抗VEGF嵌合抗体的生物学特性分析

目的：分析抗人血管内皮生长因子（VEGF）嵌合抗体VcD11的各种生物学特性,确定其人源性,特异性,中和VEGF的中和活性以及体内抑制肿瘤生长转移的抗肿瘤活性。方法：采用纯化的VcD11进行ELISA和Westerm blot实验,分析其人源性、特异性;采用抑制由VEGF刺激引起的内皮细胞增殖实验分析VcD11的中和活性;采用近交系小鼠T739的小鼠肺腺癌LA795实验动物肿瘤模型分析VcD11的抗肿瘤活性。结果VcD11在ELISA和Western blot实验中均呈阳性结果,并可抑制由VcD11的刺激引起的内皮细胞增殖,虽未能明显抑制小鼠肺腺癌LA795原发瘤的生长,但可显著抑制其肺转移灶的形成和工。结论,抗VEGF嵌合抗体VcD11具有人抗体的恒定区,可与人VEGF特异地结合,并具有良好的中和活性和抗肿瘤转移的活性。它可能在临床治疗肿瘤中有着重要、广泛的应用潜力。     

Antitumor Effect of a Neutralizing Antibody to Vascular Endothelial Growth Factor on Liver Metastasis of Endocrine Neoplasm

Distant metastasis of gastrointestinal endocrine neoplasm is resistant to currently available treatments. Because hematogenic metastasis is dominant, anti-angiogenic drugs are expected to be a novel therapy for this neoplasm. In the present study, the therapeutic effect of vascular endothelial growth factor neutralizing antibody (VEGFAb) on liver metastasis of an endocrine neoplasm was investigated experimentally. Cecal transplantation into nude mice of small pieces of EN-1, a xenotransplanted human intestinal endocrine neoplasm, resulted in liver metastasis. A treated group ( n =19) received 100 μg/mouse of VEGFAb intraperitoneally on alternate days from day 10 after tumor transplantation, and the control group ( n =19) received saline. Five of the 19 control mice died of tumor progression, of which 2 could not be evaluated. The cecal tumor weighed 6316±2333 mg ( n =17) in the control group and 1209±837 mg ( n =19) in the treated group ( P <0.01) 6 weeks after transplantation. Liver metastasis developed in 16 of 17 control mice and in 2 of 19 treated mice ( P <0.01). The VEGF level of the whole cecal tumor in the control group was significantly higher than that in the treated group (305.1±174.1 vs. 54.7±41.2 mg; P <0.001). VEGFAb did not cause any body weight loss (28.52±1.63 in the control vs. 28.44±1.71 g in the treated group). These results indicate that VEGFAb may be a novel therapeutic agent for endocrine neoplasm with distant metastasis.     

Combination Therapy with Vascular Endothelial Growth Factor Neutralizing Antibody and Mitomycin C on Human Gastric Cancer Xenograft

Antiangiogenic therapy has been proposed as a new strategy for the treatment of solid tumors. To enhance the therapeutic effect of antiangiogenic agents, combination with conventional anticancer therapy should be investigated. In the present study, we investigated the therapeutic effect of the combination of vascular endothelial growth factor neutralizing antibody (VEGF Ab) and mitomycin C (MMC) on MT-2, a human gastric cancer xenograft. When small pieces of MT-2 were transplanted orthotopically into 62 nude mice, liver metastasis developed 6 weeks after transplantation. The VEGF Ab (100 μg/mouse) was administered i.p. in the VEGF Ab group ( n =14) and the combination group ( n =16) twice a week from day 10 after transplantation. MMC (2 mg/kg) was administered in the MMC group ( n =16) and the combination group ( n =16) on days 10, 17 and 24 after transplantation. Compared with the control group, in which saline solution was administered i.p., all three treatments inhibited tumor growth significantly and the effects of MMC and combination therapy were potent. Liver metastases were also inhibited significantly by the administration of VEGF Ab alone, MMC alone or combination therapy. Liver metastasis developed in 9 mice of the control group, 3 of the VEGF Ab group, and 4 of the MMC group, but no mice had liver metastasis in the combination therapy group. However a significant body weight loss and a decrease in spleen weight were observed in the MMC and combination groups, with no significant difference between the two groups. These results suggest that combination therapy with VEGF Ab and MMC may be a potent therapy for human gastric cancer.     

