Antitumor effects of an antibody-carboxypeptidase g2 conjugate in combination with a benzoic acid mustard prodrug

The F(ab’)₂ fragment of the antitumor monoclonal antibody, A5B7, was covalently linked to the bacterial enzyme carboxypeptidase G2 (CPG2). The resulting conjugate was used in combination with a prodrug of a benzoic acid mustard alkylating agent to treat human colon tumor xenografts in a two-step targeting strategy, antibody-directed enzyme produrug therapy (ADEPT). The prodrug, 4-[(2-chloroethyl) (2-mesyloxyethyl) amino]-benzoyl-l-glutamic acid is rapidly converted by CPG2 to a drug that is at least 15x more toxic in vitro against LS174T colorectal tumor cells than the prodrug. Optimal tumor/ blood ratios of the A5B7-CPG2 were achieved 72 h after administration of the conjugate to athymic mice bearing established LS174T tumor xenografts. Significant antitumor activity was seen in LS174T tumor-bearing mice treated with the conjugate followed 3 d later by the prodrug. In contrast, prodrug, conjugate, or active drug alone did not result in any antitumor activity in this tumor model. These studies demonstrate the advantage of a two-step ADEPT system for the treatment of colorectal cancer.     

Synthesis and preliminary antitumor activity evaluation of a DHA and doxorubicin conjugate

A conjugate of DHA and doxorubicin (DHA-Dox) was synthesized, and its antitumor activity was evaluated in vitro against L1210 leukemia cells and in experimental animal tumor models including L1210 leukemia and B16 melanoma. DHA-Dox showed a greatly improved antitumor efficacy compared to free doxorubicin.     

Albumin-binding prodrugs of camptothecin and doxorubicin with an Ala-Leu-Ala-Leu-linker that are cleaved by cathepsin B: synthesis and antitumor efficacy

We have recently validated a macromolecular prodrug strategy for improved cancer chemotherapy based on two features: (a) rapid and selective binding of thiol-reactive prodrugs to the cysteine-34 position of endogenous albumin and (b) acid-sensitive promoted or enzymatic release of the drug at the tumor site [Kratz, F., Warnecke, A., Scheuemann, K., Stockmar, C., Schwab, J., Lazar, P., Druckes, P., Esser, N., Drevs, J., Rognan, D., Bissantz, C., Hinderling, C., Folkers, G., Fichtner, I., and Unger, C. (2002) J. Med. Chem. 45, 5523-33]. In the present work, we developed water-soluble camptothecin (CPT) and doxorubicin (DOXO) prodrugs that incorporate the peptide linker Ala-Leu-Ala-Leu that serves as a substrate for the tumor-associated protease, cathepsin B, which is overexpressed in several solid tumors. Consequently, two albumin-binding prodrugs were synthesized [EMC-Arg-Arg-Ala-Leu-Ala-Leu-Ala-CPT (1) and EMC-Arg-Arg-Ala-Leu-Ala-Leu-DOXO (2) (EMC = 6-maleimidocaproic acid)]. Both prodrugs exhibited excellent water-solubility and bound rapidly and selectively to the cysteine-34 position of endogenous albumin. Further in vitro studies showed that the albumin-bound form of the prodrugs was cleaved specifically by cathepsin B as well as in human tumor homogenates. Major cleavage products were CPT-peptide derivatives and CPT for the CPT prodrug and H-Leu-Ala-Leu-DOXO, H-Leu-DOXO, and DOXO for the doxorubicin prodrug. In vivo, 1 was superior to free camptothecin in an HT-29 human colon xenograft model; the antitumor efficacy of prodrug 2 was comparable to that of free doxorubicin in the M-3366 mamma carcinoma xenograft model at equimolar doses.     

