Cellular resistance to the antimelanoma immunotoxin ZME-gelonin and strategies to target resistant cells

 The development of cellular resistance to immunotoxins has been demonstrated in a variety of models and can involve a number of mechanisms. For the present study, an immunotoxin was utilized composed of an antimelanoma antibody ZME-018 recognizing a 240-kDa surface glycoprotein (gp 240) and the plant toxin gelonin. Human melanoma cells (A375-M) were grown in the presence of increasing amounts of ZME-gelonin and a clonal variant (A-375-ZR) was developed that was 100-fold resistant to ZME-gelonin compared to parental cells. Scatchard analysis showed that the A375-M parental cells had 260×10³ ZME-gelonin-binding sites/cell with relatively low affinity (5 nM). In contrast, resistant A375-ZR cells demonstrated a reduced number of low-affinity sites (160×10³/cell), but showed a small number (47×10³) of higher-affinity sites (0.8 nM). Internalization rates and degradation rates of ¹²⁵I-labeled ZME-gelonin were identical in both the parental and resistant cells. A375-ZR cells were found to be more resistant to vincristine and doxorubicin than were parental cells. Both cell lines were almost equally sensitive to native gelonin, 5-fluorouracil (5-FU), cisplatin, melphalan, carmustine, interferon γ (IFNγ) and IFNα. In addition, both cell lines were equally sensitive to another gelonin-antibody conjugate that binds to cell-surface, GD₂ (antibody 14G₂A). However, resistant cells were twice as sensitive to the cytotoxic effects of etoposide than were parental cells. Finally, a variety of agents were tested in combination with ZME-gelonin against A375-ZR cells in an attempt to identify agents to augment immunotoxin cytotoxic effects against resistant cells. The agents 5-FU, cisplatin, IFNγ, IFNα, and etoposide were the most effective in augmenting the cytotoxicity of ZME-gelonin against resistant cells. These studies suggest that development of resistance to one immunotoxin does not cause development of cross-resistance to other gelonin immunotoxins. Further, specific biological response modifiers and chemotherapeutic agents may be effective in augmenting the effectiveness of immunotoxins and specifically targeting or reducing the emergence of immunotoxin-resistant cells. Received: 15 March 1995 / Accepted: 28 November 1995     

An antimelanoma immunotoxin containing recombinant human tumor necrosis factor: tissue disposition,pharmacokinetic, and therapeutic studies in xenograft models

The ability of monoclonal antibody conjugates to re-direct plant or bacterial toxins, chemotherapeutic agents and radionuclides to selected target cells has been well-documented. Recombinant human tumor necrosis factor (TNF) is a macrophage-derived, non-glycosylated (17 kDa) peptide with a broad range of biological and immunological effects including antiviral activity, cytotoxic and cytostatic effects. A conjugate of the antimelanoma antibody ZME-018 and TNF in previous studies has shown melanoma-selective cytotoxic effects in vitro. Pharmacokinetic studies of the ZME-TNF immunotoxin showed that the agent cleared from plasma biphasically with -and -phase half-lives similar to that of ZME itself (72 min and 36 h compared to 84 min and 41 h respectively). In contrast, TNF itself was cleared rapidly from plasma with a terminalphase half-life of only 2.7 h. The clearance rate of ZME-TNF from plasma (Cl_p) was almost tenfold more rapid than for ZME (1.1 versus 0.16 ml/kg x min) but was threefold slower than the clearance for TNF itself (3.4 ml/kg x min). Tissue distribution studies in nude mice bearing human melanoma xenografts showed similar tumor localization of the immunotoxin compared to the free antibody and slightly higher concentrations in liver and kidney compared to ZME itself. Treatment of nude mice bearing well-developed A375 tumors with the immunotoxin resulted in a statistically significant (P<0.002) suppression in tumor growth rate (fivefold increase) compared to saline-treated controls, which increased 20-fold over the same period. These studies demonstrate the feasibility of this approach and suggest that TNF may represent a non-antigenic alternative to immunotoxins containing plant and bacterial toxins.Research conducted, in part, by the Clayton Foundation for Research     

Potent antitumor effects of an antitumor endothelial cell immunotoxin in a murine vascular targeting model

Cell Biochemistry and Biophysics - Immunotoxins and other antibody-based therapeutic reagents have proved effective against lymphomas and leukemias, but results with carcinomas and other solid...     