Antitumor Effect on Human Gastric Cancer and Induction of Apoptosis by Vascular Endothelial Growth Factor Neutralizing Antibody

Induction of apoptosis by antiangiogenic therapy has been suggested as a new anticancer strategy. To clarify the mechanism of the antitumor effect achieved by inhibition of vascular endothelial growth factor (VEGF), which is a major mediator of angiogenesis, we used an orthotopic transplantation model of human gastric carcinoma line (MT2) treated with a monoclonal VEGF neutralizing antibody (VEGF Ab). We histologically examined the microvessel density (MVD) and the apoptotic index (AI) in this model. Transplanted tumor growth was significantly inhibited by the VEGF Ab ( P = 0.03), and there was a significant decrease in the number of mice with liver metastasis ( P = 0.004). The MVD detected by immunohistochemical staining with ER-MP12 antibody was 33.6 ± 8.0 in the control group and 21.1 ± 5.4 in the treated group, and the difference was significant ( P < 0.0001). The AI values of the control and treated groups were 4.73 ± 1.11 and 7.26 ± 1.62, respectively, and this difference is also significant ( P < 0.0001). However, the expression of VEGF mRNA in transplanted tumors did not show a significant difference between the control and treated groups. These results suggest that the antitumor effect of the VEGF Ab on human gastric carcinoma is exerted by inducing mild hypoxia followed by apoptosis, which does not influence VEGF mRNA expression in the carcinoma.     

Targeting Vascular Endothelial Growth Factor (VEGF) for Anti-tumor Therapy, by Anti-VEGF Neutralizing Monoclonal Antibodies or by VEGF Receptor Tyrosine-kinase Inhibitors

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is an important mediator of tumor-induced angiogenesis and represents a potential target for innovative anticancer therapy. In several animal models, neutralizing anti-VEGF/VPF antibodies have shown encouraging inhibitory effects on solid tumor growth, ascites formation and metastatic dissemination. Targeting the VEGF signaling pathway by means of VEGF receptor tyrosine-kinase inhibitors has shown similar efficacy in animal tumor models. Several of these anti-VEGF therapies are currently being tested in clinical trials in cancer patients. The profiles and effects of the neutralizing anti-VEGF/VPF antibodies and the VEGF receptor tyrosine-kinase inhibitors in animal models are reviewed and of the risks and benefits of VEGF blockade by one or the other treatments are discussed.     

Expression Behavior of 85-kDa Membrane Protein in Adriamycin-resistant Tumor Cells and the Inhibition of Human Tumor Growth in Athymic Mice by MRK-20 Monoclonal Antibody against the Protein

Treatment of tumors with monoclonal antibodies against tumor antigen is one of the selective modalities for cancer therapy. We examined the therapeutic effect of MRK-20 (IgG₁), a murine monoclonal antibody against resistance-associated 85-kDa membrane protein. The 85-kDa protein is expressed on the surface of multidrug-resistant cells induced by adriamycin. This protein is also expressed in some multiple-drug-resistant cells, including atypical multidrug-resistant cells. The protein, once lost during long-term culture without adriamycin, was rapidly induced by treatment with adriamycin but not with vinblastine or etoposide, suggesting a close relationship of the protein with adriamycin resistance but not with multidrug resistance. The antibody MRK-20 suppressed the growth of subcutaneously implanted tumors expressing the 85-kDa protein. Adriamycin-resistant human ovarian tumor 2780AD and innately resistant human erythroleukemia HEL cells in athymic mice were completely cured by treatment with MRK-20 antibody when the antibody was administered i.v. 2 days after s.c. tumor implantation. On the other hand, MRK-20 did not show any effect on the growth of the 85-kDa protein-negative A2780 human ovarian tumor. These results indicate that the effect of MRK-20 is highly specific to cells expressing 85-kDa protein.     