聚苹果酸- 羟喜树碱前药的合成和性质初探

目的:化学全合成聚苹果酸(poly(β-malic acid),PMLA),将其作为高分子药物载体,制备聚苹果酸-羟喜树碱前药(PMLA-HCPT)。研究其体外释药特点和体外细胞毒性。方法:以L-天冬氨酸为原料,通过化学方法全合成PMLA,通过酰胺键键合羟基喜树碱(HCPT)。通过红外光谱、核磁共振光谱表征该前药的结构,利用体外动态透析的方法模拟体外释药特点,用高效液相色谱法测定不同pH值聚合物药物中前喜树碱的释药特性。采用人卵巢癌HO-8910细胞系研究该前药的体外毒性。结果:①经核磁共振表征PMLA-HCPT前药合成完成。②在pH 5.6、pH 6.8及pH 7.4的PBS缓冲体系16 h中,羟喜树碱药物累积释放率分别为76.8%,47.2%和18.1%,证实PMLA-HCPT中羟喜树碱的释放具有pH依赖性。③细胞实验证实PMLA-HCPT的细胞毒性和游离的HCPT相比没有降低。结论:PMLA是一种良好的药物载体材料,PMLA-HCPT有望成为具有pH敏感性的聚合物前药。     

PDEPT: polymer-directed enzyme prodrug therapy. 2. HPMA copolymer-beta-lactamase and HPMA copolymer-C-Dox as a model combination

Polymer-directed enzyme prodrug therapy (PDEPT) is a novel two-step antitumor approach that uses a combination of a polymeric prodrug and polymer-enzyme conjugate to generate a cytotoxic drug rapidly and selectively at the tumor site. Previously we have shown that N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound cathepsin B can release doxorubicin intratumorally from an HPMA copolymer conjugate PK1. Here we describe for the first time the synthesis and biological characterization of a PDEPT model combination that uses an HPMA-copolymer-methacryloyl-glycine-glycine-cephalosporin-doxorubicin (HPMA-co-MA-GG-C-Dox) as the macromolecular prodrug and an HPMA copolymer conjugate containing the nonmammalian enzyme beta-lactamase (HPMA-co-MA-GG-beta-L) as the activating component. HPMA-co-MA-GG-C-Dox had a molecular weight of approximately 31 600 Da and a C-Dox content of 5.85 wt %. Whereas free beta-L has a molecular weight of 45 kDa, the HPMA-co-MA-GG-beta-L conjugate had a molecular weight in the range of 75-150 kDa, and following purification no free enzyme was detectable. Against the cephalosporin C or HPMA-co-MA-GG-C-Dox substrates, the HPMA-co-MA-GG-beta-L conjugate retained 70% and 80% of its activity, respectively. In vivo (125)I-labeled HPMA-co-MA-GG-beta-L showed prolonged plasma concentration and greater tumor targeting than (125)I-labeled beta-L due to the enhanced permeability and retention (EPR) effect. Moreover, administration of HPMA-co-MA-GG-C-Dox iv to mice bearing sc B16F10 melanoma followed after 5 h by HPMA-co-MA-GG-beta-L led to release of free Dox. The PDEPT combination caused a significant decrease in tumor growth (T/C = 132%) whereas neither free Dox nor HPMA-co-MA-GG-C-Dox alone displayed activity. The PDEPT combination displayed no toxicity at the doses used, so further evaluation of this approach to establish the maximum tolerated dose (MTD) is recommended.     

Identification and activities of human carboxylesterases for the activation of CPT-11, a clinically approved anticancer drug.

