Optimization of radioimmunotherapy using human malignant melanoma multicell spheroids as a model

In vitro multicell spheroids from a human melanoma cell line and the human colon cancer cell line HT29, used as control, have been established as a model of poorly vascularized micrometastases in vivo. The antimelanoma monoclonal antibody 96.5 was radiolabeled with 131I at specific radioactivities from 1.85 to 3.96 GBq/mg. Cytotoxicity of 131I-96.5 to the spheroids, at an initial size of 300 microns in diameter, was investigated as a function of concentration of 131I-96.5 in the incubation medium, specific radioactivity, and treatment time. Spheroid growth delay and clonogenic survival of cells disaggregated from the spheroids at various times after treatment were used as end points. Therapeutic effects increased with the concentration of 131I-96.5 within the range 0.2 to 2 mg/liter (0.34 to 3.4 GBq/liter) at a fixed specific radioactivity. The effects increased with specific radioactivity at a fixed concentration of 131I-96.5. Difference in therapeutic effect was also observed between treatment times of 8 and 24 h. Radiation doses to the melanoma spheroids varied from 10 to 16 Gy. Unlabeled 96.5 at 2 mg/liter or 131I-iodide at 1.7 GBq/liter did not affect the growth of the melanoma spheroids. The HT29 spheroids, however, only suffered slight cytotoxicity at 1 or 2 mg/liter of 131I-96.5 and for a treatment time of 24 h despite comparable radiosensitivity of HT29 cells and melanoma cells to high-dose-rate radiation. Similar cytotoxicity was observed in the HT29 group treated with 131I-iodide at 1.7 GBq/liter. Present findings therefore demonstrate preferential and adequate killing of the melanoma spheroids by 131I-96.5 at 0.5 mg/liter and 3.96 GBq/mg in 8 h.     

Uptake kinetics of monoclonal antibodies by human malignant melanoma multicell spheroids

Detailed uptake kinetics by multicell spheroids of three tumor associated monoclonal antibodies was investigated. The spheroids were established from a human melanoma cell line and the human colon adenocarcinoma cell line HT29 as in vitro models of poorly vascularized micrometastases in vivo. The selected antibodies 96.5, 140.240, and OST15 showed a wide range of reactivity against the melanoma cell but they all had negligible binding with the colon cancer cell. Uptake of the antibodies by small spheroids (about 300 micron diameter) was generally sigmoidal in shape with respect to incubation time, and amount of uptake followed the same trend of immunoreactivity of the antibodies with single cells. The correlation was weaker for spheroids with diameter greater than 500 micron presumably due to the increasing size of the necrotic core. By varying the concentration of the antibodies in the incubation medium from tracer dose (0.2 microgram/ml) to a higher dose (3 micrograms/ml), negligible changes in the amount of antibodies bound with their target spheroids were observed. Nonspecific binding between antibodies and spheroids, however, resulted in proportional increase in uptake.     

Potentiation of ionising radiation by targeting tumour necrosis factor alpha using a bispecific antibody in human pancreatic cancer

The aim of this study was to treat carcinoembryonic antigen (CEA)-expressing pancreatic carcinoma cells with tumour necrosis factor alpha (TNFalpha) and simultaneous radiation therapy (RT), using a bispecific antibody (BAb) anti-TNFalpha/anti-CEA. TNFalpha used alone produced a dose-dependent inhibition of the clonogenic capacity of the cultured cells. Flow cytometry analysis of cell cycle progression confirmed the accumulation of cells in G(1) phase after exposure to TNFalpha. When TNFalpha was added 12 h before RT, the surviving fraction at 2 Gy was 60% lower than that obtained with irradiation alone (0.29 vs 0.73, respectively, P<0.00001). In combination treatment, cell cycle analysis demonstrated that TNFalpha reduced the number of cells in radiation-induced G(2) arrest, blocked irreversibly the cells in G(1) phase, and showed an additive decrease of the number of cells in S phase. In mice, RT as a single agent slowed tumour progression as compared with the control group (P<0.00001). BAb+TNFalpha+RT combination enhanced the delay for the tumour to reach 1500 mm(3) as compared with RT alone or with RT+TNFalpha (P=0.0011). Median delays were 90, 93, and 142 days for RT alone, RT+TNFalpha, and RT+BAb+TNFalpha groups, respectively. These results suggest that TNFalpha in combination with BAb and RT may be beneficial for the treatment of pancreatic cancer in locally advanced or adjuvant settings.     

