The effect of anti-CD3 on the induction of non-MHC restricted cytolytic activity.

The effects of an anti-CD3 mAb on induction of non-MHC restricted cytolysis was investigated. Peripheral blood mononuclear cells (PBMC) from normal donors (29) and cancer patients (18) were cultured in 100 U/ml of interleukin-2 (rIL-2) with and without anti-CD3 mAb (OKT3, 10 ng/ml) for the first 48 hours of incubation. Thereafter, both PBMC cultures were maintained on rIL-2 up to 20 days. PBMC proliferation was enhanced 17-fold in number by day 20 when anti-CD3 mAb and rIL-2 was present during the first 48 hours but only 3-fold by day 20 when rIL-2 alone was present. Concomitantly anti-CD3 mAb but not Lym-1, an isotype matched control, inhibited the induction of lytic activity against both NK sensitive (K562) and NK resistant (Raji) target cell lines. Thus the inhibitory effect is dependent on anti-CD3 mAb stimulating the CD3/TCR T-cell receptor complex. While lytic activity was dependent on the concentration of rIL-2, inhibition of the induction phase of non-MHC restricted lytic activity was independent of the concentration of rIL-2. Flow cytometry analysis indicated that treatment with the anti-CD3 mAb increased the percentage of CD3 positive cells, CD4 positive cells and especially CD25 positive cells, but decreased th percentage of CD56 positive cells. Supernatants from anti-CD3 mAb stimulated cultures also inhibited the induction of non-MHC restricted lytic activity. Lymphokine analysis showed that supernatants of anti-CD3 mAb stimulated cultures had higher levels of TNF-alpha and IFN-gamma. However, TNF-alpha and IFN-gamma alone or in combination could not mediate the inhibitory effect. The inhibitory factor(s) was partially purified by sequential chromatography on matrices of controlled pore glass and Sepharose CL-6B. The molecular weight of the inhibitory factor(s) was less than 67K. These studies have identified a novel regulatory pathway controlling non-MHC restricted cytolysis. Perturbation of the T-cell CD3/TCR complex with the anti-CD3 mAb results in the secretion of a soluble mediator that down-regulates the induction of rIL-2 dependent non-MHC restricted cytolysis.

The production of a bispecific anti-CEA, anti-hapten (4-amino-phthalate) hybrid-hybridoma.

A standard hybridoma fusion technique was used to produce a monoclonal antibody capable of binding both carcinoembryonic antigen (CEA) and the hapten 4-amino-phthalate. A hypoxanthine-aminopterin-thymidine (HAT) sensitive anti-CEA hybridoma and KLH-phthalate immunized spleen cells were hybridized to yield clones producing bispecific monoclonal antibodies. The desired bispecific antibody was identified using both enzyme-linked immunosorbent assay (ELISA) and radio-immunoassay. The resultant hybrid-hybridoma or "tridoma" was subcloned and expanded to yield a stable population. Bifunctional antibody was then isolated from the various possible recombinants by ion exchange chromatography. This general method may be used to produce bispecific monoclonals against a wide variety of tumor associative antigens and reagents for immunodetection or treatment.

Interleukin-2 with ex vivo activated killer cells: therapy of advanced non-small-cell lung cancer.

A phase II study was conducted to examine the efficacy of interleukin-2 (IL-2) with lymphokine-activated killer (LAK) cells as therapy for advanced non-small-cell lung carcinoma (NSCLC). IL-2 was administered at a fixed dose of 6 x 10(6) U/M2 per day as a 24 h continuous intravenous infusion (CIV) with LAK cells. Eleven patients were entered onto this study and six were evaluable. One patient had a near complete response of 18 months duration. Only two patients were able to complete the regimen without dose reduction. This regimen was poorly tolerated with pulmonary toxicity being the major problem. The partial responder was the only patient to undergo more than one course of therapy. IL-2/LAK therapy may have activity in NSCLC and further studies are warranted in this uniformly fatal disease. However, future studies will have to incorporate less toxic IL-2 regimens.

Interleukin-2 lymphokine-activated killer cell therapy of non-Hodgkin's lymphoma and Hodgkin's disease.

