Production, Binding and Cytotoxicity of Human/Mouse Chimeric Monoclonal Antibody-Neocarzinostatin Conjugate

A human/mouse chimeric Fab monoclonal antibody A7 (chFabA7) was covalently coupled to neocarzinostatin (NCS) by the SPDP method at various chFabA7:NCS substitution ratios. The antigen-binding activity of the conjugate, examined by ELISA using fixed antigen-positive colon cancer cells, was identical to that of the parent chFabA7 when one mole of NCS was conjugated, but was reduced with 2 or 3 moles of conjugated NCS. By means of a colony-forming assay, the cytocidal effect of the conjugate on antigen-positive cancer cells was found to be stronger than that of free NCS, whereas in antigen-negative cancer cells it was similar to that of free NCS. This effect was attenuated by adding an excess amount of monoclonal antibody A7. These findings indicate that the conjugate has an antigen-specific cytocidal action, and thus chFabA7-NCS is a promising tool for targeting cancer chemotherapy.     

Preoperative Clinical Radioimmunodetection of Pancreatic Cancer by 111In-labeled Chimeric Monoclonal Antibody Nd2

The present study was carried out with the purpose of evaluating the clinical usefulness of radioimmunodetection (RAID) with ¹¹¹In-labeled murine/human chimeric monoclonal antibody, Nd2 (c-Nd2) in patients with pancreatic cancer. Nineteen patients suspected to have pancreatic cancer were administered intravenously 74 MBq/2 mg ¹¹¹In-labeled c-Nd2 in 100 ml of saline containing 2% albumin over 30 min. A scintigram was obtained on the 3rd day after infusion by using single photon emission computed tomography (SPECT) imaging. Of the 14 patients finally diagnosed as having pancreatic cancer on the basis of surgical specimens or progress of disease, specific focal uptake at the site of the tumor was detected in 12 (true positive cases), representing a sensitivity of 85.7% (12/14), and liver metastasis was found in one case with metastasis. Of the 5 patients diagnosed with tumor-forming pancreatitis (TFP), 4 patients demonstrated true negative imaging, but one patient whose tumor demonstrated interesting findings in histology and immunostaining, showed false positive imaging. Of patients investigated for human anti-chimeric antibody (HACA) response, none showed HACA response, and no allergic reaction was seen in any of the patients administered c-Nd2. These results suggest that RAID with ¹¹¹In-labeled c-Nd2 is useful for differential preoperative diagnosis between invasive pancreatic cancer and TFP.     

Biodistribution of Neocarzinostatin Conjugated to Chimeric Fab Fragments of the Monoclonal Antibody A7 in Nude Mice Bearing Human Pancreatic Cancer Xenografts

In this study, we conjugated chimeric Fab fragments of the monoclonal antibody (MAb) A7, which reacts with pancreatic cancers, to the antitumor drug neocarzinostatin (chA7Fab-NCS) and intravenously injected ¹²⁵I-labeled chA7Fab-NCS into nude mice bearing a human pancreatic cancer xenograft. We compared the tumor localization of ¹²⁵I-labeled chA7Fab-NCS with that of conventional ¹²⁵I-labeled A7-NCS, which was produced by conjugation of MAb A7 and NCS. ¹²⁵I-Labeled chA7Fab-NCS accumulated in the tumor earlier than ¹²⁵I-labeled A7-NCS, and significantly larger amounts of ¹²⁵I-labeled chA7Fab-NCS had accumulated in the tumor 1 hour after injection. The results suggest that chA7Fab may be a suitable carrier for NCS in immunotargeting therapy against pancreatic cancer.     

