Tumor vascular targeting using a tumor-tissue endothelium-specific monoclonal antibody as an effective strategy for cancer chemotherapy.

In this study, we attempted to develop tumor vascular targeting with a tumor tissue endothelium-specific monoclonal antibody. TES-23, which strongly and selectively recognizes tumor tissue endothelial cells, was chemically conjugated with Neocarzinostatin (NCS), and the anti-tumor effect was examined. The immunoconjugate, TES-23-NCS, showed, through the use of tumor hemorrhagic necrosis, a marked anti-tumor effect on KMT-17 tumors in rats at a dosage of 17 micrograms/kg (NCS equivalent) without any side effects, probably due to specific tumor vascular injury. By contrast, TES-23 alone (107 micrograms/kg), NCS alone (17 micrograms/kg), and Mopc-NCS (Mopc, 107 micrograms/kg; NCS, 17 micrograms/kg), the immunoconjugate of control antibody, did not have any anti-tumor activities. By tissue distribution analysis, TES-23 and TES-23-NCS showed high accumulation in KMT-17 tumors 1 h after intravenous administration. Moreover TES-23 also accumulated in Sarcoma-180 tumors in mice 1 h after intravenous administration. These results suggest that TES-23 may be a candidate for a potential tumor vascular targeting agent that is applicable to a wide variety of tumor types.     

A humanized immunoenzyme with enhanced activity for glucuronide prodrug activation in the tumor microenvironment

Antibody-directed enzyme prodrug therapy (ADEPT) utilizing β-glucuronidase is a promising method to enhance the therapeutic index of cancer chemotherapy. In this approach, an immunoenzyme (antibody-β-glucuronidase fusion protein) is employed to selectively activate anticancer glucuronide prodrugs in the tumor microenvironment. A major roadblock to the clinical translation of this therapeutic strategy, however, is the low enzymatic activity and strong immunogenicity of the current generation of immunoenzymes. To overcome this problem, we fused a humanized single-chain antibody (scFv) of mAb CC49 to S2, a human β-glucuronidase (hβG) variant that displays enhanced catalytic activity for prodrug hydrolysis. Here, we show that hcc49-S2 displayed 100-fold greater binding avidity than hcc49 scFv, possessed greater enzymatic activity than wild-type hβG, and more effectively killed antigen-positive cancer cells exposed to an anticancer glucuronide prodrug as compared to an analogous hβG immunoenzyme. Treatment of tumor-bearing mice with hcc49-S2 followed by prodrug significantly delayed tumor growth as compared to hcc49-hβG. Our study shows that hcc49-S2 is a promising targeted enzyme for cancer treatment and demonstrates that enhancement of human enzyme catalytic activity is a powerful approach to improve immunoenzyme efficacy.     

Preparation and properties of the immunoconjugate composed of anti-human colon cancer monoclonal antibody and mitomycin C-dextran conjugate.

Monoclonal antibody (mAb) A7, produced against human colon cancer, was conjugated with a polymeric prodrug of mitomycin C (MMC), the MMC-dextran conjugate with an anionic charge (MMCDan) and a molecular weight of 70,000. The amino groups were introduced into the MMCDan by reacting ethylenediamine with the carboxyl group in the spacer arm of the dextran bridge by a carbodiimide-catalyzed reaction. The coupling to mAb A7 was performed using SPDP. A 15 M excess of ethylenediamine produced an optimal MMCDan with amino groups, which resulted in a homogenous conjugate (A7-MMCD) with minimal formation of high-molecular-weight aggregates in about a 30% yield of both IgG and MMC. The molar binding ratio of IgG:dextran:MMC in A7-MMCD was estimated to be 1:1.2:40. A7-MMCD, having MMC prodrug properties, released active MMC with a half-life of 29.1 h and had an almost neutral electric charge under physiological conditions. A competitive binding assay using 125I-labeled A7 revealed that the A7-MMCD almost fully retained its antibody-binding activity. The cytotoxicity of A7-MMCD was assayed by determining the degree of inhibition of [3H]-thymidine in corporation in two different ways using the human colon cancer cell line SW1116. A 48-h continuous exposure test revealed that the pharmacological activity of MMC in A7-MMCD was completely preserved. In addition, A7-MMCD exhibited about a 14-fold greater cytotoxicity than MMCDan when the IC50 values determined using a 2-h pretreatment exposure system were compared. These results suggest that A7-MMCD could be useful in immunotargeting chemotherapy for colorectal cancer.     