We conducted a phase II study utilizing interleukin-2 (IL-2) with lymphokine-activated killer (LAK) cells as therapy for non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD). IL-2 was given at a fixed dose of 3 x 10(6) U/m2/day administered as a 24-h continuous intravenous infusion with LAK cells. Nineteen extensively treated patients were entered and 15 were evaluable. In general, this regimen was reasonably well tolerated with mild toxicities that were rapidly reversible. Patients who completed therapy did so without dose attenuations. However, discontinuation of therapy was necessary in four patients due to atypical toxicities that were not clearly dose related. Two patients (one NHL and one HD) had partial remissions of brief duration, four had disease stabilization, and seven had progressive disease. While there were not sufficient numbers to evaluate critically any NHL or HD subtype, this regimen does not appear to have significant activity for either disease.

Biodistribution, pharmacokinetic, and imaging studies with 186Re-labeled NR-LU-10 whole antibody in LS174T colonic tumor-bearing mice.

Biodistribution, pharmacokinetic, and radioimaging studies were performed with 186Re-labeled NR-LU-10 whole antibody in athymic nude mice bearing the LS174T tumor growing either s.c. or in an experimental hepatic metastasis model. NR-LU-10 is an IgG2b murine monoclonal antibody (MAb) that reacts with virtually all human tumors of epithelial origin. NR-BC-1, a IgG2b murine MAb that reacts with normal human B-cell and B malignancies, was used as an isotype-matched control. These MAbs were radiolabeled with 186Re (3.7-day physical half-life; 1.07-MeV beta particle and 137-keV gamma, 9% abundance) by a preformed chelate approach by using the triamide thiolate ligand system. 186Re-labeled NR-LU-10 (50 microCi) was injected into nude mice bearing LS174T tumors growing s.c. Biodistribution studies revealed that the LS174T tumor retained the highest concentration of 186Re-labeled NR-LU-10 (5.3% injected dose/g) at day 6. The tumor:blood ratio ranged from 0.1:1 to 10.8:1 by day 6, the last day of analysis. In contrast the tumor:blood ratio of 186Re-labeled NR-BC-1, the isotype-matched MAb control, was 1:1 on day 6. Pharmacokinetic analysis indicated that the t1/2 beta of NR-LU-10 for blood and other tissues ranged from 21 to 25 h, while the t1/2 beta for the LS174T tumor averaged 52 h. The area under the curve for tumor compared to blood was 2.8- to 5.7-fold higher than the area under the curve for all other tissues and organs. The mean residence time for NR-LU-10 in blood and all other organs ranged from 23 to 26 h, while the mean residence time for NR-LU-10 in the LS174T tumor was 72 h. Scintigraphic images revealed selective uptake of the 186Re-labeled NR-LU-10, but not of the 186Re-labeled NR-BC-1, at the LS174T tumor site. Studies in an experimental model of hepatic metastasis revealed a similar selective pattern of 186Re-labeled NR-LU-10 accumulation. Scintigraphic images of the LS174T tumor growing within the athymic nude mouse liver were obtained. The biodistribution, pharmacokinetic, and scintigraphic image results suggest that 186Re-labeled NR-LU-10 shows promise as a therapeutic agent for gastrointestinal cancer.

Comparison of cisplatin and carboplatin cytotoxicity in human ovarian cancer cell lines using the MTT assay.

In this study, we compared the cytotoxicity of cisplatin and carboplatin against a panel of human ovarian cancer cell lines using the MTT assay, a rapid colorimetric test that can be used to evaluate the number of residual viable tumor cells following chemotherapy. The established human ovarian cancer cell line OVCAR-3 and the recently isolated and characterized A721, A90, A286, A1, and A121A cell lines were evaluated for chemosensitivity. Each cell line was treated separately with cisplatin and carboplatin at concentrations ranging from 500 to 0.16 micrograms/ml. Various chemotherapeutic exposure periods (1, 4, 24, and 48 hr) were tested to determine maximal efficacy. All cell lines were more susceptible to cisplatin than carboplatin at all drug concentrations and all exposure periods tested (P = 0.005). The overall median 50% inhibitory concentration (ID50) for cisplatin was 107 micrograms/ml compared with 490 micrograms/ml for carboplatin P = 0.005). For both cisplatin and carboplatin a 24-hr exposure was significantly more cytotoxic than a 1-hr exposure (P = 0.003 and P = 0.006, respectively). These in vitro results suggest that cisplatin is significantly more cytotoxic than carboplatin against human ovarian cancer cell lines and that cisplatin should not be replaced by carboplatin in the treatment of advanced epithelial ovarian cancer until randomized trials using maximum dosing of the cisplatin-containing regimen are performed.

