Enhancement of vincristine cytotoxicity in drug-resistant cells by simultaneous treatment with onconase, an antitumor ribonuclease.

BACKGROUND: Onconase, a protein isolated from oocytes and early embryos of the frog Rana pipiens, shares extensive homology with bovine pancreatic ribonuclease (RNase A) and possesses similar enzyme activity. Onconase is cytotoxic toward cancer cells in vitro and exhibits antitumor activity in animal models. In addition, Onconase has been shown to enhance the cytotoxic activity of some chemotherapeutic agents in vitro. PURPOSE: We studied interactions between the cytotoxic effects of Onconase and the chemotherapeutic agent vincristine (VCR) in the treatment of drug-sensitive and multidrug-resistant human colon carcinoma cells in vitro and in mice. METHODS: Transplantable human colon carcinoma cells (HT-29par cells) were infected with a retrovirus containing human mdr1 (also known as MDR1 and PGY1) complementary DNA (encoding P-glycoprotein [P-gp]), and clones that were cross-resistant to colchicine, doxorubicin, and vinblastine were selected (HT-29mdr1 cells). Drug-resistant HT-29mdr1 cells and drug-sensitive HT-29par parental cells were treated with Onconase and/or VCR in vitro at varying concentrations to measure the effects on protein synthesis and cell viability. The impact of Onconase on VCR accumulation in both types of cells was determined in the presence or absence of MRK-16, an anti-P-gp monoclonal antibody capable of reversing the multidrug-resistant phenotype. The antitumor effects of Onconase and/or VCR treatment were assessed in nude mice bearing established HT-29par or HT-29mdr1 intraperitoneal tumors. IC50 values (drug concentrations resulting in 50% inhibition of protein synthesis or cell viability) for Onconase and VCR were determined from semilogarithmic dose-response curves; interactions between the cytotoxic effects of these two agents were evaluated using data from protein synthesis inhibition experiments and a two-way analysis of variance. Survival distributions from in vivo experiments were compared using Cox proportional hazards models. RESULTS: The combination of Onconase and VCR yielded enhanced cytotoxicity in vitro that was independent of P-gp expression. Evaluation of the effects of these two compounds on protein synthesis over a wide range of drug concentrations indicated possible synergistic interactions (i.e., greater than additive effects) in both drug-resistant and drug-sensitive cells. The enhancement of VCR cytotoxicity was dependent on Onconase enzyme activity and was not associated with increased intracellular levels of VCR. Simultaneous treatment of mice bearing HT-29par tumors with Onconase and VCR did not extend their median survival time (MST) significantly (MST with VCR = 66 days; MST with VCR plus Onconase = 69 days; two-tailed P = .57); however, the MST of mice with HT-29mdr1 tumors was extended significantly by this treatment (MST with VCR = 44 days; MST with VCR plus Onconase = 66 days; two-tailed P<.001). CONCLUSION: Combined administration of Onconase and VCR yields enhanced cytotoxicity in vitro and in vivo against human colon carcinoma cells that overexpress the mdr1 gene.

Reversal of multidrug resistance in human colon cancer cells expressing the human MDR1 gene by liposomes in combination with monoclonal antibody or verapamil.