Selective toxicity of neocarzinostatin-monoclonal antibody conjugates to the antigen-bearing human melanoma cell line in vitro

Summary Monoclonal antibodies (IgG₁) against high molecular weight antigen A-1-43 on human melanoma cell line A-375 were successfully linked to the anti-tumour protein neocarzinostatin (NCS) using the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP). The conjugate retained both the reactivity of the antibody and the toxicity of the drug. The antigen-bearing cell line A-375, antigen-lacking cell line MeWo and normal skin fibroblasts were exposed to NCS-monoclonal antibody conjugates. As negative control, cells were also treated with free NCS and NCS coupled to normal mouse IgG₁ antibodies. Inhibition of ³H-thymidine uptake after treatment was used to measure the biological activity of the cytotoxic drug complex or substance, respectively.Comparing the inhibition dose for 50% uptake (ID₅₀) it was found that the monoclonal antibody-drug complex is about 100 times more toxic for the antigen-bearing cell line than free NCS or normal mouse IgG₁-NCS. This high toxicity is due to a local increase of drug concentration on these cells. With the two cell lines lacking the appropriate antigen no significant differences in the ID₅₀ values were observed. A selectivity factor of 40–50 was obtained by comparing the cytotoxic effect of the monoclonal antibody-NCS conjugate upon the antigen-bearing as opposed to the antigen-lacking cell type. These data demonstrate, that the toxicity of NCS can be directed by monoclonal antibodies to human tumour cells carrying the corresponding surface antigen.     

七甲川花菁染料作为肿瘤靶向和光动力治疗载体的研究进展

七甲川花菁近红外荧光染料(NIRF)可直接被肿瘤细胞特异性吸收,具有肿瘤靶向性。与化疗药物偶联后,该类染料可通过血脑屏障将药物转运至肿瘤部位,不仅可以减少化疗药物使用剂量,降低药物的毒副作用,也可通过近红外荧光成像实现对肿瘤治疗的实时监控。七甲川花菁染料所展示的线粒体毒性和光敏特性,可直接杀死肿瘤细胞,抑制肿瘤新生血管的形成。通过纳米包裹,能够显著增强该类染料的肿瘤靶向能力,实现实时跟踪药物释放情况。七甲川花菁染料特异性识别肿瘤细胞的能力与有机阴离子转运肽的作用密切相关,缺氧和线粒体膜电位也参与了染料吸收的调控。这些发现有利于将近红外荧光染料应用于肿瘤的靶向治疗。     

Bacteria in cancer therapy: a novel experimental strategy

Resistance to conventional anticancer therapies in patients with advanced solid tumors has prompted the need of alternative cancer therapies. Moreover, the success of novel cancer therapies depends on their selectivity for cancer cells with limited toxicity to normal tissues. Several decades after Coley's work a variety of natural and genetically modified non-pathogenic bacterial species are being explored as potential antitumor agents, either to provide direct tumoricidal effects or to deliver tumoricidal molecules. Live, attenuated or genetically modified non-pathogenic bacterial species are capable of multiplying selectively in tumors and inhibiting their growth. Due to their selectivity for tumor tissues, these bacteria and their spores also serve as ideal vectors for delivering therapeutic proteins to tumors. Bacterial toxins too have emerged as promising cancer treatment strategy. The most potential and promising strategy is bacteria based gene-directed enzyme prodrug therapy. Although it has shown successful results in vivo yet further investigation about the targeting mechanisms of the bacteria are required to make it a complete therapeutic approach in cancer treatment.     