Radiocurability by targeting tumor necrosis factor-alpha using a bispecific antibody in carcinoembryonic antigen transgenic mice

PURPOSE: Tumor necrosis factor-alpha (TNF-alpha) enhances radiotherapy (RT) killing of tumor cells in vitro and in vivo. To overcome systemic side effects, we used a bispecific antibody (BsAb) directed against carcinoembryonic antigen (CEA) and TNF-alpha to target this cytokine in a CEA-expressing colon carcinoma. We report the evaluation of this strategy in immunocompetent CEA-transgenic mice. METHODS AND MATERIALS: The murine CEA-transfected colon carcinoma MC-38 was used for all experiments. In vitro, clonogenic assays were performed after RT alone, TNF-alpha alone, and RT plus TNF-alpha. In vivo, the mice were randomly assigned to treatment groups: control, TNF-alpha, BsAb, BsAb plus TNF-alpha, RT, RT plus TNF-alpha, and RT plus BsAb plus TNF-alpha. Measurements of endogenous TNF-alpha mRNA levels and evaluation of necrosis (histologic evaluation) were assessed per treatment group. RESULTS: In vitro, combined RT plus TNF-alpha resulted in a significant decrease in the survival fraction at 2 Gy compared with RT alone (p < 0.00001). In vivo, we observed a complete response in 5 (50%) of 10, 2 (20%) of 10, 2 (18.2%) of 11, and 0 (0%) of 12 treated mice in the RT plus BsAb plus TNF-alpha, RT plus TNF-alpha, RT alone, and control groups, respectively. This difference was statistically significant when TNF-alpha was targeted with the BsAb (p = 0.03). The addition of exogenous TNF-alpha to RT significantly increased the endogenous TNF-alpha mRNA level, particularly when TNF-alpha was targeted with BsAb (p < 0.01). The percentages of necrotic area were significantly augmented in the RT plus BsAb plus TNF-alpha group. CONCLUSION: These results suggest that targeting TNF-alpha with the BsAb provokes RT curability in a CEA-expressing digestive tumor syngenic model and could be considered as a solid rationale for clinical trials.     

Radiation-induced apoptosis in human ovarian carcinoma cells growing as a monolayer and as multicell spheroids

Response to external gamma irradiation was studied in a human ovarian carcinoma cell line (OVCAR 3) growing as a monolayer and as multicell spheroids. Necrosis and apoptosis were documented using Trypan-blue uptake and acridine-orange staining, respectively, and apoptosis was quantified using a terminal deoxynucleotidyl transferase assay. Exposure of OVCAR 3 cells growing as a monolayer to ¹³⁷Cs gamma radiation at a dose of 10 Gy produced 30–40% apoptosis 72 hr after irradiation. Cell-cycle analysis of irradiated cells showed an accumulation of cells in G₂/M phase 24 hr after irradiation and then a decline at 48 hr in conjunction with apoptosis onset. The loss of G₀/G₁ cells in irradiated cultures suggested a preferential entry into apoptosis. No increase in apoptotic cell number was observed in OVCAR 3 spheroids after irradiation, and the cells probably died as a result of necrosis. When spheroids were disrupted immediately after irradiation to obtain a cell suspension, minor apoptosis was observed in association with a marked increase in TB-positive cell number after 96 hr of incubation following irradiation. Thus, a relationship was found between radiation-induced apoptosis and the cell cycle. Results with spheroids suggested the possible involvement of cell-to-cell interactions in apoptosis regulation. Int. J. Cancer 72:851–859, 1997. © 1997 Wiley-Liss, Inc.     