Dual Pathways to Tubular Morphogenesis of Vascular Endothelial Cells by Human Glioma Cells: Vascular Endothelial Growth Factor/Basic Fibroblast Growth Factor and Interleukin-8

In this study, we examined whether human glioma cells are angiogenic in a model using human microvascular endothelial cells, and also which factor is responsible for the glioma-dependent angiogenesis. Tubular morphogenesis in type I collagen gel by human microvascular endothelial cells was stimulated in the presence of 10 and 100 ng/ml of vascular endothelial growth factor (VEGF), 10 ng/ml basic fihroblast growth factor (bFGF) and 10 ng/ml of interleukin-8 (IL-8). Tube formation of the microvascular endothelial cells was assayed in the glioma cell lines IN157 and IN301, co-cultured using the double chamber method. IN301 cells had much higher levels of VEGF, bFGF and transforming growth factor-(mRNA than IN157 cells, whereas the two had similar levels of transforming growth factor-Alfa mRNA. By contrast, IN157 cells had much higher levels of IL-8 mRNA than IN301 cells. IN301-dependent tubular morphogenesis was inhibited by anti-VEGF or anti-bFGF antibody, and the inhibition was almost complete when anti-VEGF and anti-bFGF antibodies were present. On the other hand, IN157-dependent tubular morphogenesis was inhibited by anti-IL-8 antibody, but not by anti-VEGF or anti-bFGF antibodies. These findings demonstrated dual paracrine controls of tumor angiogenesis by human glioma cells. One is mediated through VEGF and/or bFGF, and the other, through IL-8.     

放射与抗VEGF抗体对人肝癌移植瘤生长的抑制作用

目的观察放射与抗VEGF单抗对人肝癌裸鼠移植瘤生长的抑制作用。方法SMMC-7721肝癌细胞种植于裸鼠后肢皮下,成瘤动物分为放射+抗体组、放射组、抗体组和对照组,单抗每次每只50μg隔日腹腔给药,共6次,放射剂量20Gy一次给予,测肿瘤直径并计算瘤体积。抗体给药结束后2周处死动物,称瘤重,免疫组化法测肿瘤微血管密度。结果放射和抗VEGF单抗明显抑制肿瘤生长,减少肿瘤微血管密度,但两组之间差异无显著性。放射与抗VEGF单抗联合较单纯放射或抗体抑瘤作用显著,三组瘤重抑制率分别为89.2%、75.3%和65.9%,差异有显著性。结论放射结合抗VEGF单抗显著抑制肝癌移植瘤生长,是肝癌综合治疗的有效途径。     

Establishment of a Human Monoclonal Antibody to Hanganutziu-Deicher Antigen as a Tumor-associated Carbohydrate Antigen

We have established a human-human hybridoma producing a monoclonal antibody against a tumor-associated carbohydrate-specific antigen, Hanganutziu-Deicher (HD) antigen. Human spleen lymphocytes from a patient with esophageal varices complicated with liver cirrhosis were cultured in serum-free medium and co-stimulated with both anti-human μ-chain antibodies and supernatants of concanavalin A-stimulated human spleen cell culture (ConA sup). The activated lymphocytes were subsequently primed in vitro with particulate HD3 antigen and fused with a parent hybrid myeloma cell line, KR-12. A hybridoma, 1F43E31G7 produced anti-HD human monoclonal antibody (IgM λ). This monoclonal antibody reacted strongly with N-glycolyl-neuraminyl α2-3 lactosylceramide (HD3) and slightly with N-glycolylneuraminyl α2-3 lactoneotetraosylceramide (HD3), but did not react with N-glycolylneuraminyl α2-3 lactoneohexaosylceramide (HD7), N-acetylneuraminyl α2-3 lactosylceramide (GM3) and other derivatives of HD3 prepared by chemical modification of the sialic acid residue of HD3, which indicates that the monoclonal antibody is directed precisely toward the terminal sialic acid and whole structure of HD3.     