CPT-11 is a clinically approved anticancer drug used for the treatment of advanced colorectal cancer. Upon administration, the carbamate side chain of the drug is hydrolyzed, resulting in the release of SN-38, an agent that has approximately 1000-fold increased cytotoxic activity. Since only a very small percentage of the injected dose of CPT-11 is converted to SN-38, there is a significant opportunity to improve its therapeutic efficacy and to diminish its systemic toxicity by selectively activating the drug within tumor sites. We envisioned that a mAb-human enzyme conjugate for CPT-11 activation would be of interest, particularly since the conjugate would likely be minimally immunogenic, and the prodrug is clinically approved. Toward this end, it was necessary to identify the most active human enzyme that could convert CPT-11 to SN-38. We isolated enzymes from human liver microsomes based on their abilities to effect the conversion and identified human carboxylesterase 2 (hCE-2) as having the greatest specific activity. hCE-2 was 26-fold more active than human carboxylesterase 1 and was 65% as active as rabbit liver carboxylesterase, the most active CPT-11 hydrolyzing enzyme known. The anti-p97 mAb 96.5 was linked to hCE-2, forming a conjugate that could bind to antigen-positive cancer cells and convert CPT-11 to SN-38. Cytotoxicity assays established that the conjugate led to the generation of active drug, but the kinetics of prodrug activation (48 pmol x min(-1) x mg(-1) was insufficient for immunologically specific prodrug activation. These results confirm the importance of hCE-2 for CPT-11 activation and underscore the importance of enzyme kinetics for selective prodrug activation.     

Polyethylene glycol conjugates of methotrexate varying in their molecular weight from MW 750 to MW 40000: synthesis,characterization, and structure-activity relationships in vitro and in vivo

Poly(ethylene glycol)s (PEGs) are potential drug carriers for improving the therapeutic index of anticancer agents. In this work, the anticancer drug methotrexate (MTX) was activated with N,N'-dicyclohexylcarbodiimide (DCC) and coupled to amino group bearing PEGs of MW 750, 2000, 5000, 10 000, 20,000, and 40,000. First, the activation process of MTX with DCC in the presence and absence of N-hydroxysuccinimide was analyzed through HPLC. Preincubation of methotrexate with DCC alone at 0 degrees C proved to be favorable with respect to the amount of activated species and the formation of byproducts. MTX-PEG conjugates were synthesized according to this procedure, isolated through size-exclusion chromatography, and characterized through analytical HPLC, MALDI-TOF spectrometry, and gel permeation chromatography. In a cell-free assay, all of the drug polymer conjugates inhibited the target enzyme of MTX, dihydrofolate reductase (DHFR), to a similar extent, but were not as active as free MTX. Additionally, incubation of the MTX-PEG40000 conjugate for 6 days at 37 degrees C in phosphate buffered saline (pH 7.4), in cell-conditioned medium, or in human serum revealed no significant release of methotrexate. These results, taken together, indicate that release of MTX from polymer conjugates is not necessary for an effective interaction with the active site of dihydrofolate reductase. Evaluation of the in vitro cytotoxicity of the MTX-PEG conjugates in two adherent and three suspension human tumor cell lines revealed that the IC(50) values of the tested compounds increased with the size of the drug-polymer conjugates. The most effective compound tested in these assays was the free drug MTX itself (IC(50) value ranging from approximately 0.01 to 0.05 microM), while the IC(50) values of the polymer conjugates were higher (IC(50) value for MTX-PEG750, 2000 and 5000: approximately 0.6-3 microM; for MTX-PEG10000 and 20000: approximately 2-7 microM; and for MTX-PEG40000: > 6 microM). Subsequently, MTX-PEG5000, MTX-PEG20000, and MTX-PEG40000 were evaluated in a human mesothelioma MSTO-211H xenograft model, and their antitumor effects were compared with free methotrexate and the albumin conjugate MTX-HSA, a conjugate that is currently in phase II clinical trials. In contrast to the in vitro results, the high molecular weight MTX-PEG conjugates exhibited the highest in vivo antitumor activity: At a dose of 40 and 80 mg/kg MTX-PEG5000 was less active than MTX at its optimal dose of 100 mg/kg; MTX-PEG20000 at a dose of 40 mg/kg showed antitumor efficacy comparable to MTX, but MTX-PEG40000 at a dose of 20 mg/kg was superior to MTX and demonstrated antitumor activity of the same order as MTX-HSA (20 mg/kg).     