Mutated SEA-D227A-conjugated antibodies greatly enhance antitumor activity against MUC1-expressing bile duct carcinoma

For the purpose of establishing a new adoptive immunotherapy for bile duct carcinoma (BDC), we have directed our attention to superantigens (SAgs), the most potent known activators of T lymphocytes. In our previous study, staphylococcal enterotoxin A (SEA) was conjugated chemically with MUSE11 mAb, which recognizes the MUC1 cancer-associated antigen, and shown to enhance the specific cytotoxic activity of T-LAK cells against MUC1-expressing BDC cells (TFK-1) in vitro and in vivo. However, it is probable that SEA might cause side-effects because of nonspecific binding to class II positive cells. In order to overcome these, we generated mutated SEA (mSEA) by changing Asp at position 227 of native SEA to Ala, which has reduced affinity to MHC class II molecules, but retains the potential for T cell activation. When mSEA-D227A was administered to rabbits to examine effects on blood pressure, 500 times more mSEA-D227A was tolerated than native SEA. This prompted us to construct a mSEA-D227A-conjugated mAb, reactive with MUC1. It augmented the antitumor activity of T-LAK cells significantly, and furthermore, mSEA-D227A could be conjugated to two bispecific antibodies, BsAb (anti-MUC1 x anti-CD3) and BsAb (anti-MUC1 x anti-CD28), which in combination had greater enhancing effects than mSEA-D227A-conjugated anti-MUC1 mAb, and combination of unconjugated BsAbs. These findings indicate a utility of mSEA-D227A-conjugated antibodies for targeted cancer immunotherapy.     

Development of potent monoclonal antibody auristatin conjugates for cancer therapy

We describe the in vitro and in vivo properties of monoclonal antibody (mAb)-drug conjugates consisting of the potent synthetic dolastatin 10 analogs auristatin E (AE) and monomethylauristatin E (MMAE), linked to the chimeric mAbs cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specific to CD30 on hematological malignancies). The linkers used for conjugate formation included an acid-labile hydrazone and protease-sensitive dipeptides, leading to uniformly substituted conjugates that efficiently released active drug in the lysosomes of antigen-positive (Ag+) tumor cells. The peptide-linked mAb-valine-citrulline-MMAE and mAb-phenylalanine-lysine-MMAE conjugates were much more stable in buffers and plasma than the conjugates of mAb and the hydrazone of 5-benzoylvaleric acid-AE ester (AEVB). As a result, the mAb-Val-Cit-MMAE conjugates exhibited greater in vitro specificity and lower in vivo toxicity than corresponding hydrazone conjugates. In vivo studies demonstrated that the peptide-linked conjugates induced regressions and cures of established tumor xenografts with therapeutic indices as high as 60-fold. These conjugates illustrate the importance of linker technology, drug potency and conjugation methodology in developing safe and efficacious mAb-drug conjugates for cancer therapy.     

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.     

Activation of human lymphocytes by a monoclonal antibody to B lymphoblastoid cells; molecular mass and distribution of binding protein

A novel monoclonal antibody (BAT) to the B-lymphoblastoid cell line activates murine lymphocytes and exhibits a striking antitumor activity in mice. In order to evaluate the potential use of this antibody against human cancer, we have investigated its immuno-stimulatory properties on human peripheral blood lymphocytes (PBL). Our findings demonstrate that BAT mAb induces proliferation and cytotoxicity in human PBL against natural-killer-cell-sensitive and natural-killer-cell-resistant tumor cell lines. Interleukin-2 at a low concentration synergizes with BAT mAb in eliciting these effects. BAT mAb binds to human peripheral T cells as revealed by a double-labelling technique using anti-CD3 and BAT mAb. The molecular mass of the antigen recognized by BAT mAb was 48–50 kDa under reducing and non-reducing conditions. This study provides a basis for future experiments to evaluate the use of BAT mAb in the immunotherapy of cancer.Supported by research grant (to B.H.) from the Chief Scientist, Ministry of Health, Israel