Antibody-dependent, cell-mediated cytotoxicity by an anti-class II murine monoclonal antibody: effects of recombinant interleukin 2 on human effector cell lysis of human B-cell tumors.

Lym-1 is an IgG2a murine monoclonal antibody that reacts with variant Class II molecules expressed on B-cell malignancies. Lym-1 was shown to mediate antibody-dependent cellular cytotoxicity (ADCC) of human effector cells against a variety of malignant B-cell lines. Tumor cell lysis was Lym-1 specific because (a) the reaction was dose dependent with significant ADCC detectable at Lym-1 concentrations as low as 1 microgram/ml; (b) tumor targets not expressing the Lym-1 antigen were unaffected; (c) an isotype-matched irrelevant monoclonal antibody and an IgG1 anti-Class II monoclonal antibody failed to mediate ADCC; and (d) addition of Protein A (which binds avidly to Lym-1) blocked ADCC by 90 to 100%. Peripheral blood mononuclear cells obtained from normal donors as well as from cancer patients were able to interact with Lym-1 to elicit ADCC. Recombinant interleukin 2 (rIL-2) enhanced non-antibody-mediated tumor lysis and Lym-1 ADCC with an optimal concentration of 100 units/ml. Pulse treatment of normal peripheral blood mononuclear cells with rIL-2 was able to augment Lym-1 ADCC but was less effective than having the rIL-2 present through the assay. Peripheral blood mononuclear cells obtained from patients being treated with high doses of rIL-2 administered by continuous i.v. infusion demonstrated Lym-1 ADCC levels which were higher than normal individuals and which were further augmented by in vitro incubation with rIL-2.

Role of asialo-GM1 positive liver cells from athymic nude or polyinosinic-polycytidylic acid-treated mice in suppressing colon-derived experimental hepatic metastasis.

Liver-derived (LD) murine colon adenocarcinoma MCA-38 cells injected into the ileocolic vein (ICV) of C57BL/6 mice developed distinct hepatic foci within 14-21 days and survived for an average of 19-35 days. In contrast, C57BL/6-nu/nu mice given injections of LD-MCA cells by the same route did not develop hepatic lesions. Furthermore, 111In-labeled LD-MCA-38 tumor cells were rapidly taken up by the liver of conventional mice within 1 h and 73% of the radioactivity remained after 24 h. However, about 60% of the 111In-labeled LD-MCA-38 tumor cells were cleared from the liver of nude mice after 24 h. Nonparenchymal liver cells isolated from untreated conventional mice displayed little cytotoxicity against freshly excised 51Cr-labeled LD-MCA-38 cells but did lyse the standard natural killer target, YAC-1 tumor cells, in 4 h chromium release assays. On the other hand, nonparenchymal liver cells but not spleen cells from nude mice were cytotoxic to 51CR-labeled LD-MCA-38 in vitro. The nonparenchymal liver cell population responsible for tumor killing was phenotypically nonadherent and asialo-GM1 (AsGM1) positive. C57BL/6 mice treated with polyinosinic-polycytidylic acid [poly(IC)] also displayed cytotoxic activity against LD-MCA-38 tumor cells in vitro. Furthermore, poly(IC) treatment of mice 1-8 days after tumor inoculation suppressed the number of hepatic foci and also significantly increased the life span of tumor-bearing mice. Treatment of athymic nude mice or poly(IC)-treated conventional mice with anti-AsGM1 induced significant numbers of foci and significantly decreased the life span of MCA-38-bearing mice suggesting that AsGM1-positive cells in the liver of these mice may inhibit tumor growth in vivo. In conclusion, the host defense system of the liver from athymic nude or poly(IC)-treated mice possess AsGM1-positive cells that can suppress tumor implantation or tumor growth in the early stages of metastasis in liver.

Small animal MRI at 0.35 Tesla: growth and morphology of intra-organ murine tumors.