靶向抗PD-1/CD19双特异性抗体的表达与活性鉴定

目的:重组表达和纯化靶向抗PD-1/CD19双特异性抗体(bispecific antibody, BsAb)并验证其活性.方法:以pCAR1为载体,利用分子克隆技术构建抗PD-1/CD19 BsAb真核表达载体,通过PEI试剂转染哺乳动物细胞株CHO-S瞬时表达抗体.利用亲和层析法对BsAb进行纯化,用SDS-PAG...     

Lethal effect of bleomycin and peplomycin on HeLa cells in multicell tumor spheroids

The lethal effects of bleomycin and its derivative, peplomycin, were determined for HeLa S3 cells grown in multicell spheroids cocultured with human diploid fibroblasts. Both drugs were less effective for cells in spheroids than for cells grown exponentially in monolayers. However, HeLa cells from spheroids exposed to the drugs in single-cell suspension were more sensitive to the drugs than were cells in monolayers. Sequential trypsinization of spheroids after exposure to both drugs showed that the surviving fraction increased sharply with increasing depth of cell layers in the spheroids. The presence of 0.5 mM misonidazole, a hypoxic radiosensitizer, enhanced the lethal effect of peplomycin only for the cells in the deeper layer. These findings suggest that the drug resistance of cells in spheroids was due, at least in part, to the microenvironment of the deeper layers. When spheroids were incubated in fresh medium following exposure to both drugs, the cells recovered from the potentially lethal damage within 1 hr. The extent of the recovery from a fixed drug concentration was higher in cells of the superficial layers than in cells of the deeper layers. It is suggested that the limitation of the lethal effects of bleomycin and peplomycin in solid tumors may be overcome by improving the state of oxygenation of hypoxic cells and by combining either drug with one which inhibits recovery from potentially lethal damage.     

Antitumor effects of a bispecific antibody targeting CA19-9 antigen and CD16.

Bispecific murine monoclonal antibodies that target tumor and Fc gamma RIII (CD16) can promote relevant tumor lysis by large granular lymphocytes. For these antibodies to be clinically useful, their properties should be maintained in vivo, where competing human immunoglobulin, shed target antigen, and shed CD16 may be encountered. At a minimum, bispecific antibody antitumor effects should be preserved in whole blood. Furthermore, potentiation of tumor lysis should be reflected by demonstrating the ability of bispecific antibody-retargeted effector cells to infiltrate and mediate lysis of organized tumor. If these characteristics are demonstrated, and there is evidence of in vivo efficacy of bispecific antibody-based therapy in a relevant animal model, further clinical development of such antibodies would be warranted. In this report the ability of CL158 bispecific antibody supernatants to mediate lysis of SW948 tumor growing in monolayer is shown to be preserved in the presence of interleukin 2-activated whole blood. When SW948 cells were grown in vitro as multicellular human tumor spheroids, incubation with interleukin 2-activated lymphocytes (LAK cells) and CL158 led to structural and widespread necrosis. This was dependent on CL158 and resistant to competition by pooled human immunoglobulin or interleukin 2-exposed whole blood. These effects were not promoted by the monospecific antibodies produced by the parent clones of CL158 and were not observed when the IgG2a variant of CA19-9 antibody, which mediates conventional antibody-dependent cellular cytotoxicity, was used instead of its bispecific derivative. To examine the efficacy of bispecific antibody-based treatments on in vivo tumor, scid mice bearing early s.c. SW948 xenografts were treated with interleukin 2 for 5 consecutive days, supplemented by three i.v. injections of 10(7) human LAK cells and various antibodies. Treatment of mice bearing SW948 tumors with LAK cells did not retard tumor growth, but when CL158 was added, significant delays in tumor growth were observed. Tumor growth delay required treatment with both LAK cells and the bispecific antibody. Treatment with the IgG2a variant of CA19-9 antibody, alone or with LAK cells, had no effects on tumor growth. Although the mechanisms of these antitumor effects require further study, it is clear that human LAK cell treatment of animals bearing early, established s.c. tumors is enhanced by the addition of bispecific antibodies with relevant binding characteristics. When compared with the IgG2a isotype variant of CA19-9 monoclonal antibody, this bispecific antibody offers the advantages of preservation of activity in physiological conditions, infiltration and disruption of organized tumor in vitro, and antitumor effects in a relevant xenograft model.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tumor location and drug targeting using a monoclonal antibody (anti-SSEA-1) and antigen-binding fragments