Antibody-dependent Cytotoxicity Mediated by Chimeric Monoclonal Antibody Nd2 and Experimental Immunotherapy for Pancreatic Cancer

In a previous study, mouse monoclonal antibody (MoAb) Nd2 (m-Nd2, mouse IgGl) labeled with ¹³¹ I exhibited efficacy in in vivo radioimmunotherapy against pancreatic cancer. In this study we prepared mouse/human chimeric antibody Nd2 (c-Nd2, human IgG1) for clinical use and examined whether c-Nd2 induced antibody-dependent cell-mediated cytotoxicity (ADCC). Cytotoxicity to pancreatic cancer (PC) cell lines, including Nd2 antigen-positive (SW1990, RWP-1, Capan-1) and Nd2 antigen-negative (Panc-1, MiaPaca-2, Capan-2) lines, was evaluated by mixed human leukocyte and tumor cell culture (MLTC) at an effector cell to target cell (E/T) ratio of 50 with or without Nd2. Cytotoxicities to SW1990 with no antibody, m-Nd2 and c-Nd2 (1 μg/ml) were 26.7%, 38.0% and 55%, respectively; to RWP-1, 28%, 41% and 70%; to Capan-1, 26%, 30% and 52%; to Panc-1, 24%, 28% and 30%; to MiaPaca-2, 18%, 20% and 27% and to Capan-2, 29.7%, 35.0% and 40.6%. Cytotoxic capacity during MLTC with c-Nd2 was significantly higher than during MLTC with m-Nd2 or with no antibody. These findings indicated that cytotoxicity to Nd2-positive PC cells during MLTC is induced by ADCC. Intraperitoneal injection of c-Nd2 inhibited the tumor growth of SW1990 xenografted subcutaneously in nude mice and prolonged the survival of nude mice in which SW1990 tumor was transplanted orthotopically at the tail of the pancreas. These findings suggested that, because of its ability to induce ADCC, c-Nd2 may be clinically useful for the immunotherapeutic treatment of pancreatic cancer.     

Therapy and Imaging of Pancreatic Carcinoma Xenografts with Radioiodine-labeled Chimeric Monoclonal Antibody A10 and Its Fab Fragment

Recombinant mouse/human chimeric monoclonal antibody A10 (ch-A10) and its Fab fragment (ch-Fab) react with carcinoembryonic antigen on various gastrointestinal carcinomas. We performed biodistribution studies with ¹²⁵I-labeled ch-Al0 and ch-Fab in an antigen-positive human pancreatic carcinoma (BxPC-3) xenograft model. We also evaluated the anti-tumor effect of ¹³¹I-labeled ch-Al0 and studied the detection of BxPC-3 xenografts with ¹²³I-labeIed ch-Fab in whole body scintigraphy. In comparative biodistribution studies, the tumor uptake of ¹²⁵I-labeled ch-Al0 was significantly greater than that of ¹²⁵I-labeIed ch-Fab 24 h post-injection. However, the tumor-to-blood ratio was 46.8 for ch-Fab at 24 h post-injection, while it was only 1.4 for ch-Al0. Microautoradiography studies showed that ch-Fab penetrated more uniformly into the tumor nodules than did ch-Al0. In mice given a therapeutic dose of ¹³¹I-labeled ch-AlO, a significant inhibition of tumor growth was seen, while control ^I31l-labeled human IgG did not affect tumor growth. Leukocyte toxicity was observed within 3 weeks after injection of ¹³¹I-labeled ch-Al0, but leukocyte counts recovered to normal levels at 8 weeks post-injection. In whole-body scintigraphy, clear and rapid tumor imaging was obtained with 200 (Ci of ¹²³I-labeled ch-Fab 24 h post-injection. These results suggest that radioiodine-labeled chimeric A10 antibodies could potentially be useful candidates for radioimmunotherapy and radio-immunodetection of pancreatic carcinomas.     

Establishment and Characterization of Mouse-Human Chimeric Monoclonal Antibody to erbB-2 Product

A mouse-human chimeric antibody for erbB -2 product was established by a new procedure using heavy chain loss mouse mutant hybridoma and human immunoglobulin expression vector. The E401 hybridoma secreted anti- erbB -2 product monoclonal antibody (MoAb) (IgG1, k ). The gene of the mouse variable regions of heavy chain was amplified and cloned by the polymerase chain reaction technique directly from the E401 hybridoma RNA. The variable region of heavy chain was joined with the expression vector, which contains human γ1 constant gene. The expression vector was transfected into heavy chain loss mutant cells E401-12, which produced only murine immunoglobulin light chains. A chimeric monoclonal antibody CH401 retained full binding reactivity to erbB -2 product, compared with murine E401 parental antibody. Furthermore, the chimeric MoAb CH401 was much more efficient in supporting antibody dependent cell-mediated cytotoxicity activity against erbB -2-bearing human adenocarcinoma cells than murine MoAb E401. These suggest that a chimeric monoclonal antibody CH401 may be a potent reagent for therapy of human adenocarcinomas.     