BACKGROUND: Colorectal cancer is a major cause of cancer-related mortality in the world and the second leading cause of neoplastic death in the United States. A major obstacle in the chemotherapy of this neoplasm is the emergence of multidrug resistance that is frequently associated with the expression of P-glycoprotein (p170) encoded by MDR1 (also known as PGY1) genes. Previously, we demonstrated that liposome-encapsulated doxorubicin is more cytotoxic than free doxorubicin in human promyelocytic leukemia and human breast cancer cells with the multidrug-resistant phenotype. PURPOSE: Our purpose was to investigate modulation of multidrug resistance by liposome-encapsulated vincristine (VCR) in a drug-resistant human colon cancer cell line HT-29mdr1 and the potentiation of this modulation in combination with monoclonal antibody MRK-16 or verapamil. METHODS: HT-29 parental cells and HT-29mdr1 cells were exposed to free VCR or liposome-encapsulated VCR alone or in combination with MRK-16 or verapamil. Cytotoxicity of cells after various treatments was determined by neutral red staining, and cellular content of VCR was measured by using radiolabeled VCR; p170 expression of cells was assessed by azidopine. RESULTS: HT-29mdr1 cells express a high amount of p170, thus conferring sixfold to sevenfold resistance to VCR compared with the parent cell line. Liposome-encapsulated VCR lowers drug resistance in HT-29mdr1 cells fourfold; IC50 values (concentration that causes 50% reduction in cell number) were 12.5 +/- 2.5 ng/mL compared with 42.5 +/- 5.0 ng/mL with free VCR. IC50 values for free VCR with empty liposomes were 25 +/- 1.25 ng/mL. The combination of MRK-16 and free VCR produced a twofold increase in cytotoxicity over free VCR in p170-expressing cells; the combination of MRK-16 and liposome-encapsulated VCR produced a 10-fold potentiation of cytotoxicity. toxicity. Nonspecific monoclonal antibody NR-LU-10 had no effect on cytotoxicity of HT-29mdr1 cells with free VCR or liposome-encapsulated VCR. The combination of 1.5 microM verapamil potentiated the cytotoxicity of free VCR ninefold to 10-fold, IC50 values reduced to 5.0 +/- 1.5 ng/mL, and in combination with liposome-encapsulated VCR, IC50 values reduced to 2.5 +/- 1.0 ng/mL, demonstrating a 15- to 17-fold potentiation of cytotoxicity. There were no significant differences in drug accumulation in HT-29mdr1 cells when treated with liposome-encapsulated VCR or free VCR. Liposomes inhibited the photoaffinity labeling of azidopine to p170 HT-29mdr1 cells. CONCLUSIONS: Liposome encapsulation of VCR effectively modulates multidrug resistance in human colon cancer cells and may become an important modality in treatment for colon cancers.

Enhancement by recombinant human interferon alfa of the reversal of multidrug resistance by MRK-16 monoclonal antibody.

BACKGROUND: The anti-P-glycoprotein monoclonal antibody MRK-16 mediates the reversal of multidrug resistance. Recombinant human interferon alfa (rHuIFN alpha) enhances the cytotoxic activity of diverse chemotherapeutics and may modulate multidrug resistance. PURPOSE: Our purpose was to determine the outcome of combination treatment with MRK-16, rHuIFN alpha-2a, and cytotoxic agents on tumor cells that express P-glycoprotein (Pgp). METHODS: Three Pgp-expressing, multidrug-resistant human tumor cell lines were used: the MDR1 retrovirus-infected HT-29 colon adenocarcinoma (HT-29mdr1), the doxorubicin (Adriamycin)-resistant MCF-7 (AdrR MCF-7) breast carcinoma, and the de novo Pgp-acquired, HCT-15 colon carcinoma. The parental cell lines HT-29par and MCF-7 were used as controls. The in vitro effects of MRK-16 and rHuIFN alpha-2a were studied on: (a) chemosensitivity of parental and multidrug-resistant cell lines to vincristine, doxorubicin, or paclitaxel (Taxol); (b) intracellular drug concentrations; and (c) Pgp expression. The efficacy of vincristine alone or in combination with MRK-16 and/or rHuIFN alpha-2a was assessed against HT-29mdr1 cells in female, athymic NCr-nu/nu mice. RESULTS: For vincristine, the IC50 (i.e., the concentration that causes 50% inhibition of cell growth) was 7.0 ng/mL in HT-29mdr1 cells. Pretreatment of HT-29mdr1 cells with MRK-16 partially restored vincristine sensitivity (IC50 = 4.8 ng/mL), which was enhanced by noncytotoxic concentrations of rHuIFN alpha-2a (IC50 = 2.9 ng/mL) via a mechanism independent of Pgp modulation or [3H]vincristine efflux. rHuIFN alpha-2a potentiated MRK-16 reversal of multidrug resistance with both doxorubicin and paclitaxel on HT-29mdr1 cells and with vincristine on AdrR MCF-7 and HCT-15 tumor cells. Treatment of mice with 1 mg/kg vincristine weekly for 3 weeks, beginning 10 days after tumor injection, significantly increased the median survival times of the HT-29par tumor-bearing mice (60 days versus 35 days; P < .0001) but was only marginally therapeutic for HT-29mdr1 tumor-bearing mice (52 days versus 46 days). Pretreatment with MRK-16 (500 micrograms) and rHuIFN alpha-2a (5 x 10(4) U), alone or in combination, 24 hours before vincristine therapy did not affect the survival of HT-29par tumor-bearing mice. In contrast, the survival of mice bearing HT-29mdr1 tumors was significantly increased following treatment with MRK-16 before vincristine (80 days; P < .0001). Administration of a nontherapeutic dose of rHuIFN alpha-2a (5 x 10(4) U) with MRK-16 before vincristine treatment further increased the median survival times of HT-29mdr1 tumor-bearing mice (116 days; P < .0001). CONCLUSIONS: MRK-16 used in combination with rHuIFN alpha-2a was significantly more effective than MRK-16 in overcoming multidrug resistance.