Enhanced intracellular delivery of methotrexate by a receptor-mediated process

In the present investigation we have described a method of enhancing the uptake of methotrexate by macrophages. This enhanced uptake was mediated by endocytosis through the "scavenger receptor" system which recognized maleylated bovine serum albumin. Experimental evidence showed that macrophages internalized methotrexate coupled to maleylated bovine serum albumin through a saturable process at 37 degrees C leading to an eightfold higher concentration of cell-associated methotrexate compared to the free drug. Following uptake, the drug conjugate was degraded in the lysosomes leading to intracellular release of a pharmacologically active form of methotrexate. When administered to macrophages infected with Leishmania mexicana amazonensis, the drug conjugate could eliminate the intracellular amastigotes more efficiently than the free drug. The leishmanicidal effect of the drug conjugate was inhibited in the presence of excess maleylated bovine serum albumin and lysosomal inhibitors such as chloroquine and monensin. Addition of folinic acid to the medium also prevented the elimination of the amastigotes by the drug conjugate. These results suggested that the scavenger receptor-mediated endocytosis of the drug conjugate led to enhanced transport and intracellular release of a pharmacologically active form of methotrexate resulting in more efficient killing of the amastigotes compared to the free drug. This modality of delivering drugs selectively to macrophages might have utility in the chemotherapy of macrophage-associated disorders in general.     

Selective in vitro and in vivo growth inhibition against human yolk sac tumor cell lines by purified antibody against human α-fetoprotein conjugated with mitomycin C via human serum albumin

Summary The anticancer drug mitomycin C (MMC) was conjugated with an affinity-purified horse antibody to human -fetoprotein (aAFP) with human serum albumin (HSA) as the intermediate drug carrier. The conjugate (aAFP:HSA:MMC molar ratio, 1:1:30) retained full antibody binding activity as determined by a competitive binding radioimmunoassay. In a cytotoxicity test in which the AFP-producing human yolk sac tumor TG-1 cells were preincubated with test materials for 2 h followed by an additional 48-h culture in fresh medium, the conjugate was 20-fold more cytotoxic than free MMC at an equivalent MMC concentration of 100 ng/ml. The in vivo antitumor effect of the conjugate was tested against the human yolk sac tumor JOG-9 growing in athymic nude mice. When the tumor-bearing mice were treated with a total of 6 injections given on 2 consecutive days and then every other day starting 8 days after SC tumor inoculation [2 (equivalent MMC) g/head per injection], the conjugate retarded tumor growth more effectively than free MMC and normal horse immunoglobulin conjugate.     

Multifunctional human serum albumin-therapeutic nucleotide conjugate with redox and pH-sensitive drug release mechanism for cancer theranostics

We report on the synthesis and properties of a new multimodal theranostic conjugate based on an anticancer fluorinated nucleotide conjugated with a dual-labeled albumin. A fluorine-labeled homocysteine thiolactone has been used as functional handle to synthesize the fluorinated albumin and couple it with a chemotherapeutic agent 5-trifluoromethyl-2′-deoxyuridine 5′-monophosphate (pTFT). The conjugate allows for direct optical and ¹⁹F magnetic resonance cancer imaging and release of the drug upon addition of glutathione. Interestingly, the pTFT release from albumin conjugate could only be promoted by the increased acidity (pH 5.4). The in vitro study and primary in vivo investigations showed stronger antitumor activity than free pTFT.     

Polymeric prodrug for release of an antitumoral agent by specific enzymes

The clinical usefulness of antitumor chemotherapy has been strongly limited by the lack of specificity of most anticancer drugs, which act also against healthy cells. The aim of this work was to design, synthesize, and evaluate a macromolecular prodrug of Cytarabine, a known antitumor drug, which is a specific substrate for plasmin enzyme whose concentration is high in various kinds of tumor mass as a result of plasminogen activator secretion. alpha,beta-Poly(N-hydroxyethyl)-DL-aspartamide (PHEA), a known synthetic and biocompatible polyamino acid, was used as a drug carrier, and Cytarabine was linked to PHEA by D-Val-Leu-Lys spacer synthesized beginning from Cbz-D-Val-LeuOH dipeptide and N6-CbzLys methyl ester. The content of Cytarabine in the purified PHEA-D-Val-Leu-Lys-Cytarabine conjugate was equal to 3% w/w. In vitro experiments in the presence of plasmin evidenced the ability of this enzyme to strongly increase drug release from the macromolecular prodrug, as well as plasma incubation shows high stability of drug-polymer linkage. The direct linkage of Cytarabine to PHEA was also performed and, like PHEA-D-Val-Leu-Lys-Cytarabine conjugate, the obtained PHEA-Cytarabine conjugate showed high stability in plasma, but no release of Cytarabine was revealed in the presence of plasmin.     