Development and Characterization of Chimeric Anti-carcinoembryonic Antigen Monoclonal Antibodies and Their Fab Fragments

In an attempt to reduce the immunogenicity of two different murine anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAbs), KM10 and A10, we produced recombinant mouse/human chimeric MAbs and the respective Fab fragments carrying the variable regions of the murine MAbs. Chimeric A10 Fab fragment was expressed in Escherichia coli , and produced in large quantities in a mini-jar fermentation system. In competitive binding assays, chimeric MAbs and their Fab fragments showed identical specificity to human CEA epitopes, as compared to the parental MAbs or Fab fragments. Both chimeric Fab fragments exhibited strong immunohistochemical reactivity with various gastrointestinal carcinomas and no reactivity with CEA-related antigens, such as NCA (nonspecific cross-reacting antigen) or BGPI (biliary glycoprotein I). Furthermore, chimeric KM10 MAb elicited substantially higher antibody-dependent cellular cytotoxicity than the murine MAb. Complement-dependent cytotoxicity in vitro was much weaker with chimeric KM10 MAb. These results indicate that chimeric MAbs or Fab fragments could potentially replace the parental murine antibodies or their Fab fragments in therapy or diagnosis of human gastrointestinal carcinomas.     

A Human/Mouse Chimeric Monoclonal Antibody against Intercellular Adhesion Molecule-1 for Tumor Radioimmunoimaging

A mouse-human chimeric antibody for intercellular adhesion molecule-1 (ICAM-1) was established by using heavy chain loss mouse mutant hybridoma and human immunoglobulin expression vector. The HA58 hybridoma secreted anti-ICAM-1 monoclonal antibody (MoAb) (IgG1, k ). The gene of the mouse variable region of heavy chain was amplified and cloned by the polymerase chain reaction technique directly from the HA58 hybridoma RNA. The variable region of heavy chain was joined with an expression vector which contains human γ1 constant gene. The expression vector was transfected into heavy chain loss mutant cells HA58-7, which produced only murine immunoglobulin light chains. The resultant chimeric MoAb HA58, chHA58, retained full-binding reactivity to ICAM-1 compared with murine HA58 parental antibody. The chimeric MoAb chHA58 showed little antibody dependent cell-mediated cytotoxic activity against cultured tumor cells. Biodistribution studies with ^99mTc-labeled chHA58 in nude mice bearing human gastric carcinoma JRST cells demonstrated that the tumor-blood ratio was 1.55 at 18 h after injection, when the tumors were clearly visible in gamma scintigraphy. These data suggest that chHA58 may be of practical use for radioimmunoimaging of a wide variety of tumors.     

Comparison of the Chase Effects of Avidin, Streptavidin, Neutravidin, and Avidin-Ferritin on a Radiolabeled Biotinylated Anti-tumor Monoclonal Antibody

Injection of avidin can decrease the background radioactivity due to a radiolabeled biotinylated monoclonal antibody. We compared the chase effects of avidin, streptavidin, neutravidin, and avidin-conjugated ferritin on a radiolabeled antitumor monoclonal antibody in tumor-bearing nude mice. A radioiodine-labeled biotinylated monoclonal antibody (OST7) was administered to athymic mice bearing osteogenic sarcomas. After 24 h, an avidin, streptavidin, neutravidin or avidin-conjugated ferritin chaser was intravenously injected into the mice. At 2 h after the chase, the biodistribution of the radiolabeled monoclonal antibody was determined. Clearance from the blood was dose-dependently accelerated by avidin and its effect was 10-fold stronger than that of neutravidin or avidinferritin. Streptavidin did not promote clearance of the biotinylated antibody. Avidin was the most effective chasing agent for improving the biodistribution of the radiolabeled biotinylated monoclonal antibody among the four avidin derivatives tested.     