Increased epidermal growth factor receptor in an estrogen-responsive, adriamycin-resistant MCF-7 cell line.

We examined the expression of the estrogen and epidermal growth factor (EGF) receptors in a drug-resistant subline of MCF-7 cells in order to study potential alterations in hormone dependence or in the growth factor pathway that could be related to the development of drug resistance in human breast cancer. The drug-resistant subline was derived from MCF-7 cells by selection with Adriamycin in the presence of the P-glycoprotein antagonist, verapamil, to prevent acquisition of the classical multidrug resistance phenotype. The Adriamycin-resistant cells retain estrogen-binding, estrogen-responsive monolayer growth, and estrogen-dependent tumorigenesis. Estrogen-binding studies demonstrate 1.4 x 10(6) sites per cell with unaltered affinity when compared to parental MCF-7 cells, which have 2.7 x 10(5) sites per cell. An increase in expression of EGF receptor, eight to 12-fold, occurred early in the selection for drug resistance, and appears to be unrelated to verapamil exposure, since cells maintained in Adriamycin without verapamil also have increased EGF receptor expression. Partially drug-sensitive revertants carried a verapamil, but out of Adriamycin, demonstrate a decline in EGF receptor expression. We postulate that activation of growth factor pathways in drug-resistant cells may enhance mechanisms of drug resistance, or provide mitogenic stimuli for cells to recover after damage by drug exposure.

Restoration of interferon alpha potentiation of a recombinant ricin A chain immunotoxin following cytoreduction of xenografts of advanced ovarian tumors.

BACKGROUND: We have demonstrated that, in the human ovarian carcinoma cell line (OVCAR-3), recombinant human interferon alpha (rHuIFN-alpha) potentiated in vitro inhibition of protein synthesis by immunotoxins. The antitumor activity of intracavitary immunotoxin administered to nude mice 5 days after tumor cell injection was enhanced by a nontherapeutic dose of rHuIFN-alpha, as evidenced by increased survival time. PURPOSE: Our purpose was to determine the outcome of treatment with immunotoxin and rHuIFN-alpha in xenografts of more advanced tumors. METHODS: At 10 or 15 days after tumor cell injection, nude mice with peritoneal OVCAR-3 xenografts were treated intraperitoneally with immunotoxin or with 454A12 monoclonal antibody (MAb) recombinant ricin A chain (rRA), alone or combined with a nontherapeutic dose of rHuIFN-alpha. The immunotoxin was composed of rRA covalently bound to an anti-CD71 (transferrin receptor) MAb. In other experiments, mice were treated intraperitoneally with cyclophosphamide and cisplatin to reduce tumor size on days 20 and 27 after tumor cell inoculation and then, beginning on day 40, with immunotoxin alone or combined with rHuIFN-alpha. RESULTS: Initiation of treatment 10 days after OVCAR-3 transplantation significantly increased median survival from 41 to 89 days (10% survivors on day 120) with 454A12 MAb rRA alone and to more than 120 days (70% survivors) with 454A12 MAb rRA combined with rHuIFN-alpha (P < .0001). The increase in survival time between tumor-bearing mice treated with immunotoxin combined with rHuIFN-alpha and those treated with immunotoxin alone was statistically significant (P = .017). In contrast, the 15-day transplant tumors were not curable with immunotoxin therapy (survival, 72 days; 0% survivors) and were refractory to rHuIFN-alpha potentiation (survival, 75 days; 0% survivors). After the second course of chemotherapy to reduce the size of the advanced tumors (day 40), during the ascites cell count nadir, initiation of treatment with 454A12 MAb rRA alone or combined with rHuIFN-alpha resulted in significantly different survival times of 129 and 162 days, respectively (P = .0037). Pathologic examination of surviving mice treated with chemotherapy and 454A12 MAb rRA alone or in combination with rHuIFN-alpha revealed that one (17%) of six mice and 11 (65%) of 17 were tumor free, respectively. CONCLUSIONS: The synergy between immunotoxins and IFN-alpha is dependent on tumor burden. These agents are less effective against large tumor burdens (i.e., advanced stage disease), but their beneficial effects re-emerge after cytoreduction by combination chemotherapy. IMPLICATIONS: The ideal setting for testing the efficacy of intracavitary immunotoxin combined with rHuIFN-alpha after front-line chemotherapy is in patients with residual tumor refractory to additional chemotherapy or in those with toxic effects that prevent delivery of effective doses.