Both murine and heterotransplanted human nonseminomatous germ-cell tumors have been successfully located by external scintigraphy using radioiodinated anti-SSEA-1, a monoclonal IgM, and its pepsin-derived antigen-binding fragment, F(ab')2 mu. Antibody localization in the tumor is mainly due to antigenic specificity, rather than to nonspecific trapping, and depends strongly on the amount of time after injection. The antibody has been used for drug targeting in vitro and in vivo.     

Establishment and evaluation of cancer-specific human monoclonal antibody GAH for targeting chemotherapy using immunoliposomes

To establish human monoclonal antibodies suitable for targeting chemotherapy, we prepared a panel of human-mouse hybridomas, using mouse myelomas and lymphocytes of regional lymph nodes excised from cancer patients, and selected antibodies on the basis of their specificity of binding to the surface of viable cancer cells derived from fresh cancer tissues. A selected antibody, named GAH, was found to react with viable cancer cells from 21/22 stomach and 13/20 colon cancer tissues. As for further analysis, complementary DNAs encoding GAH were cloned and recombinant GAH (rGAH) was obtained from established CHO cells transfected with GAH expression vectors. rGAH selectively stained cancer cells in human tissue sections from 13/14 stomach, 4/11 colon, 5/11 mammary, and 0/7 lung cancers, while no positive staining was observed in those of non-tumor and various normal specimens. Notably, using confocal fluorescence microscopy, rGAH was not only bound to the surface of cancer cells, but was also internalized by the cells. The potential of rGAH for intracellular drug delivery was subsequently evaluated using rGAH-conjugated, doxorubicin (DXR)-encapsulated immunoliposomes. The immunoliposomes were also internalized into the cancer cells and finally DXR was delivered to the cell nucleus. Furthermore, the immunoliposomes could inhibit the growth of DXR-insensitive stomach cancer cells (B37) in an in vivo model. These results suggest that a GAH-utilized liposome-targeting technique will provide a potent and useful cancer chemotherapy with broad applications for cancer patients.     

Enhancement of radiation therapy by tumor necrosis factor alpha in human colon cancer using a bispecific antibody

PURPOSE: To overcome the systemic side effects of tumor necrosis factor alpha (TNFalpha) injected i.v., we used a bispecific antibody (BAb) directed against carcinoembryonic antigen (CEA) and TNFalpha to target this cytokine in human CEA-expressing colorectal carcinoma treated simultaneously with radiation therapy (RT). METHODS, MATERIALS AND RESULTS: LS174T cell line was used to study the interaction of TNFalpha and radiation on clonogenic cytotoxicity. When TNFalpha (2500 U/mL) was added 12 h before RT, the surviving fraction at 2 Gy was 54% lower than that obtained with irradiation alone (0.23 vs. 0.42, respectively, p = 0.001). At 20%, 50%, or 70% survival, data points were within the envelope of additivity. Concerning in vivo experiments, RT as a single agent slowed tumor progression as compared with the control group (p = 0.027), whereas TNFalpha, BAb, or BAb + TNFalpha had no effect. BAb + TNFalpha + RT combination enhanced the delay for the tumor to reach 2000 mm(3) as compared with RT alone (p = 0.033, for BAb + TNFalpha + RT group vs. RT group). CONCLUSION: These results suggest that TNFalpha in combination with BAb and RT may be beneficial for the treatment of locally advanced colorectal cancer.     

Therapeutic advantage of pretargeted radioimmunotherapy using a recombinant bispecific antibody in a human colon cancer xenograft.