In vivo Efficacy of Neocarzinostatin Coupled with Fab Human/Mouse Chimeric Monoclonal Antibody A7 against Human Colorectal Cancer

The anticancer polypeptide neocarzinostatin (NCS) was covalently coupled to a human/mouse chimeric Fab A7 monoclonal antibody (chFabA7) and the in vivo efficacy of this conjugate was examined. NCS concentration assay was carried out, and acute toxicity and tumoricidal effects were examined. The concentration assay, using anti-NCS monoclonal antibody, revealed that administration of the chA7Fab conjugate leads to a greater blood retention and a higher tumor accumulation of NCS, when compared to free NCS administration. The tumoricidal effect of chA7Fab-NCS was higher than that of either free NCS or the saline control, against antigen-positive tumors. In antigen-negative tumors there was no difference in toxic effect among the three preparations. Values of LD₅₀, reflecting acute toxicity, were 5050 U/kg and 3600 U/kg for the chA7Fab-NCS and the free NCS, respectively. These results suggest that chFahA7-NCS may be a promising tool for targeting cancer chemotherapy.     

Human Tumor Growth Suppression by Apoptosis Induced with Anti-ErbB-2 Chimeric Monoclonal Antibody

We established an anti-ErbB-2 mouse-human chimeric monoclonal antibody (MoAb), CH401, which was able to kill cancer cells overexpressing the ErbB-2 protein in vitro . The analysis of the killing mechanism indicated that MoAb CH401 might be the first anti-ErbB-2 mouse-human chimeric MoAb which can induce the apoptosis of cancer cells, since morphological changes and DNA fragmentation were recognized in MoAb CH401-treated cells. The ErbB-2 receptor appears to have two opposing functions: acting as a receptor both for a growth factor and for an apoptotic factor. Our results indicate that MoAb CH401 treatment may prove to be very useful for cancer therapy.     

Follow-up Study of Patients Treated with Monoclonal Antibody-Drug Conjugate: Report of 77 Cases with Colorectal Cancer

A total of 77 patients with advanced colorectal cancer, including postoperative patients with liver, lung and peritoneal metastases, were treated with single or multiple injections of monoclonal antibody A7-neocarzinostatin (A7-NCS). A follow-up study of the patients treated with A7-NCS was done and the clinical outcome was compared with that of patients given other chemotherapies. In the postoperative patients with liver metastasis, the A7-NCS treatment prolonged survival time when compared with systemic administration of anticancer drugs, while it showed a similar survival time to chemoembolization using multiple anticancer agents suspended in a lipid contrast medium. Among the patients who underwent surgical resection of primary cancer, with or without liver metastasis, there was no difference in overall 5-year survival rate between the group treated with A7-NCS and the group treated with the other chemotherapies. However, the survival time of the patients treated with A7-NCS was longer than that of the patients treated with the other chemotherapies. In addition, the patients given a higher dose of A7-NCS had a longer survival time than the patients given a lower dose of A7-NCS. Human anti-mouse antibody was detected in all the A7-NCS-treated patients examined. There were no serious side effects in any of the patients given A7-NCS. Thus, this study indicates that the A7-NCS treatment is safe and useful for colorectal cancer patients, though some problems remain, such as optimization of injection dose, route, interval, etc., and overcoming human anti-mouse antibody development.     

Effector Cell Analysis of Human Multidrug-resistant Cell Killing by Mouse-Human Chimeric Antibody against P-Glycoprotein