In vivo effects in mice of an anti-T cell immunotoxin

We have evaluated both the proliferative response as well as the Thy-1 Ag expression of lymphocytes from mice treated in vivo with an anti-Thy-1 immunotoxin (IT). The IT was a rat IgG2c mAb recognizing the Thy-1 Ag, disulfide-linked to a ribosome-inactivating protein isolated from the seeds of the plant Saponaria officinalis (soapwort). Toxicity studies showed that a single i.v. injection of doses up to 20 micrograms IT/mouse was well tolerated and allowed indefinite survival. The Con A-induced proliferative response of spleen cells from mice killed 1 day after treatment with sublethal doses of IT was inhibited in a dose-dependent manner, with complete inhibition observed at doses of greater than or equal to 5 micrograms IT/mouse. Control experiments showed that the inhibition was due to the IT and not to its single components. Moreover, the IT effect was abolished by a large (100-fold) excess of anti-Thy-1 mAb alone given concurrently, but not by an unrelated, isotype-matched rat mAb. At all IT doses, the proliferative response to a B cell mitogen (LPS) was normal. Kinetic studies showed a time- and dose-dependent reconstitution of Con A responsiveness. In limiting dilution cultures of spleen cells from mice treated with 5 micrograms IT 1 or 4 days before death, a 97% depletion of T lymphocytes capable of proliferation was observed. Limiting dilution cultures showed that also the thymus of IT-treated mice was depleted by more than 90% of growth-competent T lymphocytes. Cytofluorographic studies of Thy-1+ cells from the spleens of IT-treated mice gave results which did not correlate with those obtained in functional assays. Thus, a dose-dependent reduction, followed by a time-dependent reconstitution of Thy-1+ cells was observed in this case too, but the depletion occurred at later time points and was less complete than that observed in functional assays. Moreover, the mean fluorescence intensity of the residual Thy-1+ cells decreased below normal levels.     

Human antibody responses to components of the monoclonal antimelanoma antibody ricin A chain immunotoxin XomaZyme-MEL

Human antibody responses to immunotoxin components were evaluated in 21 melanoma patients who were treated with XomaZyme-MEL, a murine monoclonal antimelanoma antibody-ricin A chain conjugate. Twenty of the 21 melanoma patients produced antibodies against ricin A chain, while 15 of 21 produced antibodies reactive with the murine monoclonal antibody component. Both IgM and IgG antibody responses were produced. Immunoglobulin responses were usually detected 1 to 2 weeks following initiation of therapy, with peak levels generally attained 2 to 4 weeks posttherapy. Titers of the anti-ricin A chain antibodies were generally higher than those of the antimurine monoclonal antibodies for the dose range tested. There was no clear correlation between the dose of immunotoxin administered and the antibody titer. By use of a competitive flow cytometry assay, antiidiotype responses were demonstrated in eight of 10 melanoma patients who had antimurine antibodies. Both the kinetics of appearance and the relative titers of the antiidiotype responses generally corresponded to the antimurine responses. The development of antimmunotoxin antibodies can reduce the therapeutic potential of immunotoxins through several mechanisms. The development of antibodies in a significant number of patients suggests that optimally effective, repeated courses of therapy will require some procedure for suppressing or abrogating the response against the immunotoxin.     

Pharmacokinetics of intrathecal transferrin-ricin a chain immunotoxin.

We have studied the pharmacokinetics of an anti-transferrin receptor immunotoxin following intrathecal (i.t.) and intravenous (i.v.) bolus inoculation in healthy rats. After i.t. inoculation of 4.9 microg transferrin-ricin A-chain (Tfn-RTA) we have measured the immunotoxin concentration in the cerebrospinal fluid (CSF), in the brain tissue and in the peripheral blood. After i.v. administration of 4.9 microg Tfn-RTA the concentration of Tfn-RTA immunotoxin was evaluated in the peripheral blood. We found that the clearance of Tfn-RTA from the CSF is rapid (9.1 microLmin(-1)), the immunotoxin then diffuses into the brain tissue and in the peripheral blood where it reaches concentrations below the MTC50 (Minimum Toxin Concentration 50%). The rate of immunotoxin elimination from the peripheral blood following either i.v. or i.t. administration are similar (kel = 0.0021 min(-1) vs. 0.0025 min(-1)). Thus, in the healthy rat the immunotoxin does not accumulate following i.t. inoculation, reaching non toxic concentrations in the brain tissue and in the peripheral blood, whereas in the CSF as well as at the interface CSF/brain tissue the immunotoxin may reach potentially therapeutic concentrations. In conclusion we believe that the i.t. inoculation of an immunotoxin could be considered a potentially useful route of administration in the treatment of leptomeningeal carcinomatosis.     