Preclinical evaluation of bryostatin as an anticancer agent against several murine tumor cell lines: in vitro versus in vivo activity.

We have examined the ability of bryostatin 1 to inhibit the in vitro growth and in vivo development of a panel of four murine tumors of diverse tissue origins. A wide range of antiproliferative responses was observed for the four tumors. At 100 ng/ml the in vitro growth of the Renca renal adenocarcinoma, the B16 melanoma, the M5076 reticulum cell sarcoma, and the L10A B-cell lymphoma were inhibited by 0, 40, 40, and 94% respectively. All three cell lines sensitive to bryostatin in vitro responded to multiple dose, 1 microgram/injection/day in vivo i.p., bryostatin therapy. Only the in vitro resistant Renca tumor failed to respond to bryostatin in vivo. The correlation between in vitro and in vivo antitumor efficacy suggests a direct mechanism of antitumor activity for bryostatin. Both local regional therapy (M5076 i.p.) and systemic therapy (B16 lung metastases and L10A s.c. tumors) with bryostatin were successful at prolonging survival time. Multiple i.p. doses of bryostatin at a minimum level of 0.5-1.0 microgram/injection were required to observe significant in vivo antitumor effects. The success of in vivo administration of bryostatin in mice bearing 8-10-mm s.c. masses of L10A lymphoma (5-10 x 10(9)) and our further observation that five of a panel of six human B-cell lymphoma cell lines were sensitive to the growth inhibitory effects of bryostatin in vitro suggest that bryostatin may be effective in treating lymphoid malignancies in humans.

Reversal of drug resistance in a human colon cancer xenograft expressing MDR1 complementary DNA by in vivo administration of MRK-16 monoclonal antibody.