A mouse-human chimeric monoclonal antibody (mAb), MH162, against P-glycoprotein was previously found to be more effective than an all-mouse mAb (MRK16) in lysis of multidrug-resistant (MDR) tumor cells by blood mononuclear cells. The present study was performed to identify the effector cells responsible for the chimeric mAb-dependent cell-mediated cytotoxicity (ADCC) against MDR cells. The ADCC reaction was assessed by a 6-h ⁵¹Cr release assay. Highly purified lymphocytes (>99%), monocytes (>99%) and neutrophils (>96%) were obtained from peripheral blood of the same healthy donors. A comparison of these three effector cell populations showed no difference between MH162 and its all-murine counterpart MRK16 in MDR cell lysis by monocytes or neutrophils. But MH162 was more effective than MRK16 in lymphocyte-mediated lysis of the MDR cells. The lymphocytes responsible for this ADCC had CD16+ Fc receptors. Pretreatment of monocytes with colony-stimulating factors (IL-3, GM-CSF and M-CSF) caused significant increase in their MH162-mediated lysis of MDR cells. Another anti-P-glycoprotein chimeric mAb (MH171) was also more effective than its murine counterpart MRK17 in lymphocyte-mediated lysis of MDR cells. These findings suggest that mouse-human chimeric mAbs may be useful therapeutically for in vivo destruction of MDR cancer cells by the ADCC reaction.     

胃癌单克隆抗体用于氨甲喋呤导向治疗的实验研究

本文报道胃癌单克隆抗体MGb2-BSA-MTX结合物的制备及其对肿瘤靶细胞的杀伤作用。首先将MTX引入BSA,继与单抗连结得到结合物。结合物中IgG:BSA:MTX克分子经为1:1.5:62。偶联过程中抗体活性无明显丧失,结合物与肿瘤靶细胞的结合能力与未修饰抗体相近。噻唑蓝活细胞染色的检测结果表明结合物对胃癌靶细胞KATOⅢ具有较强的选择性杀伤作用。     

抗人血管内皮生长因子嵌合抗体在真核细胞中的高效表达

目的：在中国仓鼠卵巢（Chinese hamster ovary,CHO)细胞中高效表达有活性的抗人血管内皮生长因子（vascular endothelial growth factor,VEGF)嵌合抗体,并探索获得最佳表达的途径。方法：采用一种新型的基因工程抗体真核高效表达载体系统,将抗VEGF嵌合抗体轻,将抗VEGF嵌合抗体轻、重链基因导入二氢叶酸还原酶缺陷型CHO细胞,筛选表达抗VEGF嵌合抗体的克隆,再进行递增逍度的氨甲喋呤（methotrexate,MTX)加压扩增表达,采用ELISA检测所表达的嵌合抗体的生物学特性和产量。结果：采用三种不同的筛选加压扩增表达方法获得的结果有差异,其中采用每轮加压扩增表达后进行亚克隆的方法获得了高表达产量的克隆,产量可达28ug/ml。ELISA结果证实所表达的抗体为特异地与VEGF结合的、含人抗体恒定区的抗VEGF嵌合抗体。结论：成功地在真核细胞中高效表达了有活性的抗VEGF嵌合抗体,为下一步该嵌合抗体的临床试用奠定了重要的基础。并探索出该表达系统最佳的筛选加压扩增表达方法。     

Enhanced Tumor Localization of Radiolabeled Fab Fragments of Monoclonal Antibody A7 in Nude Mice Bearing Human Pancreatic Carcinoma Xenografts

Much recent research has been directed toward the use of monoclonal antibodies (MAb) for the inimunodetection of solid tumors. In pancreatic cancer, the results of conventional immunoscintigraphy using intact MAb remain disappointing. Clear immunoseintigrapliy with radiolabeled MAb requires a high tumor tissue/blood ratio of radioactivity and a low normal tissue/blood ratio of radioactivity. In this study, ¹²⁵I-labeled Fab fragments produced by papain digestion of MAb A7 were injected intravenously into nude mice bearing a human pancreatic cancer (HPC-YS) xenograft previously shown to react specifically with MAb A7. The radioactivity of tumors and normal organs was subsequently measured. The tumor tissue/blood ratio of ¹²⁵I-labeled Fab fragments of MAb A7 was 1.00±0.24 and 9.68±2.54 at 2 and 24 h after injection, respectively. The tumor tissue/blood ratio of radioactivity was significantly higher than those of normal organs at 24 h after injection. Moreover, the tumor tissue/blood ratio of ¹²⁵I-labeled Fab fragments of MAb A7 was greater than that of intact MAb A7, although the ¹²⁵I-labeled Fab accumulation level was much less than that of ¹²⁵I-labeled intact MAb A7 in the tumor. When mice bearing tumors which did not react with MAb A7 were studied, ¹²⁵I-labeled Fab fragments did not specifically localize to the tumors. These results suggest that Fab fragments of MAb A7 may be suitable carriers of radionuclides for the immunodetection of human pancreatic cancer.     