PURPOSE: To assess if pretargeting, using a combination of a recombinant bispecific antibody (bsMAb) that binds divalently to carcinoembryonic antigen (CEA) and monovalently to the hapten histamine-succinyl-glycine and a (90)Y-peptide, improves therapeutic efficacy in a human colon cancer-nude mouse xenograft compared with control animals given (90)Y-humanized anti-CEA immunoglobulin G (IgG). EXPERIMENTAL DESIGN: Clearance and biodistribution were monitored by whole-body readings and necropsy. Animals were monitored for 34 weeks with a determination of residual disease and renal pathology in survivors. Hematologic toxicity was assessed separately in non-tumor-bearing NIH Swiss mice. RESULTS: Hematologic toxicity was severe at doses of 100 to 200 microCi of (90)Y-IgG, yet mild in the pretargeted animals given 500 or 700 microCi of the (90)Y-peptide. Evidence of end-stage renal disease was found at 900 microCi of the pretargeted (90)Y-peptide whereas animals given 700 microCi showed only mild renal pathology, similar to that seen in control animals given (90)Y-IgG. Biodistribution data indicated that the average amount of tumor radioactivity by a 700-microCi dose of the pretargeted peptide over a 96-hour period was increased 2.5-fold (48 microCi/g) compared with 150 microCi of (90)Y-IgG (18.9 microCi/g). At these doses, survival (i.e., time to progression to 2.5 cm(3)) was significantly improved (P < 0.04) compared with (90)Y-IgG, with ablation of about one third of the tumors, whereas viable tumor was present in all of the (90)Y-IgG-treated animals. CONCLUSION: Pretargeting increases the amount of radioactivity delivered to colorectal tumors sufficiently to improve the therapeutic index and responses as compared with conventional radioimmunotherapy.     

A universal pretargeting system for cancer detection and therapy using bispecific antibody

Multistep targeting systems represent highly selective alternatives to targeting systems using directly radiolabeled antibodies for diagnostic and therapeutic applications. A flexible bispecific antibody (bsMAb) multistep, pretargeting system that potentially can be developed for use with a variety of different imaging or therapeutic agents is described herein. The flexibility of this system is based on use of an antibody directed against histamine-succinyl-glycine (HSG) and the development of peptides containing the HSG residue. HSG-containing peptides were synthesized with either 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid for the chelation of 111In, 90Y, or 177Lu, or a technetium/rhenium chelate. The peptides can be radiolabeled to a high specific activity in a facile manner that avoids the need for purification. In vivo studies in nude mice bearing human colon tumor xenografts showed that the radiolabeled peptides cleared rapidly from the body with minimal retention in tumor or normal tissues. For pretargeting, these peptides were used in combination with a bsMAb composed of the anti-HSG Fab' that was covalently coupled with the Fab' of either an anticarcinoembryonic antigen or an anticolon-specific antigen-p antibody to provide tumor targeting capability. When the radiolabeled peptides were administered 1-2 days after a pretargeting dose of the bsMAbs, tumor uptake of the radiolabeled peptides increased as much as 28-175-fold over that seen with the peptides alone with tumor:nontumor ratios exceeding 2:1 to 8:1 within just 3 h of the peptide injection, which was a marked improvement over the tumor:nontumor ratios seen with a directly radiolabeled 99mTc-anti-anticarcinoembryonic antigen Fab' at this same time. The anticolon-specific antigen-p x anti-HSG F(ab')2 bsMAb had the highest and longest retention in the tumor, and when used in combination with the 111In-labeled peptide, radiation dose estimates for therapeutic radionuclides, such as 90Y and 177Lu, suggested that antitumor effects would be expected with tolerable radiation exposure to the normal tissues. These results suggest that this multistep, pretargeting system has diagnostic imaging and therapeutic potential.     

HPLC and flow cytometric analyses of uptake of adriamycin and menogaril by monolayers and multicell spheroids