抗VEGF抗体及抗KDR抗体对人胃癌生长的抑制作用

目的:观察抗VEGF及抗KDR抗体对胃癌生长和转移的抑制作用。方法:1)将不同浓度的抗VEGF及抗KDR抗体分别加入胃癌BGC-823细胞,培养5天后测细胞活性(MTT法)。2)采用原位种植裸鼠模型,观察抗VEGF抗体及抗KDR抗体的作用。实验分为4组:对照组,抗VEGF组,抗KDR组及抗VEGF+抗KDR组。自肿瘤种植后第7天起,经腹腔注射连续给药8周,每周2次,第10周末处死裸鼠,测定肿瘤重量和肝转移状况,计数微血管密度。结果:1)抗VEGF抗体和抗KDR抗体浓度由高至低对BGC-823细胞生长的抑制作用减弱。2)抗VEGF抗体和抗KDR抗体对原位肿瘤生长有明显抑制作用,两者合用时作用更明显;对照组肝转移率80.0%,而抗VEGF组、抗KDR组和二者合用组分别为16.7%、25.0%、0,与对照组相比其它3组的微血管密度(MVD)明显为低。结论:通过中和VEGF抗体和封闭KDR受体抑制血管形成,可能为胃癌的治疗提供一个新的途径。     

Anti—Epidermal Growth Factor Receptor Monoclonal Antibody Cetuximab Plus Doxorubicin in the Treatment of Metastatic Castration-Resistant Prostate Cancer

Purpose

An open-label, dose-escalating phase Ib/IIa trial was performed to establish a safety profile of ascending doses of cetuximab (IMC C225) in combination with doxorubicin administered weekly for 6 treatments in patients with metastatic castration-resistant prostate cancer. The secondary endpoint was to assess the efficacy of cetuximab in combination with doxorubicin as well as to determine the optimal biologic dose and the maximum tolerated dose.

Patients and Methods

Patients in 8 groups received escalating doses of cetuximab 20-300 mg/m² plus doxorubicin 15 or 20 mg/m² given intravenously weekly for 6 consecutive weeks, followed by a 1-week observation period. A treatment response was defined as a > 50% decline in prostate-specific antigen (PSA) or regression of radiographically measurable disease.

Results

Of the 36 treated patients, 25% had grade 2 neutropenia, 39% had leukopenia, and 44% had stomatitis at doxorubicin 20 mg/m². Erythematous skin exanthema was seen in 38% of the patients. There was no significant regression of bone or soft tissue disease, but stable disease was observed in 20 (65%) of the 31 patients with bone disease and 14 (61%) of the 23 patients with lymph node disease. Declines in PSA were modest in the 36 patients, with 1 (2.7%) with an 80% decline from baseline, 2 (5.6%) with > 50% to < 80% declines, and 14 (39%) with progression. Median survival was approximately 18 months.

Conclusion

In a heavily pretreated population of men with metastatic castration-resistant prostate cancer, this study of cetuximab/doxorubicin was associated with minimal PSA declines posttherapy, though median survival was longer compared to historical control groups. Further studies with cetuximab combined with more contemporary chemotherapy for castration-resistant prostate cancer might be warranted.     

Tumor Localization and in vivo Antitumor Activity of the Immunoconjugate Composed of Anti-human Colon Cancer Monoclonal Antibody and Mitomycin C-Dextran Conjugate

The tissue distribution and in vivo antitumor activity of a novel monoclonal antibody-mitomycin C conjugate (A7-MMCD) composed of anti-human MAb A7 and MMC-dextran conjugate were investigated using tumor-bearing mice. A7-MMCD was prepared via an anionic dextran intermediate for the purpose of keeping the non-specific uptake by the reticuloendothelial system to a minimum, ¹¹¹n-labeled A7-MMCD showed about a 5-times-greater accumulation in SW1116 (targeted tumor) than in S180 (non-targeted tumor) 48 h after injection, and produced a tumor-to-blood ratio which was 3 times higher in SW1116-bearing mice than in S180-bearing mice 96 h after injection. Accumulations in the liver, spleen, and kidney were also observed to some extent. Pharmacokinetic analysis revealed that A7-MMCD had nearly the same properties in the body as MMCD_an (MMCD with an anionic charge), i.e. those of a negatively charged macromolecule. Both A7-MMCD and MMCD_an had relatively similar tissue uptake rate indices for the liver and spleen. The tumor uptake rate index for SW1116 was about 2.5 times greater than that for S180, and the total amount of ¹¹¹In-A7-MMCD accumulated in SW1116 was calculated to be approximately 5 times greater than the amount in S180. These results indicated that A7-MMCD could achieve site-specific targeting in the body. Furthermore, in the therapeutic experiment using SW1116 implanted subcutaneously, A7-MMCD suppressed tumor growth significantly, compared to free MMC and MMCD_an. These results suggest that in designing an monoclonal antibody-drug conjugate via an intermediary, the physicochemical properties of intermediate macromolecules must also be taken into consideration to obtain a high degree of efficacy in vivo.     