Construction and expression of a human-mouse chimeric antibody against human bladder cancer

Human bladder cancer is one of the most common malignant diseases in urogenital system. Traditional therapies are far from successful, especially in advanced cases[1,2]. Targeted therapy with specific antibody is considered a promising strategy for the treatment of diseases as carcinoma, immune disorders and infectious diseases, and so on[3-5]. BDI-1 is an anti-human bladder cancer monoclonal antibody produced through hybridoma technique. It has undergone a series of trials with good results…     

嵌合抗原受体T细胞治疗胰腺癌的现状及未来策略

嵌合抗原受体（chimeric antigen receptor,CAR）T细胞作为肿瘤免疫治疗领域的新兴疗法在B细胞系血液系统肿瘤中已经获得了较为满意的疗效,因此,CAR-T细胞疗法也被列为包括胰腺癌在内的实体肿瘤治疗的新探索方向。尽管实体瘤本身的异质性、微环境的复杂性导致单纯的CAR-T细胞治疗效果不尽如人意,但随着新技术的开发,CAR结构的优化,各种免疫佐剂的联合应用使CAR-T细胞疗法值得进一步研究。该文就CAR-T细胞疗法的特点、治疗胰腺癌的现状及未来方向进行概述。     

Anti-ganglioside GM2 Monoclonal Antibody-dependent Killing of Human Lung Cancer Cells by Lymphocytes and Monocytes

Ganglioside GM₂ (GM₂) frequently appears on the cell surface of human cancers of neuroendocrine origin. A mouse-human chimeric monoclonal antibody (mAb), KM966, against GM₂ was previously found to promote the lysis of various cancer cells by human blood mononnclear cells (MNC). In this study, we analyzed the effector cells responsible for the chimeric mAb-dependent cell-mediated cytotoxicity (ADCC) against small cell lung cancer (SCLC) cells and examined the enhancing effect of various cytokines on the ADCC activity. The ADCC activity was assessed by 4-h ⁵¹Cr release assay. Highly purified lymphocytes (>99%) and monocytes (>90%) were separated by centrifugal elutriation from peripheral blood MNC of the same healthy donor. KM966 induced lysis of SCLC cells mediated by both lymphocytes and monocytes to similar extents, in a dose-dependent manner. Pretreatment of lymphocytes with various cytokines [interleukin (IL)-2, IL-12 and interferon-γ] and that of monocytes with macrophage-colony-stimulating factor significantly augmented the killer activity against SCLC cells in the presence of KM966 mAb. KM966 was also effective for the lysis of non-small cell lung cancer cells in direct proportion to the GM₂ expression levels. These findings suggest that combined treatment of KM966 mAb with cytokines may be therapeutically useful for in vivo killing of lung cancer cells expressing GM₂ through the ADCC reaction.     

McAb3D3与平阳霉素交联物的制备及其细胞杀伤实验

用ＤｅｘｔｒａｎＴ４０为中介体的方法交联抗人肺腺癌单抗３Ｄ３和平阳霉素（Ａ５），每克分子抗体可携带６８克分子的药物，交联物中抗体活性仍能达到１０－４水平，能较好地与靶细胞Ｌ－３４２结合。体外细胞毒实验，显示交联物和游离Ａ５对靶细胞５０％杀伤浓度分别为０．９μｍｏｌ／Ｌ和４．６μｍｏｌ／Ｌ，杀伤效果比游离Ａ５强５倍多。单抗对靶细胞几乎没有杀伤作用；无关抗体交联物的作用也很弱。交联物和游离Ａ５对非靶细胞ＭＧｃ－８０３的杀伤作用无明显差别。结果提示ＭＣＡｂ３Ｄ３具有良好的导向作用，能携带结合的Ａ５特异地杀伤肺腺癌Ｌ－３４２细胞，可作为临床抗肿瘤药物导向治疗的一种载体。