A whole-body small animal radiofrequency coil was designed and built for use with a 0.35 Tesla clinical magnetic resonance imager. The primary motivation for this work was to evaluate the effectiveness of this system for small animal magnetic resonance imaging of tumor-bearing mice. This noninvasive technique is shown to provide high resolution whole-body images of mice, to be capable of detecting intra-organ tumors, and to be useful for evaluating tumor size and growth. Its potential for monitoring response to experimental therapeutic regimens is also noted. Two tumor models were examined--colon adenocarcinoma MCA-38 and human ASPC-1 pancreatic adenocarcinoma.

Monoclonal antibody to a human pancreatic carcinoma cell line recognizes gastrointestinal neoplasms.

A monoclonal antibody designated RWP1.1 was produced against a human pancreatic carcinoma cell line RWP1. This antibody was shown to recognize an epitope of carcinoembryonic antigen. The reactivity of the antibody was evaluated on formalin-fixed normal tissues, 86 malignant neoplasms, 10 colonic polyps, and 6 cases of chronic pancreatitis using the peroxidase anti-peroxidase technique. RWP1.1 did not react with normal tissues apart from weak staining of fetal pancreatic ducts. This antibody preferentially reacted with primary and metastatic adenocarcinomas of the colon, stomach, pancreas, and colonic polyps but not with most adenocarcinomas from other sites nor with other types of tumors or cases of chronic pancreatitis. It also reacted with colon adenocarcinomas on frozen sections. The restricted specificity of this antibody could be used in differentiating gastrointestinal adenocarcinomas from other types of tumors including adenocarcinomas from other sites and most pancreatic adenocarcinomas from chronic pancreatitis.

Partial hepatectomy augments the liver's antitumor response.

Despite adequate locoregional control, colorectal metastasis to the liver remains a significant cause of death. Resection of hepatic metastasis improves five-year survival 18% to 34%. A study of the impact of 40% partial hepatectomy on cytokine production in the liver was undertaken. Nonparenchymal liver cells (NPCs) were prepared by collagenase perfusion and metrizamide gradient from partially hepatectomized and laparotomized control C57BL/6Ros mice. Nonparenchymal cell from partially hepatectomized mice compared with laparotomized mice showed a twofold to threefold increase in interferon (IFN) activity. Both interferon alpha/beta and supernatants from cultured NPCs of partially hepatectomized mice suppressed the proliferation of liver-derived MCA-38 colon adenocarcinoma cells in vitro. This tumor has been shown to metastasize to the liver of C57BL/6Ros mice. The production of various cytokines by NPCs induced by partial hepatectomy may provide a possible antimetastatic mechanism.

Hepatectomy prolongs survival of mice with induced liver metastases.

Resection of hepatic metastases from colorectal cancer has been shown to prolong survival in some patients. Whether this results from a reduction of tumor burden or is an indirect effect mediated by hepatectomy is questionable. Male C57BL/6Ros 8-week-old mice underwent ileocolic vein injection of a suspension of 0.3 mL of 2 x 10(5) viable liver-derived murine (MCA-38) colonic adenocarcinoma cells. This model produces hepatic metastases in all lobes of the liver. At 7, 14, or 21 days after tumor injection, mice were randomized to receive either 42% resection of the liver or laparotomy alone. Survival in the animals with hepatectomy was significantly prolonged when the hepatectomy was performed 14 or 21 days after tumor injection.

Modulation of colon-derived experimental hepatic metastasis by murine nonparenchymal liver cells.

Although numerous animal tumor models have been used to study colon carcinoma, few display hepatic metastasis. C57B1/6Ros mice inoculated with liver-derived murine colon adenocarcinoma MCA-38 in the ileocolic vein develop distinct hepatic foci within 21 days and survive an average of 35 days. Furthermore, 111In-labeled LD-MCA-38 tumor cells were rapidly taken up by the liver within 60 min and 73% of the label remained in the liver after 24 h. Isolated nonparenchymal liver cells from untreated mice displayed little cytotoxicity against freshly excised 51Cr-labeled MCA-38 cells but did inhibit tumor growth in vitro as measured by inhibition of 3H-thymidine incorporation. Treatment with anti-asialo-GM1 decreased the lifespan of MCA-38 tumor bearing mice suggesting that asialo-GM1 positive cells in the liver may inhibit tumor growth in vivo. Nonparenchymal liver cells from mice treated with polyinosinic-polycytidylic acid showed augmented cytotoxic and cytostatic activity against LD-MCA-38 tumor cells in vitro. Polyinosinic-polycytidylic acid treatment also significantly increased the lifespan of MCA-38 tumor bearing mice. In conclusion, the host defense system of the liver can be modulated to enhance or inhibit colon-derived experimental hepatic metastasis.