Antitumor Activity of ME2303, a Fluorine-containing Anthracycline, against Human Tumors Implanted in Nude Mice

A fluorine-containing anthracyctine, ME2303, given intravenously once a week for 4 weeks at the maximum tolerated dose showed better therapeutic effects against 2 gastric, 3 lung and 2 human breast tumor xenografts than did adriamycin (ADM) at the maximum tolerated dose. Among the tumors, ME2303 showed a better effect against St-40, a well-differentiated human gastric adenocarcinoma, against which ADM showed only a marginal effect. Likewise, ME2303 was more effective against Lu-24 human small cell carcinoma and MX-1 human medullary tubular adenocarcinoma than ADM. Notably, the Lu-24 tumor, developed in nude mice, disappeared after the treatment in 3 out of 6 mice. ME2303 would be an interesting compound for phase I and II clinical studies in the future     

抗血管内皮生长因子抗体抑制成骨肉瘤生长及血管生成的实验探讨

目的:观察抗血管内皮生长因子(VEGF)抗体对人成骨肉瘤生长的影响。方法:用抗VEGF单克隆抗体100!g隔天于治疗组荷瘤裸鼠肿瘤周围及瘤块内注射,对照组用等量PBS注射,共给药6次。测量两组裸鼠肿瘤体积及肿瘤湿重,肿瘤组织行免疫组织化学染色观察微血管密度(MVD),光镜下病理学检查。结果:治疗组与对照组的肿瘤体积、重量、MVD值两组间差异有显著性,抑瘤率达54.05%。病检发现治疗组肿瘤内有散在点状或线状坏死灶,甚至小片状坏死灶,对照组很少出现坏死灶。结论:抗VEGF单克隆抗体能够有效抑制裸鼠体内人成骨肉瘤移植瘤的血管生成抑制肿瘤生长。     

Inhibition of Multidrug-resistant Human Tumor Growth in Athymic Mice by Anti-P-glycoprotein Monoclonal Antibodies

In an effort to devise an effective treatment for human drug-resistant cancers, we have developed monoclonal antibodies, MRK16 and 17, reactive to the multidrug transporter protein, P-glycoprotein. The monoclonal antibodies given intravenously effectively prevented tumor development in athymic mice inoculated subcutaneously with drug-resistant human ovarian cancer cells 2780^AD. Treatment with MRK16 induced rapid regression of established subcutaneous tumors and apparent cures of some animals. Complement-dependent cytotoxicity (MRK16) and antibody-dependent cell-mediated cytolysis (MRK16 and 17) were observed with these antibodies. These monoclonal antibodies may have potential as treatment tools against multidrug resistant human tumors possessing the P-glycoprotein.     

人脑胶质细胞瘤中血管内皮生长因子基因的表达研究

目的　研究血管内皮生长因子 (vascularendothelialgrowthfactor ,VEGF)在脑胶质细胞瘤中的表达及其与肿瘤病理分级、新生血管形成的关系。方法　采用Northern杂交和免疫组织化学染色检测 3 0例脑胶质瘤标本 ,2例脑胶质瘤细胞株 ,4例正常脑组织的VEGFmRNA和蛋白表达水平。结果　正常脑组织中VEGFmRNA的表达水平极低 ,而脑胶质瘤组织和细胞系中VEGFmRNA的表达水平高 ,P <0 .0 5。在正常脑组织中未见VEGF免疫组化染色阳性细胞 ,脑胶质瘤组织和细胞系中VEGF高表达 ,P <0 .0 5。随着脑胶质瘤恶性程度的增加 ,VEGFmRNA的表达增高 (γ =0 .85 ,P <0 .0 1)。VEGF的表达还与肿瘤的血管密度相关 (γ =0 .88,P <0 .0 1)。结论　脑胶质瘤中VEGF的异常表达 ,是判断脑胶质细胞瘤恶性程度和血管丰富程度的 1项客观指标。     