Secondary lymphoid tissue chemokine mediates T cell-dependent antitumor responses in vivo

Secondary lymphoid tissue chemokine (SLC, also referred to as Exodus 2 or 6Ckine) is a recently identified high endothelial-derived CC chemokine. The ability of SLC to chemoattract both Th1 lymphocytes and dendritic cells formed the rationale to evaluate this chemokine in cancer immunotherapy. Intratumoral injection of recombinant SLC evidenced potent antitumor responses and led to complete tumor eradication in 40% of treated mice. SLC-mediated antitumor responses were lymphocyte dependent as evidenced by the fact that this therapy did not alter tumor growth in SCID mice. Studies performed in CD4 and CD8 knockout mice also revealed a requirement for both CD4 and CD8 lymphocyte subsets for SLC-mediated tumor regression. In immunocompetent mice, intratumoral SLC injection led to a significant increase in CD4 and CD8 T lymphocytes and dendritic cells, infiltrating both the tumor and the draining lymph nodes. These cell infiltrates were accompanied by the enhanced elaboration of Th1 cytokines and chemokines monokine induced by IFN-gamma and IFN-gamma-inducible protein 10 but a concomitant decrease in immunosuppressive cytokines at the tumor site. In response to irradiated autologous tumor, splenic and lymph node-derived cells from SLC-treated tumor-bearing mice secreted significantly more IFN-gamma, GM-CSF, and IL-12 and reduced levels of IL-10 than did diluent-treated tumor-bearing mice. After stimulation with irradiated autologous tumor, lymph node-derived lymphocytes from SLC-treated tumor-bearing mice demonstrated enhanced cytolytic capacity, suggesting the generation of systemic immune responses. These findings provide a strong rationale for further evaluation of SLC in tumor immunity and its use in cancer immunotherapy.     

Pharmacokinetics and tissue distribution of enrofloxacin and its metabolite ciprofloxacin in the Chinese mitten-handed crab, Eriocheir sinensis

The pharmacokinetics of enrofloxacin and its active metabolite ciprofloxacin were investigated in the Chinese mitten-handed crab after a single intramuscular injection of enrofloxacin at 5.0mg/kg body weight. The tissue concentrations of enrofloxacin and ciprofloxacin were determined simultaneously by a high-performance liquid chromatography (HPLC) method. The data were analyzed with Practical Pharmacokinetic Program 3P97. The highest average concentrations of enrofloxacin in liver, muscle, gill, and hemolymph were 3.93, 12.42, 16.73, and 11.04 microg/g (ml), respectively. The elimination half-lives (t(1/2beta)) for enrofloxacin were 92.42, 64.86, 38.80, and 52.39 h, respectively. The AUC(0-infinity) values for enrofloxacin were 304.80, 260.74, 288.30, and 269.24 microg h/ml, respectively. Ciprofloxacin could be detected in all four tissues. The respective values of main pharmacokinetics parameters Cmax, t(1/2beta), and AUC(0-infinity) were 0.52 microg/g (ml), 38.38 h, and 35.06 microg h/ml for liver; 0.24 microg/g (ml), 65.36 h, and 25.64 microg h/ml for muscle; 0.10 microg/g (ml), 112.88 h, and 11.57 microg h/ml for gill; and 0.30 microg/g (ml), 93.33 h, and 39.99 microg h/ml for hemolymph.     

Pharmacokinetics and tissue distribution of novel traditional Chinese medicine-platinum anticancer agents in rats