Synthesis and evaluation of a DHA and 10-hydroxycamptothecin conjugate

We have synthesized a conjugate of cis-4,7,10,13,16,19-docosahexenoic acid (DHA) and 10-hydroxycamptothecin (HCPT), DHA-HCPT. The antitumor activity of DHA-HCPT was evaluated in vitro against L1210 leukemia cells and in experimental animal tumor models including L1210 leukemia, Lewis lung carcinoma, and colon 38 adenocarcinoma. DHA-HCPT showed a greatly improved antitumor efficacy compared to HCPT.     

Synthesis and preclinical characterization of a paclitaxel prodrug with improved antitumor activity and water solubility

The development of novel chemotherapy strategies based on prodrugs remains a major challenge for effective treatment of malignancies. We tested the hypothesis that this can be achieved by a prodrug of paclitaxel where one biologically active center, represented by the C7 hydroxyl group, was blocked by a dihydroxypropyl side chain which can be hydrolytically cleaved by a pH-dependent, slow-release mechanism. The prodrug was synthesized by condensation of solketal chloroformate with the C7 hydroxyl group of paclitaxel followed by a ring-opening reaction to the dihydroxyl derivative. The cytotoxicity of the prodrug was similar to paclitaxel, when tested in vitro against a variety of human tumor cell lines. In vitro cell cycle analysis indicated that concentrations within the micromolar range of both drug and prodrug are required to induce sufficient G2M arrest. The hydrophilic paclitaxel prodrug proved to be more than 50-fold more water soluble than the parental drug and effectively converted to paclitaxel by pH dependent hydrolysis. Importantly, the prodrug could be used at a 3-fold higher maximum tolerated dose (MTD) and revealed a markedly improved antitumor activity in mice compared to paclitaxel. Taken together, our results demonstrate, that a hydrolytically activated paclitaxel prodrug exhibits greater water solubility and superior antitumor activity than the parental drug.     

A new acivicin prodrug designed for tumor-targeted delivery

Acivicin is an antitumor agent known to inhibit cell growth. A new prodrug 9b of acivicin 10 was synthesized, based on a p-hydroxybenzylcarbamate self-immolative spacer capable to release acivicin under esterase activity. The prodrug includes a maleimide-containing arm for linkage with thiol-containing macromolecules such as antibodies. This molecule is intended for the conception of bioconjugates to target an inactive acivicin precursor to tumor cells, when linked to a monoclonal antibody (mAb) which recognizes a tumor-specific antigen. Prodrug cleavage by plasmatic esterases will then restore the acivicin's activity toward tumor cells. We report here the synthesis and the in vitro characteristics of the prodrug. As expected, its inhibitory activity against the gamma-glutamyl transpeptidase (gamma-GT) enzyme and its cytotoxicity towards HL-60 cells were highly reduced compared to the parent drug. The chemical and plasmatic hydrolysis kinetics of the compound was studied by HPLC. The prodrug is stable, being slowly hydrolyzed in pH 7.6 buffer at 37 degrees C with a half-life of 37 h. It is converted into an active acivicin under the effect of pig liver esterase, and its half-life in human plasma is 3 h. These results indicate this compound may be further used as a prodrug-antibody conjugate, to target acivicin to malignant cells.     