抗乳腺癌单克隆抗体的制备及其应用

目的：制备抗人乳腺癌单克隆抗体（McAb），用于临床肿瘤的免疫病理诊断与分型研究，为药物导向诊断和治疗奠定基础。方法：以乳腺癌细胞系ZR75免疫Balb/c小鼠，取其脾细胞与小鼠骨髓瘤细胞SP2／0进行融合，经选择培养、筛选及克隆化，获得恒定分泌抗ZR76单克隆抗体的杂交瘤细胞系BM109。结果：单抗BM109的Ig亚类为IgG1，BM109抗原主要表达在靶细胞膜上，为分子量37KD的蛋白质抗原。在乳腺癌的免疫反应率为86．7％（26／30），与胃癌、食管癌、卵巢癌等6种肿瘤的免疫反应率为9．5％（4／42），而对乳腺纤维瘤、囊性增生症和周围正常乳腺组织未见阳性反应。结论：所获一株抗乳腺癌单克隆抗体，具有较好的特异性，可望进一步用于乳腺癌的诊断、治疗及其发生发展的研究。     

抗胃癌单抗在肾包膜下荷人胃癌移植瘤裸小鼠体内生物学分布

 本人报道胃癌单抗MGb₂和MG7用¹²⁵I标记后, 观察它们在左右肾包膜下分别接种SGC-7901与GAⅡ移植瘤的裸小鼠体内的生物学分布。 结果表明, 腹腔注射¹²⁵I-MGb₂(40uci/7.4ugMGb₂/小鼠)后96小时．SGC－7901和GAⅡ移植瘤与血的T/NT值分虽为1.03和1.57。 而腹腔注射¹²⁵I－MG₇(40uci/7.4ugMG7/小鼠后96小时, 两移植瘤组织与血的T／NT值分别为0.31和1.53。     

人胃癌VEGF和NOS的表达及其相关性

目的：研究VEGF、iNOS和eNOS在人胃癌的表达及其相关性；研究N0和VEGF的相互作用及在促肿瘤生长中的作用机制。方法：应用免疫组化方法检测34例胃癌标本VEGF、iNOS和eNOS的表达及分布。结果：1)34例胃癌组织中，表达iNOS的为73．5％，表达eNOS的为82．4％，表达VEGF的占91．2％。2)VEGF与iNOS的表达具有明显相关性(入0．005)，VEGF与eNOS的表达无明显相关性(Pb0．05)。结论：VEGF与iNOS的表达具有明显相关性，iNOS在VEGF的生成和发挥过程中起重要作用。     

人脑膜瘤血管内皮生长因子表达及生物学意义

[目的]研究血管内皮生长因子(VEGF)在人脑膜瘤中的表达及其生物学意义.[方法]采用免疫组织化学方法检测51例脑膜瘤(WHO分级良性38例,非典型11例,恶性2例)VEGF蛋白的表达水平和微血管密度(MVD).[结果]良性脑膜瘤VEGF阳性表达率86.8%(33/38),不典型、恶性脑膜瘤VEGF阳性表达率为84.6%(11/13)(P＞0.05).而3例正常脑膜组织未见VEGF表达(P＜0.05).良性、非典型及恶性脑膜瘤的MVD分别为131.08±115.93和145.12±99.21(P＞O.05),VEGF表达阴性的脑膜瘤其MVD为88.25±59.86,VEGF表达呈 、 、 的MVD为102.25±149.55,108.12±54.43,172.36±118.58,两者表达成正相关(r=O.45,P＜0.05).VEGF表达与脑膜瘤的侵袭性、脑浸润性无关.[结论]VEGF在脑膜瘤的血管形成中起重要作用,可能参与脑膜瘤的形成,但与脑膜瘤的良性向非典型转变、脑浸润性、侵袭性等生物学特性无关.