The pharmacokinetics and tissue distribution profiles of a novel series of traditional Chinese medicine-platinum (TCM-Pt) compounds [Pt(C(8)H(8)O(5))(NH(2)R)(2)]: 1 (where R=H), 3 (R=CH(3)) and 5 (R=C(6)H(10)), were studied in Sprague-Dawley rats following a single bolus intravenous (i.v.) injection. Platinum concentrations in total plasma, plasma ultrafiltrate, urine and tissues were measured by flameless atomic absorption spectroscopy. Pharmacokinetic studies showed that plasma concentrations of total and free platinum for the novel TCM-Pt compounds as well as cisplatin and carboplatin declined in a biexponential manner with a short distribution half-life (t(1/2alpha): 0.12-0.34h). Compared with cisplatin, the novel TCM-Pt compounds had a longer elimination half-life (t(1/2beta)), larger dose normalized area under the curve (AUC/D), larger volume of distribution at steady-state (V(ss)), slower clearance (CL) of free platinum and higher percentage of cumulative urinary excretion (CUE), which can be attributed to their lower chemical reactivities. In tissues, the highest Pt concentrations were found in the kidney, followed by the liver and the lowest in the heart; no Pt was detected in the brain. Twenty-four hours after drug administration, platinum concentrations in tissues were significantly lower for the novel TCM-Pt compounds. These findings suggest that the novel compounds might afford higher clinical efficacy and reduced systemic side effects, when compared with cisplatin.     

Synergistic antitumor effects of novel HDAC inhibitors and paclitaxel in vitro and in vivo

Preclinical studies support the therapeutic potential of histone deacetylases inhibitors (HDACi) in combination with taxanes. The efficacy of combination has been mainly ascribed to a cooperative effect on microtubule stabilization following tubulin acetylation. In the present study we investigated the effect of paclitaxel in combination with two novel HDACi, ST2782 or ST3595, able to induce p53 and tubulin hyperacetylation. A synergistic effect of the paclitaxel/ST2782 (or ST3595) combination was found in wild-type p53 ovarian carcinoma cells, but not in a p53 mutant subline, in spite of a marked tubulin acetylation. Such a synergistic interaction was confirmed in additional human solid tumor cell lines harboring wild-type p53 but not in those expressing mutant or null p53. In addition, a synergistic cytotoxic effect was found when ST2782 was combined with the depolymerising agent vinorelbine. In contrast to SAHA, which was substantially less effective in sensitizing cells to paclitaxel-induced apoptosis, ST2782 prevented up-regulation of p21(WAF1/Cip1) by paclitaxel, which has a protective role in response to taxanes, and caused p53 down-regulation, acetylation and mitochondrial localization of acetylated p53. The synergistic antitumor effects of the paclitaxel/ST3595 combination were confirmed in two tumor xenograft models. Our results support the relevance of p53 modulation as a major determinant of the synergistic interaction observed between paclitaxel and novel HDACi and emphasize the therapeutic interest of this combination.     

Pharmacokinetics of systemically administered tyrosine: a comparison of serum, brain tissue and in vivo microdialysate levels in the rat

Tyrosine uptake has been reported to differ across brain regions. However, such studies have typically been conducted over brief intervals and in anesthetized rats; anesthesia itself affects amino acid transport across the blood-brain barrier. To address these concerns, serum, brain tissue and in vivo microdialysate tyrosine levels were compared for 0-3 h after administration of tyrosine [0.138-1.10 mmol/kg intraperitoneally (i.p.)] to groups of awake rats. Serum and brain tissue tyrosine levels increased linearly with respect to dose. Basal tissue tyrosine levels varied significantly across brain regions [medial prefrontal cortex (MPFC), striatum, hypothalamus, and cerebellum], but the rate of tyrosine uptake was similar for hypothalamus, striatum and MPFC. For brain regions in which tyrosine levels in both microdialysate and tissue were assayed, namely MPFC and striatum, there was a high degree of correlation between tyrosine levels in tissue and in microdialysate. Increasing brain tyrosine levels had no effect on DA levels in MPFC microdialysate. We conclude that (i) regional differences in the response of dopamine neurons to systemic tyrosine administration cannot be attributed to pharmacokinetic factors; (ii) in vivo microdialysate provides an excellent index over time and across a wide range of tyrosine doses, of brain tissue tyrosine levels; and (iii) increases in brain tyrosine levels do not affect basal DA release in the MPFC.     