One strategy to overcome multidrug resistance in neoplasia is to inhibit the gp170 glycoprotein (relative molecular mass, 170,000) that functions as a plasma membrane, energy-dependent, drug-efflux pump. The human colon cancer cell line HT-29, which grows as an ascitic tumor in athymic NCr-nu/nu nude mice, was made multidrug resistant by infection with an MDR1 (also known as PGY1) retrovirus. Referred to as HT-29mdr1, it was used to study reversal of drug resistance in vivo by the anti-P-glycoprotein monoclonal antibody MRK-16. Flow cytometry and radioimmunoassay demonstrated a marked increase in MRK-16 reactivity on HT-29mdr1 cells as compared with its reactivity on the parental, uninfected cell line (HT-29par). The 50% inhibitory concentrations (IC50) of vincristine on HT-29par and HT-29mdr1 cells were 2.5 and 15 ng/mL, respectively. The MRK-16 monoclonal antibody did not affect the vincristine sensitivity of the HT-29par cells. Pretreatment of HT-29mdr1 cells with 10 micrograms/mL MRK-16 in tissue culture partially restored the vincristine sensitivity (IC50 = 7 ng/mL). This modulation of vincristine sensitivity by MRK-16 was then tested in vivo. The median survival times of mice given intraperitoneal transplants of 5 x 10(6) HT-29par or HT-29mdr1 were 37 and 39 days, respectively. Treatment of mice with 1 mg/kg vincristine weekly for 3 weeks, beginning 10 days after tumor injection, resulted in a significant increase in the median survival time of the HT-29par tumor-bearing mice (68 days, P less than .0001), but it had no effect on the HT-29mdr1 tumor-bearing mice. However, treatment of mice bearing the HT-29mdr1 tumor with MRK-16 before vincristine therapy reversed the resistance to the drug (median survival time = 64 days, P less than .0001). The MRK-16 monoclonal antibody alone had no effect on the median survival time of mice given an injection of either HT-29par or HT-29mdr1 cells. These results suggest that strategies employing monoclonal antibody against gp170 may be clinically useful to reverse multidrug resistance.

Immunoconjugates of Pseudomonas exotoxin A: evaluation in mice, monkeys, and man.

Immunotoxins of PE were constructed with stable thioether linkages using two monoclonal antibodies to ovarian cancer, OVB-3 and NR-LU-10. Antigens recognized by both antibodies have limited normal tissue distribution and are expressed on virtually all ovarian cancers. Both antibodies form highly potent conjugates (ID50 = 100 pg/ml) with high selectivity (greater than or equal to 4 logs) and can eliminate greater than or equal to 5 logs of tumor cells in vitro. The conjugates have been evaluated for efficacy in both ovarian and colon carcinoma ascites xenografts. In the ovarian model, the conjugates produce an increase in life span (ILS) of 200 to 300 with some cures against established but low tumor burden ascites. Increasing the tumor burden decreases efficacy and duration of responses. A lower ILS of 150 to 200 is achieved in the more aggressive colon model. However, the combination of immunotoxin with chemotherapy, which is ineffective on its own, demonstrated enhanced activity (ILS = 300). Toxicity of the conjugates is hepatic and easily monitored by liver function tests (LDH). Antitoxin responses are highly variable, but typically have a rapid onset and appear to be predicted by preexisting levels. Pilot clinical evaluation in ovarian cancer (intraperitoneal) is ongoing.

Enhanced therapeutic efficacy against an ovarian tumor xenograft of immunotoxins used in conjunction with recombinant alpha-interferon.

The antitumor effects of two immunotoxins were evaluated in vitro and in vivo against the human ovarian carcinoma cell line, OVCAR-3. The immunotoxins used were composed of recombinant ricin A chain (rRTA) covalently attached to a monoclonal antibody directed toward the human transferrin receptor (45412/rRTA, also called 454A12 MAB-rRTA by Cetus Corporation) or Pseudomonas exotoxin coupled to an anticarcinoma monoclonal antibody (NR-LU-10/PE). Preliminary characterization of the NR-LU-10 antigen by immunoprecipitation and cellular fluorescence demonstrated two dominant cell surface polypeptide moieties with molecular weights of 40,000 and 45,000 and a minor component with a molecular weight of 33,000. The immunotoxins were used alone or in combination with recombinant human alpha-interferon (rhIFN-alpha). Protein synthesis was inhibited in a dose-dependent manner in OVCA-3 cells incubated in vitro with either NR-LU-10/PE or 454A12/rRTA (50% inhibitory concentrations, 1 and 75 ng/ml, respectively). Unconjugated NR-LU-10 or 454A12 abrogated the activity of the relevant immunotoxins. Concomitant incubation in vitro of OVCAR-3 cells with NR-LU-10/PE or 454A12/rRTA and a noncytotoxic concentration of rhIFN-alpha potentiated the inhibitory activity of the immunotoxins via a mechanism independent of antigenic upregulation. This potentially synergistic combination was then tested in vivo. The median survival time (MST) of mice given injections i.p. of 4 x 10(6) OVCAR-3 cells was 46 days. Cohorts of mice that received intracavitary treatment beginning 5 days posttumor cell inoculation with either 0.25 or 0.5 microgram of NR-LU-10/PE every other day for a total of 10 treatments exhibited a significantly increased MST of 63 and 104 days, respectively (P less than 0.0001). Likewise, the i.p. injection of either 2.5 or 10 micrograms of 454A12/rRTA given in an identical schedule resulted in a MST of 89 and greater than 120 days, respectively (P less than 0.0001). When rhIFN-alpha was administered i.p. in conjunction with those doses of either immunotoxin, a significant increase in the MST was observed in comparison with mice given immunotoxin alone. The combination of 5 x 10(4) units of rhIFN-alpha and 0.25 microgram of NR-LU-10/PE resulted in 67% long-term survivors (greater than 120 days) compared with only 13% survival of mice given the immunotoxin alone. Similarly, 2.5 micrograms of 454A12/rRTA plus rhIFN-alpha resulted in an enhanced therapeutic response (89% long-term survivors) when compared with 454A12/rRTA alone (29%).(ABSTRACT TRUNCATED AT 400 WORDS)