Novel synthesis and in vitro drug release of polymeric prodrug: Chitosan–O-isopropyl-5′-O-d4T monophosphate conjugate

A novel approach to synthesize chitosan–O-isopropyl-5′-O-d4T monophosphate conjugate was developed. Chitosan-d4T monophosphate prodrug with a phosphoramidate linkage was efficiently synthesized through Atherton-Todd reaction. In vitro drug release studies in pH 1.1 and 7.4 indicated that chitosan–O-isopropyl-5′-O-d4T monophosphate conjugate prefers to release the d4T 5′-(O-isopropyl)monophosphate than free d4T for a prolonged period. The results suggested that chitosan–O-isopropyl-5′-O-d4T monophosphate conjugate may be used as a sustained polymeric prodrug for improving therapy efficacy and reducing side effects in antiretroviral treatment.     

Preparation,characterization and in vitro activity of a docetaxel–albumin conjugate

Docetaxel is one of the most effective anticancer drugs. However, the current formulation of docetaxel contains Tween 80 and ethanol as the solvent, which can cause severe side effects. Consequently, the development of new type of formulation of docetaxel with high efficiency and low side effects is a very important issue. In this study, we explored the covalent linking of docetaxel and albumin via one organic linker. 6-Maleimidocaproic acid was applied to link the C2′ hydroxyl group of docetaxel with the cysteine-34 of albumin to obtain 1:1 docetaxel–albumin conjugate. The synthesized conjugate can control the release of docetaxel in the bovine serum. Furthermore, in vitro cell cytotoxicity experiments indicated that the docetaxel–albumin conjugate have high activities for human prostate cancer cell line PC3 and human breast cancer cell line MCF-7. The present study provides a valuable strategy for further development of a new type of docetaxel–albumin prodrug.     

Conjugates of COL-1 monoclonal antibody and β-D-galactosidase can specifically kill tumor cells by generation of 5-fluorouridine from the prodrug β-D-galactosyl-5-fluorouridine

5′-O-β-d-galactosyl-5-fluorouridine is a prodrug that can be converted by the enzyme β-d-galactosidase to the potent antineoplastic drug 5-fluorouridine. The prodrug is more than 100x less toxic than the drug to bone marrow cells in Balb/c mice. The ratio of the IC₅₀ of the prodrug to that of the drug determined on a variety of tumor cell lines in vitro ranged from 500∶1–1000∶1. An antibody-enzyme conjugate (AEC) was synthesized and purified. Maleimide-substituted COL-1 anti-CEA monoclonal antibody was linked to free thiol groups of β-d-galactosidase. The conjugate was purified by size exclusion and ion exchange chromatography. It retained full immunoreactivity and enzyme activity. After binding to antigen-positive tumor cells, the conjugate was able to activate the prodrug and specifically kill the cells. We are continuing to investigate this model for its potential use in antibody-directed enzyme prodrug therapy (ADEPT).     

A Novel Anti-HIV Dextrin–Zidovudine Conjugate Improving the Pharmacokinetics of Zidovudine in Rats

The aim of this study was to investigate a newly synthesized dextrin–zidovudine (AZT) conjugate designed as a sustained release prodrug of AZT for parenteral administration. AZT was first reacted with succinic anhydride to form a succinoylated AZT which was subsequently coupled with dextrin to yield the dextrin–AZT conjugate. The structure of the conjugate was characterized by FT-IR and ¹H-NMR spectroscopy. The drug content of the conjugate was 18.9 wt.%. The release in vitro of free AZT and succinoylated AZT was investigated in buffer solutions at pH 5.5 and 7.4 and in human plasma. AZT and succinoylated AZT release from the conjugate was 1.4% (pH 5.5), 41.7% (pH 7.4) and 78.4% in human plasma after 24 h. Release was complete in human plasma after 48 h. A pharmacokinetic study in rats following intravenous administration of the conjugate showed prolonged plasma levels of AZT compared to free AZT. The use of the conjugate extended the plasma half-life of AZT from 1.3 to 19.3 h and the mean residence time from 0.4 to 23.6 h. Furthermore, the conjugate provided a significant greater area under the plasma concentration-time curve and reduced the systemic clearance of AZT. This study suggested the potential of this novel dextrin–AZT conjugate as a new intravenous preparation of AZT.     