Detection and characterization of a pancreatic tumor inhibitory factor.

Numerous growth factors which stimulate cellular proliferation have been identified and characterized. Little information is available on autocrine factors that inhibit growth. The objective of this study was to determine if an established human pancreatic cancer cell line, RWP-1, produced such a tumor growth inhibitory factor (TIF). RWP-1 serum-free conditioned media (CM) was concentrated and its effect on DNA synthesis was determined by measuring incorporation of [3H]thymidine. CM inhibited [3H]thymidine incorporation in a dose-dependent and nontoxic manner with all cell lines tested. TIF activity was stable at 56 degrees C, labile at 95 degrees C and partially inactivated by acid, reduction/alkylation, and enzymatic digestion. The TIF exhibited differential activity against the tested cell lines and was found to be active against a normal murine fibroblast line. The TIF appears to be a polypeptide with a molecular weight greater than 5000 Da. Attempts at isolation of TIF with affinity and ion-exchange chromatography have thus far been unsuccessful, implying that more than one factor may be involved. RWP-1 TIF is a potentially important factor because of its role in the regulation of cellular proliferation.

Relationship of in vitro cell motility and colonization potential in a mouse colon adenocarcinoma (MCA-38) cell line.

Cell motility is an important factor in the metastatic process that can be affected by environmental conditions. A quantitative study was made of the relationship between cell motility and the colonization potential of a mouse colon adenocarcinoma cell line (MCA-38). MCA-38 cells grown in culture did not produce hepatic or pulmonary colonies following ileocolic or tail vein injection, respectively. In contrast, MCA-38 cells adapted to grow in the mouse produced colonies in both organs. The motility of the MCA-38 cells that did not produce colonies, as determined by the depth of penetration into cellulose nitrate filters (8 micron pore size), was significantly less than that of MCA-38 cells with colony-forming potential. Return to in vitro growth resulted in both a loss of colonization potential and a reduction in motility. In this system, secondary organ colonization and in vitro cell motility are positively correlated, suggesting an association between cell motility and metastatic potential.

Effects of liposome-entrapped doxorubicin on liver metastases of mouse colon carcinomas 26 and 38.

The effects of doxorubicin (DOX) and DOX entrapped in standardized liposomes [mean diameter, 0.15 micron; (DOX-Lip)] on the survival of mice bearing liver metastases of mouse colon carcinoma CT38LD (C57BL/6J mice) or CT26 (BALB/c mice) were investigated. In vitro cultured CT38LD cells were more sensitive to DOX than CT26. In vivo DOX and DOX-Lip, administered iv 10 mg/kg weekly to a maximum of five injections, increased the life-spans of mice bearing CT38LD liver metastases 32% (P less than .05) and 64% (P less than .05), respectively. DOX-Lip was more effective than DOX in prolonging survival (P less than .05). Free DOX did not significantly increase the life-spans of mice bearing CT26 liver metastases (P greater than .5), whereas DOX-Lip increased the life-spans 35% (P less than .05). The results suggest that liposomal delivery of agents to the liver can enhance therapeutic activity and could be used as an arm of protocols for adjuvant therapy of liver metastases.

Characterization of a murine tumor of spontaneous origin with selective hepatic metastasis.

The primary objective of this study was to establish the identity and characteristics of a murine uterine tumor of spontaneous origin in a C57Bl/6 Ros mouse that selectively metastasized to the liver. The primary tumor contained a minimum of two different cell types. One cell type was elongated and spindly and readily adhered to a plastic coated surface. From it, a permanent cell line, termed RCS-1 was established. The RCS-1 cell line was poorly tumorigenic in normal C57Bl/6 Ros mice. The other cell type, designated RCS-2, was more rounded in structure. It was maintained in vivo, enzymatically dissociated and used after short-term in vitro cell culture. The RCS-2 cells had phagocytic vacuoles, Fc, and C3 receptors. These cells phagocytosed antibody coated SRBCs and opsonized zymosan. Furthermore, these cells generated the superoxide anion. Thus, the RCS-2 cells are of macrophage origin. The spontaneous and experimental pattern of metastasis of the RCS-2 tumor cells was established. The RCS-2 tumor cells selectively metastasized to the liver by the hematogenous route. Metastatic RCS-2 tumor cells obtained from the liver (RCS-2M) retained their macrophage characteristics. These studies indicate that this murine uterine tumor of spontaneous origin consists of two cell types. One cell type is a reticulum cell sarcoma.