Enhanced therapeutic efficacy of an immunotoxin in combination with chemotherapy against an intraperitoneal human tumor xenograft in athymic mice.

A mouse IgG2b anti-pan carcinoma monoclonal antibody, NR-LU-10, was shown to bind homogeneously to ascites xenografts of both ovarian and colon carcinoma. Following linkage to a highly potent holotoxin, Pseudomonas exotoxin A (PE), NR-LU-10 demonstrated high potency and selectivity in vitro (ID50 = 100 pg/ml; elimination of greater than or equal to 4.5 logs of cells). The conjugate was evaluated for therapeutic efficacy against a human colon tumor (HT-29) transplantable in the peritoneal cavity of nude mice. Beginning 3 days after HT-29 injection, mice received either three or six i.p. injections of 0.5 micrograms of unconjugated NR-LU-10 or immunotoxin conjugate (NR-LU-10/PE) every other day. Mice that received three or six treatments of NR-LU-10 alone had median survival times (MSTs) of 39 and 40 days, respectively, which did not differ significantly from the MST observed for the untreated control groups (MST = 35 days). In contrast, treatment with three or six injections of 0.5 micrograms NR-LU-10/PE exhibited significantly increased MSTs (P = 0.002) of 50 and 60 days, respectively. Coinjection of unconjugated NR-LU-10 (20 micrograms) and 0.5 micrograms of NR-LU-10/PE blocked the therapeutic effect of the immunotoxin (MST = 33 days). The therapeutic efficacy of NR-LU-10/PE was further enhanced against HT-29 when administered i.p. during and after cytoreductive chemotherapy. The i.p. administration of 300 mg/lg of cyclophosphamide plus 100 mg/kg of the chemoprotective drug, WR-2721, 10 and 17 days posttumor cell inoculation induced a significant increase in MST from 36 days to 59 days (P = 0.002). Interestingly, groups of mice that received either two, four, or seven treatments of NR-LU-10/PE following cytoreductive therapy exhibited a further significant increase (P = 0.001) in MSTs of 89, 97, and 105 days, respectively. Therefore, the use of immunotoxin therapy following cytoreductive chemotherapy significantly prolonged survival time of mice bearing the HT-29 colon tumor over that observed with chemotherapy or NR-LU-10/PE alone.

Chemoimmunotoxin therapy against a human colon tumor (HT-29) xenografted into nude mice.