Pharmacokinetics and tissue distribution of a peptide nucleic acid after intravenous administration

Peptide nucleic acids (PNAs) are DNA analogs that hybridize to complementary nucleic sequences with high affinity and stability. In our previous work, we showed that a PNA complementary to a 12-base pair (bp) sequence of the coding region of the rat neurotensin receptor (rNTR1) mRNA is effective in significantly blocking a rat's central responses to neurotensin (NT), even when the PNA is injected intraperitoneally (i.p.). Using a novel gel shift detection assay to detect PNA, we have now used this same PNA sequence to derive its pharmacokinetic variables and its tissue distribution in the rat. The PNA has a distribution half-life of 3 +/- 3 minutes and an elimination half-life of 17 +/- 3 minutes. The total plasma clearance and volume of distribution of this PNA were 3.4 +/- 0.9 ml/min x kg and 60 +/- 30 ml/kg. Two hours after dosing, the PNA was found at detectable but low levels in all organs examined-in order of decreasing concentration: kidney, liver, heart, brain, and spleen. Approximately 90% of the PNA dose was recovered as unchanged parent compound in the urine 24 hours after administration.     

Studies in vivo of the tissue uptake, cellular distribution and catabolic turnover of exogenous glucocerebrosidase in rat.

The kinetics of plasma clearance of highly purified human placental glucocerebrosidase in rats were biphasic with 75% of the infused dose showing a rapid clearance (t1/2 = 11 min) and the remaining 25% a considerably lower rate (t1/2 = 60 min). The majority of the enzyme (60%) was taken up by the liver. Although saturation kinetics for the clearance or uptake were not observed, the very high hepatic endocytic index (217 microliter/min) of glucocerebrosidase uptake indicated that liver uptake was mediated by an adsorptive endocytic process. Analysis of the cellular distribution of recovered glucocerebrosidase revealed predominantly parenchymal cell uptake with 38% of the exogenous enzyme in hepatocytes and only 2% in sinusoidal cells. High-mannose glycoproteins blocked hepatocyte and sinusoidal cell uptake of glucocerebrosidase equally. Kinetic experiments failed to demonstrate a transfer or shuttle of exogenous glucocerebrosidase from sinusoidal cells to hepatocytes. The possibility was raised that uptake of enzyme by the liver may be mediated by a common receptor that functions in both hepatocytes and sinusoidal cells. The catabolic turnover of exogenous glucocerebrosidase in rat liver was biphasic and the rate of decline was similar in hepatocytes and sinusoidal cells.     

Syngeneic monoclonal antimelanoma antibodies and their application for analysis of tumor antigens, gene cloning, and in vitro/in vivo diagnosis

In this article, we summarized syngeneic monoclonal antimelanoma antibodies and their application for chemical characterization of mouse melanoma antigens, cloning of genomic DNA controlling antigen expression, and in vivo/in vitro tumor diagnosis. The melanoma antigen is composed of a protein complex in association with GM3(NeuAc)-like sugar moiety. The GM3 structure expresses the cross-species epitopes shared in various mammalian species, whereas the mouse specific melanoma epitope is present on protein molecules. By using the monoclonal antimelanoma reactive with GM3 epitope, we developed a very sensitive sandwich radioimmunoassay system detecting soluble melanoma antigens equivalent to 10(2)-10(3) cells/ml. The antibody was also useful in imaging tumor in vivo. These results indicate that the antibody with cross-species reactivity has a potential for tumor targeting. The monoclonal antibody M562 recognizing protein molecule with species specific epitope but not other antimelanoma antibodies, however, effectively inhibited experimental lung metastasis of melanoma cells, indicating that the M562 epitope seems to possess important biological functions. Recently, the genomic DNA controlling the antigen expression was successfully isolated by DNA transfection and expression technique with monoclonal anti-melanoma M562 and the fluorescence-activated cell sorter. We also found that genomic DNA possesses transformation-related activity in NIH3T3 cells.     

Selective in vivo antitumor effects of monoclonal anti-I-A antibody on B cell lymphoma

In a study of the biologic consequences of using monoclonal antibodies (mAb) with specificity for I-A for the elimination of an I-A-bearing B cell lymphoma, it was found that, despite the presence of I-A on a number of normal cell types and the propensity of anti-I-A to induce modulation of I-A and I-E on normal cells in vivo, a substantial effect on lymphoma growth could be measured in mAb-treated hosts. Unlike I-A on normal cells, tumor I-A failed to modulate in vivo, and 50% of animals could be cured of lymphoma by multiple doses of anti-I-A mAb. With a sensitive spleen tumor colonization assay, it was shown that neither T lymphocytes nor natural killer cells were involved in tumor elimination by anti-I-A mAb. In addition, C3 depletion only minimally affected the ability of anti-I-A to inhibit tumor growth, suggesting that complement-dependent lysis of tumor cells was not a major mechanism. Spleen cells from long term survivors of tumor challenge and mAb treatment functioned normally as antigen-presenting cells and in the recognition of alloantigens, and serum Ig levels were somewhat higher than in untreated mice; thus, such therapy can be carried out without compromising the immune reactivity of long term survivors.     