We have used both HPLC and flow cytometry to measure and compare the uptake of two anthracyclines, menogaril (MEN) and Adriamycin (ADR), in V79 Chinese hamster lung fibroblasts grown as monolayers and as 650 microns multicell spheroids. In order to compare intracellular drug accumulation in spheroid cells measured by the two methods, we converted mean channel fluorescence of the flow cytometer to drug uptake expressed as ng/10(6) cells by using a standard curve. The standard curve related the flow cytometric mean channel fluorescence, of monolayer cells exposed to either drug, to the intracellular drug accumulation determined by HPLC. This standard curve was then used to convert the mean channel fluorescence of cells from drug-exposed spheroids to ng/10(6) cells. Our results show that equal intracellular drug accumulation (determined by HPLC) in spheroids and monolayers does not result in equal cellular fluorescence emission (determined by flow cytometry) by these 2 cell populations. For example, monolayer cells with an intracellular MEN accumulation of 650 ng/10(6) cells, emit 40 units of fluorescence as measured by flow cytometry. However, spheroid cells with the same intracellular accumulation emit about 80 units of fluorescence. This results in the intracellular MEN uptake in spheroids measured by flow cytometry being as much as 2- to 3-fold higher than that measured by HPLC. Intracellular ADR accumulation measured by flow cytometry was also higher than that obtained by HPLC. In spite of the quantitative difference between the two methods, qualitatively both methods gave similar results. Thus, both techniques showed that at equal drug concentration in medium drug uptake in monolayers was much greater than in spheroids.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro and in vivo antitumor effect of a trivalent bispecific antibody targeting ErbB2 and CD16

In order to optimize the structure of bispecific antibody (BsAb) and minimize its toxicity, we developed a trivalent anti-ErbB2/anti-CD16 BsAb. This BsAb possesses three antigen binding sites, two antigen binding sites in the form of scFvs targeting the tumor cells overexpressing ErbB2 and a monovalent Fab fragment redirecting NK cells. Critical for this BsAb is its capacity to trigger cytotoxicity of the effector cells in vitro and in vivo. In the present study, we demonstrated that the BsAb is capable of binding to ErbB2 extracellular domain on SKBR3 cells and effectively direct the cytotoxic activities of effector cells to SKBR3 cells even at a low concentration. The BsAb was more effective to SKBR3 cells than to MCF-7 cells, indicating that the killing of tumor cells was dependent on the density of ErbB2 molecules on cell surface. Furthermore, the BsAb was more potent than anti-ErbB2 single chain antibody (scFv)-Fc fusion proteins in the cytotoxicity assay and in SKOV3 xenograft animals. Improved efficacy demonstrates that the BsAb may be valuable to be further modified and optimized for the treatment of malignant cells in a minimal residual disease.     

Construction and characterization of bispecific costimulatory molecules containing a minimized CD86 (B7-2) domain and single-chain antibody fragments for tumor targeting.

Efficient T-cell activation requires two signals. The first signal, which confers specificity, is provided by interaction of the T-cell receptor with peptides presented by MHC molecules. One of the second costimulatory signals is induced by binding of B7 proteins on the surface of antigen-presenting cells to CD28 on the T-cell surface. Expression of B7 molecules on tumor cells can result in the activation of tumor specific T lymphocytes and induce protective antitumor immunity. However, at present such gene-therapeutic approaches are limited by the inability to selectively target B7 gene expression to cancer cells. As an alternative approach we exploited recombinant antibody fragments to localize a costimulatory B7 molecule to the surface of tumor cells. We constructed chimeric proteins that contain in a single polypeptide chain a portion of human B7-2 (CD86) genetically fused to single-chain (sc) Fv antibody domains specific for the tumor-associated antigens epidermal growth factor receptor and the closely related ErbB2 receptor tyrosine kinase. A small recombinant fragment of human CD86 was characterized that corresponds to amino acid residues 1-111 (CD86(111)) of the mature protein. CD86(111) produced in the yeast Pichia pastoris and CD86(111) expressed in bacteria was functionally active and displayed specific binding to B7 counter receptors. Bacterially expressed CD86(111)-scFv fusion proteins specifically localized to the respective target antigens on the surface of tumor cells and markedly enhanced the proliferation of primary T cells when bound to immobilized tumor antigen.     

Therapy of human non-small-cell lung carcinoma using antibody targeting of a modified superantigen.