The efficacy of intracavitary chemoimmunotoxin therapy for cancer treatment was evaluated using the human colon carcinoma (HT-29) which had been xenografted i.p. into nude mice. Mice bearing HT-29 were treated with an immunotoxin consisting of the monoclonal antibody OVB3 coupled to Pseudomonas exotoxin (OVB3-PE), with cyclophosphamide (Cy), or with both OVB3-PE plus Cy. Mice given injections i.p. of 3 x 10(6) HT-29 ascites cells developed a localized disease that presented as both malignant ascites and solid tumor confined to the peritoneal cavity. All mice died within 30 to 40 days. Mice that received either three or six injections of OVB3-PE at a dose of 0.5 micrograms every other day beginning 3 days post-tumor inoculation exhibited significantly increased median survival times (MSTs) (P = 0.002) of 62 and 68 days, respectively, as compared to a MST of 33 days for the controls. OVB3 alone or an irrelevant monoclonal antibody conjugated to PE exhibited no antitumor activity. The therapeutic effects of the immunotoxin could be blocked by giving a large amount of unconjugated OVB3 at the same time. Treatment of mice with Cy alone at the maximal tolerated dose (250 mg/kg) on Days 10 and 17 after tumor inoculation increased the MST from 33 days to 54 days. The maximum tolerated dose could be increased to 300 mg/kg per injection if the Cy treatment was preceded by 100 mg/kg of S-2-(3-aminopropylamino)ethyl phosphorothioic acid (WR-2721), a sulfhydryl compound that selectively protects normal tissue against the toxicity of radiation and alkylating agents. Cy plus WR-2721 treatment on Days 10 and 17 increased the MST from 35 to 61 days (P = 0.002). Interestingly, groups of mice that received either two, four, or seven treatments of OVB3-PE following Cy plus WR-2721 therapy exhibited a further increase (P less than 0.002) in MSTs to 81, 87, and 96 days, respectively. Thus, the combination of cytoreductive chemotherapy with the OVB3-PE was significantly more effective for the intracavitary treatment of established HT-29 colon cancer xenografts than either chemotherapy or immunotoxin therapy alone.

Immunotoxins to a human melanoma-associated antigen: comparison of gelonin with ricin and other A chain conjugates.

Gelonin, a ribosome-inactivating protein from the seeds of Gelonium multiflorum, has been conjugated to antibodies. Previous reports have indicated variable potency of such immunotoxins. The lack of toxicity of gelonin, however, makes it attractive for immunoconjugate production. The ribosome-inactivating protein was covalently linked (using N-succinimidyl-3-(2-pyridyldithio)propionate) to monoclonal antibody, 9.2.27, directed to a human melanoma-associated glycoprotein/proteoglycan. The immunoconjugate showed high selectivity with dose-dependent cytotoxic activity to cultured human melanoma cells (50% inhibitory dose; 1-3 X 10(-11) M versus antigen-positive cells; 1-3 X 10(-7) M versus antigen-negative cells). Specificity and immunoreactivity of the conjugate were similar to those of unconjugated antibody. Biodistribution studies with iodine trace-labeled conjugate in nude mice indicated that tumor localization of the gelonin conjugate was decreased compared to unconjugated antibody. However, a significant therapeutic effect of the conjugate was found with multiple but not single dose i.v. treatment in nude mice bearing established palpable melanoma. These in vivo experiments showed that gelonin conjugates are not toxic up to 2 mg total dose/mouse and significantly retarded the growth of established s.c. tumor. Comparison of gelonin conjugates in vitro and in vivo with other A-chain conjugates of 9.2.27 (abrin and ricin) indicated that gelonin had similar potency, better selectivity, better tumor localization, and more significant therapeutic effects.

Immunotoxins to a human melanoma-associated antigen: resistance to pokeweed antiviral protein conjugates in vitro.

Pokeweed antiviral protein (PAP) from the summer leaves of Phytolacca americana was purified and conjugated via N-succinimidyl-3-(2-pyridyldithio)propionate to 9.2.27 anti-melanoma antibody to a glycoprotein-proteoglycan complex. The conjugate was highly potent (50% inhibition dose of 5 X 10(-11)-10(-13) M on antigen-positive melanoma) and highly selective (5 X 10(-8) on antigen-negative melanoma). Human melanoma cells were selected for resistance to in vitro killing of the PAP conjugate by cycling through a killing and recovery sequence. Resultant cultures were shown to be more than 2 logs less sensitive to the killing of the PAP conjugate than untreated cultures. Isolation of clones by limiting dilution and reanalysis indicated that the resistant polyclonal culture contained clones with a range of sensitivities. Resistant cultures were also resistant to other A-chain conjugates of 9.2.27, but not to intact toxins like ricin and abrin. Resistant cultures showed no change in antigen expression after selection with the PAP conjugate of 9.2.27. Thus, just as with many other chemotherapeutic agents, tumor cells can become resistant to agents inhibiting protein synthesis even when targeted with monoclonal antibody. The mechanisms of this resistance and modalities to minimize resistance are currently being explored.