Interactions of the antitumor drug, etoposide, with reduced thiols in vitro and in vivo

The interaction of activated etoposide, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside) (VP-16), with thiols has been studied both in vitro and in vivo in mice. We have found that both glutathione (GSH) and cysteine rapidly reduce the VP-16 free radical, which results in the regeneration of the parent drug and the oxidation of the thiol. Using spin-trapping and electron spin resonance (ESR) techniques, we have shown that this one-electron/hydrogen donation by thiols forms thiyl radicals (RS.) which are intermediates for the formation of the oxidized thiols. The administration of VP-16 in vivo to mice decreased the total thiol levels in liver and concomitantly increased the formation of oxidized thiols. Furthermore, VP-16 stimulated glutathione reductase in liver. While administration of VP-16 also increased the total thiol pools in kidney, in contrast, no significant effects were observed on lung and heart thiol pools.     

Pharmacokinetics, biological effects, and distribution of (1-->3)-beta-D-glucan in blood and organs in rabbits

The pharmacokinetics, biological effects and distribution in blood and organs of 125I-labeled (1-->3)-beta-D-glucan purified from Candida albicans were analyzed in rabbits during the 24-h period following an intravenous administration.The intravascular half-life of (1-->3)-beta- D-glucan was 1.8 min in the low-dose group (9.3 mug/kg) and 1.4 min in the high-dose group (222 mug/kg), and the mean (+/-SD) total body clearance was 1.12 +/- 0.30 and 1.17 +/- 0.16 ml/min, respectively. The rabbits remained well and (1-->3)-beta-D-glucan failed to alter blood cell counts. Less than 3% of the (125)I-(1-->3)-beta-D-glucan was initially associated with the cellular compartment, and this value decreased further during the 2-h period following administration (P = 0.0001). Over 97% of (125)I-(1-->3)-beta-D-glucan was associated with cell-free plasma, and the majority in plasma appeared to be present in the unbound form (not associated with lipoproteins or plasma proteins). The liver contained more than 80% of the (125)I-(1-->3)-beta-D-glucan detected in the six major organs analyzed.     

The in vitro antitumor effect and in vivo tumor-specificity distribution of human-mouse chimeric antibody against transferrin receptor

Transferrin receptor (TfR/CD71) deserves attention as a selective target for cancer therapy due to its higher expression in tumors versus normal tissues. Also, it has been shown the mouse-derived monoclonal antibody against TfR can significantly inhibit the proliferation of tumor cells. Through constructing the chimeric antibody against TfR, the antigenicity of antibody can be weakened, and most importantly, the antitumor effect of antibody can be strengthened by the introduction of the human Fc fragment. In previous studies, we successfully constructed the human-mouse chimeric antibody against TfR (D2C) and demonstrated that its Fab fragment could specially recognize the TfR on the surface of target cells. In this study, through labeling the chimeric antibody D2C with ¹²⁵I, we calculated the affinity constant (Ka) of 9.34–9.62×10⁹ l/mol for this antibody according to the Scatchard drawing method. Moreover, in vivo studies in nude mice-bearing human liver cancer (SMMC-7721) xenografts have shown that the radioactivity distribution ratio of ¹³¹I-D2C on T/NT was 2–14:1 or 3–21:1 on the seventh day after intraperitoneal or intratumoral injection of ¹³¹I-labeled D2C (¹³¹I-D2C). These evidences indicated that the in vivo distribution of D2C display the characteristics of certain tumor-specificity localization. In vitro studies, D2C can induce the apoptosis of K562 through the mitochondria death pathway and arrest the cell at G₁ phase, as determined by cell cycle analysis. Using the human tumor cells (K562, CEM, and SMMC-7721) expressing TfR as target cells, and normal human PBMC as effector cells, Fc fragment of D2C can perform both the antibody-dependent cell-mediated cytotoxicity and the complement-dependent cytotoxicity. Together, it was demonstrated that the D2C display a tumor-specificity distribution, and has a strong antitumor effect. Thus, it has the potential therapeutic significance.Ye Qing and Wang Shuo contributed equally to this work.