Superantigens activate T-cells by linking the T-cell receptor to MHC class II on antigen-presenting cells, and novel reactivity can be introduced by fusing the superantigen to a targeting molecule. Thus, an antibody-targeted superantigen, which activates T cells to destroy tumour cells, might be used as cancer therapy. A suitable target is the 5T4 oncofetal antigen, which is expressed on many carcinomas. We constructed a fusion protein from a Fab of a monoclonal antibody recognizing the 5T4 antigen, and an engineered superantigen. The recombinant product 5T4FabV13-SEA(D227A)bound the 5T4 antigen expressed on the human non-small-cell lung cancer cell line Calu-1 with a Kd of 1.2 nM while the substitution of Asp227 to Ala in the superantigen moiety reduced binding activity to MHC class II. 5T4FabV13-SEA(D227A)tumour reactivity was demonstrated in 7/7 NSCLC samples by immunohistochemistry, while normal tissue reactivity was low to moderate. 5T4FabV13-SEA(D227A)induced significant T-cell-dependent in vitro killing of sensitive 5T4 bearing Calu-1 cells, with maximum lysis at 10(-10)M, while the capacity to lyse MHC class II expressing cells was approximately 1000 times less effective. Immunotherapy of 5T4FabV13-SEA(D227A)against human NSCLC was investigated in SCID mice reconstituted with human peripheral blood mononuclear cells. Mice carrying intreperitoneally growing Calu-1 cells showed significant reduction in tumour mass and number after intravenous therapy with 5T4FabV13-SEA(D227A). Thus, 5T4FabV13-SEA(D227A)has highly attractive properties for therapy of human NSCLC.     

131碘标记双功能单克隆抗体的制备鉴定与纯化及质量控制

目的:本实验在于研究BiMcAB介导免疫治疗的可能性,因双抗是生物制剂,有必要进行质量控制。方法:采用硫酸胺盐析法纯化蛋白,及氯胺法标记蛋白,并进行灭菌检查。结果:BiMcAB的理化特性,生物学鉴定,放射化学指标,生物及免疫学指标均符合临床应用标准。结论:BiMcAB有可能成为一种既能定向杀伤癌细胞,而对正常细胞又无损害作用的安全的新的治疗途径。     

Antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC) of breast cancer cells mediated by bispecific antibody, MDX-210

Background: MDX-210 is a bispecific antibody (BsAb) with specificity for both the proto-oncogene product of HER-2/neu (c-erbB-2) and FcRI (CD64). HER-2/neu is overexpressed in malignant tissue of approximately 30% of patients with breast cancer, and FcRI is expressed on human monocytes, macrophages, and IFN- activated granulocytes. We investigated phagocytosis and cytolysis of cultured human breast cancer cells by human monocyte-derived macrophages (MDM) mediated by BsAb MDX-210, its partially humanized derivative (MDX-H210), and its parent MoAb 520C9 (anti-HER-2/neu) under various conditions.Materials and Methods: Purified monocytes were cultured with GM-CSF, M-CSF, or no cytokine for five or six days. Antibody dependent cellular phagocytosis (ADCP) and cytolysis (ADCC) assays were performed with the MDM and HER-2/neu positive target cells (SK-BR-3). ADCP was measured by two-color fluorescence flow cytometry using PKH2 (green fluorescent dye) and phycoerythrin-conjugated (red) monoclonal antibodies (MoAb) against human CD14 and CD11b. ADCC was measured with a non-radioactive LDH detection kit.Results: Both BsAb MDX-210 (via FcRI) and MoAb 520C9 (mouse IgG1, via FcRII) mediated similar levels of ADCP and ADCC. ADCP mediated by BsAb MDX-H210 was identical to that mediated by BsAb MDX-210. Confocal microscopy demonstrated that dual-labeled cells represented true phagocytosis. Both ADCP and ADCC were higher when MDM were pre-incubated with GM-CSF than when incubated with M-CSF.Conclusions: BsAb MDX-210 is as active in vitro as the parent MoAb 520C9 in inducing both phagocytosis and cytolysis of MDM. MDX-210 and its partially humanized derivative, MDX-H210, mediated similar levels of ADCP. GM-CSF appears to superior to M-CSF in inducing MDM-mediated ADCC and ADCP. These studies support the ongoing clinical investigations of BsAb MDX-210 and its partially humanized derivative.