Endorphins and parturition.

Endorphins and beta-lipotropin have been implicated as the modulators of pain during the labor process. To investigate their possible role during parturition, the present study was undertaken to determine the levels of beta-endorphin, beta-endorphinlike immunoactivity, and beta-lipotropin during labor and delivery. Fourteen patients were evaluated at 4 cm dilatation, complete dilatation, at the time of delivery, and immediately postpartum. In addition, samples were obtained from nine nonpregnant control subjects. The venous levels of beta-endorphinlike immunoactivity and beta-lipotropin were determined, and the beta-endorphin level was calculated. The maternal cerebrospinal fluid (CSF) levels and the newborn umbilical venous level also were determined in the delivery room. A gradual rise was noted in the beta-endorphin, beta-lipotropin, and beta-endorphinlike immunoactivity values up to the time of delivery. There was noted to be a modest decrease in their levels in the immediate postpartum period. These levels were significantly elevated over control samples for both beta-endorphinlike immunoactivity and beta-lipotropin (P less than .05). Values for beta-endorphin were not significantly different from control levels. In addition, maternal CSF levels were found to show modest elevations above the simultaneously drawn venous specimens. The umbilical cord venous samples drawn at delivery were also higher than the simultaneous maternal venous levels. These values, although elevated over control values, were not statistically different from simultaneously drawn maternal venous levels.

Selective antitumor effect on L10 hepatocarcinoma cells of a potent immunoconjugate composed of the A chain of abrin and a monoclonal antibody to a hepatoma-associated antigen.

The toxic A chain of abrin was isolated by affinity chromatography and was demonstrated to be a potent inhibitor of protein synthesis in a cell-free rabbit reticulocyte system with a complete inhibitory dose at 1 X 10(-9) M. This A chain was coupled by a disulfide linkage to a purified monoclonal antibody directed against a tumor-associated antigen found on the line 10 hepatocarcinoma tumor in strain two guinea pigs. The immunoconjugate retained the functions of the individual components, i.e., antigen binding to the intact cell in vitro and inhibition of its protein synthesis. This conjugate was a selective antineoplastic agent with a cytocidal dose at 5 X 10(-9) M toward antigen-bearing cells in vitro. Several antigen-negative cells were much less susceptible to its cytotoxic effect. The cytotoxicity of the conjugate appeared to be by antibody-mediated delivery of toxic A chain into the target cell. When cells were pretreated with excess free antibody followed by a brief exposure to conjugate, there was a reversal of the cytotoxicity to antigen-positive cells but not to the antigen-negative cells. The therapeutic efficacy of the conjugate was assayed by injecting a single dose s.c. or i.v. into syngeneic guinea pigs bearing established line 10 tumors. These in vivo studies showed that (a) the conjugate was not toxic at a dosage of 60 to 1120 micrograms/guinea pig, (b) the conjugate decreased or abolished the growth of established solid tumors, (c) the conjugate delayed or inhibited tumor metastases to lymph nodes, and (d) 20 to 40% of the animals in selective groups had a long-term complete regression.

Tumor models to investigate the therapeutic efficiency of immunomodulators.

The development of successful approaches to immunotherapy is dependent on the proper consideration of the pathobiology of metastasis and a better understanding of the principles of biological response modification. Immunomodulation as a therapeutic modality has not proven notably effective in animals with preexistent palpable disease, an observation in agreement with the results from clinical protocols. Therefore, rather than developing experimental immunotherapy models of immunoprophylaxis or models with minimal tumor burden, we believe it more appropriate to develop models based on preexistent metastatic disease in a tumor-conditioned host. The most rigorous test of a biological response modifier's efficiency prior to clinical trials will be provided from immunotherapeutic trials of palpable autochthonous tumors.