by IL-2. During daily intrapleural administration of IL-2, significant induction of LAK activity in vivo was observed after 3 days, but then this LAK activity in pleural MNC decreased almost to zero by day 15. Daily injections of IL-2 resulted in reduction in the up-regulation of LAK induction by pleural macrophages and also in increases in the levels of soluble IL-2 receptors in pleural effusions. These findings indicate that in vivo LAK induction of lymphocytes in malignant effusions by IL-2 may be regulated by macrophages in the effusions.