Expression of a soluble TGF-beta receptor by tumor cells enhances dendritic cell/tumor fusion vaccine efficacy

Dendritic cell (DC)-based antitumor immunotherapy is a promising cancer therapy. We have previously shown that tumor-derived TGF-beta limits the efficacy of the DC/tumor fusion vaccine in mice. In the current study we investigated the effect of neutralizing tumor-derived TGF-beta on the efficacy of the DC/tumor fusion vaccine. An adenovirus encoding human TGF-beta receptor type II fused to the Fc region of human IgM (Adv-TGF-beta-R) or a control adenovirus encoding LacZ (Adv-LacZ) was used to express a soluble form of the neutralizing TGF-beta receptor (TGF-beta-R). Murine breast carcinoma cells, 4T1, but not bone marrow-derived DCs, were successfully transfected with Adv-TGF-beta-R (4T1+Adv-TGF-beta-R) using a multiplicity of infection of 300. Immunization with irradiated 4T1+Adv-TGF-beta-R tumor cells conferred enhanced antitumor immunity compared with immunization with irradiated 4T1+Adv-LacZ tumor cells. The DC/4T1+Adv-TGF-beta-R fusion vaccine offered enhanced protective and therapeutic efficacy compared with the DC/4T1-Adv-LacZ fusion vaccine. Because TGF-beta is known to induce regulatory T cells (Tregs), we further showed that the DC/4T1+Adv-TGF-beta-R fusion vaccine induced fewer CD4(+)CD25(+)Foxp3(+) Tregs than the DC/4T1+Adv-LacZ fusion vaccine in vitro and in vivo. The suppressive role of splenic CD4(+)CD25(+) Tregs isolated from mice immunized with DC/4T1+Adv-LacZ was demonstrated using a CTL killing assay. Similar enhanced therapeutic efficacy was observed in murine renal cell carcinoma, RenCa, which expresses a high level of TGF-beta. We conclude that the blockade of tumor-derived TGF-beta reduces Treg induction by the DC/tumor fusion vaccine and enhances antitumor immunity. This may be an effective strategy to enhance human DC-based antitumor vaccines.     

In vivo antitumor activity of a panel of four monoclonal antibody-vinca alkaloid immunoconjugates which bind to three distinct epitopes of carcinoembryonic antigen.

A panel of four murine monoclonal antibodies apparently directed against three distinct epitopes of carcinoembryonic antigen (CEA) was conjugated via oxidized carbohydrate groups to 4-desacetylvinblastine-3-carboxyhydrazide. The resulting antibody-vinca conjugates were evaluated for antitumor activity against 2-9-day-established LS174T human colorectal carcinoma xenografts. The antibodies (immunoglobulin G, IgG) employed in this study were 11.285.14 (IgG1), 14.95.55 (IgG2a), CEM231 (IgG1), ZCE025 (IgG1). Additive immunofluorescence studies indicated that CEM231 and ZCE025 recognized the same or a closely related epitope(s) on CEA which was distinct from the two epitopes bound by 11.285.14 and 14.95.55. The in vivo antitumor efficacy studies demonstrated that chemoimmunoconjugates prepared from 14.95.55 and ZCE025 were more active than the conjugates constructed from the 11.285.14 and CEM231 antibodies. The 14.95.55 and ZCE025 immunoconjugates were also more efficacious than free drug or drug conjugated to irrelevant murine IgG. The presence of increased carbohydrate content on the light chain of ZCE025 may have been responsible for the ability to construct ZCE025-vinca conjugates with about twice the drug content (approximately 10 mol of vinca/mol of IgG) than was achieved with the other antibodies. The highly conjugated form of ZCE025 demonstrated similar efficacy but was much less toxic than a ZCE025 conjugate containing 5 mol of vinca/mol of IgG. These data indicated that significant differences existed in the ability of monoclonal antibodies to target a cytotoxic agent for effective antitumor activity even when the immunoconjugates recognized the same antigen or even the same or closely related antigen epitope(s). Furthermore, these differences could not have been identified without extensive in vivo evaluation for antitumor efficacy.