Description of a murine model of experimental hepatic metastases.

A murine model of experimental hepatic metastases has been developed. The cecum is exteriorized through a midline incision, and 1.5 X 10(5) MCA-38 liver-derived (LD) tumor cells in 0.1 ml was injected into the ileocolic vein (ICV). Ninety-eight percent of injected mice developed hepatic foci. The operative mortality was 6.1%. Micrometastases could first be detected on day 11. Laparotomy of 21 days revealed the presence of a mean of 18 hepatic foci. Experimental hepatic metastases could be palpated 35 days following ICV injection. Mice bearing MCA-38 LD foci survived an average of 53.3 days. This model will be of use in the development of novel approaches for the treatment of hepatic metastasis.

Characterization of a renal cell carcinoma cell line suitable as a target for immunological studies in vitro and in the nu/nu mouse.

A continuous human renal carcinoma cell line designated RPMI-SE was established from a patient with a poorly to moderately differentiated renal cell carcinoma. The cells are anchorage dependent, have a well defined globular shape with pseudopod-like structures, a doubling time in vitro of 24 hr. and are able to grow subcutaneously in female ICR Swiss nu/nu mice. RPMI-SE cells obtained from tissue culture and the nude mouse had the chromosome number of near-tetraploidy. Common morphologic rearrangements were present in both the cell line and nu/nu mouse tumor. RPMI-SE was evaluated as a target cell line in an in vitro Indium-111 release assay. Three patterns of cytotoxicity were observed at an effector to target cell ratio of 100:1. The highest degree of cytotoxicity (75 per cent) was obtained with autologous lymphocytes. An intermediate level of cytotoxicity (50 per cent) was obtained with allogeneic lymphocytes. The lowest level of cytotoxicity (27 per cent) was obtained with normal lymphocytes and was comparable to the level of cytotoxicity observed with the NK sensitive K562 target cell line. Morphologic data and chromosomal analysis indicate that the RPMI-SE cell line has maintained characteristics of the original tumor. This cell line will be useful for immunological studies as a target cell line in vitro as well as in vivo in the nude mouse.

Humoral immune response of patients receiving specific active immunotherapy for renal cell carcinoma.

The object of this study was to characterize the antigens evoking an immune response in renal cell carcinoma (RCC) patients receiving specific active immunotherapy with irradiated autologous tumor cells and Corynebacterium parvum as adjuvant. Seventy serum samples from 11 patients with RCC undergoing specific active immunotherapy were evaluated. Fifty of the 70 serum specimens (71%) had immunoglobulin G antibody directed to autologous tumor cells. Absorption studies were completed on 4 patients (S.E., M.M., R.N., S.V.) with 2 patients (S.V. and M.M.) demonstrating reactivity to a RCC-associated antigen present on their autologous tumor cells. One patient's serum (R.N.) was absorbed not only with autologous tumor cells but also with an allogeneic RCC cell line. The fourth patient's (S. E.) serum reactivity was able to be absorbed only with autologous tumor cells and several, but not all, of the clones of that autologous cell line. Patient S.E. serum binding by clones of RCC cell line RPMI-SE was seen to vary from no ability to bind RPMI-SE in some clones to double the parental binding in others. Consistent with this finding was the demonstration that high serum-binding clones could absorb Patient S.E. serum reactivity to autologous RCC cells, while low binding clones could not. These data suggest a measure of heterogeneity among the parental RCC cell line, as demonstrated by its clones. This study has shown that the autologous tumor vaccine with adjuvant used here was an immunogenic therapeutic agent. The response mounted by these patients was a response to a RCC-associated antigen with the level of reactivity changing with the number of immunizations and disease status. Also suggested by this work is the possible primary tumor heterogeneity, as demonstrated by the differential reactivity seen among clones of a RCC cell line established from such a primary tumor.