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.     

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.     

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.     

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.     

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.     

乳腺癌单链抗体制备及其在荷人乳腺癌裸鼠中的放免显像

目的：制备抗乳腺癌单链抗体,用于荷瘤裸鼠的放免显像研究。方法：提取乳腺癌膜抗原,制备抗乳腺癌单克隆抗体M4G3,用Pharmacia噬菌体单链抗体制备系统得到可溶性具有抗乳腺癌活性的ScFv抗体,利用环DTPA法标记核素99^mTc,进行体外培养的乳腺癌细胞系MCF-7和荷瘤小鼠体内定位显像。结果：免疫组化显示单链抗体与乳腺癌膜抗原有较强的亲和力,并在体外培养的乳腺癌细胞及荷瘤小鼠体内显像成功,且在抗体注射第3-5天显像最清晰。结论：提示M4G3的ScFv可在乳腺癌的放射免疫显像和定位诊断研究中发挥作用,值得进一步研究。     

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.     

Immunosuppressive Effect of Shedding Intercellular Adhesion Molecule 1 Antigen on Cell-mediated Cytotoxicity against Tumor Cells

We have examined whether shedding intercellular adhesion molecule-1 (ICAM-1) antigen from cultured tumors is able to inhibit the leukocyte function-associated antigen-1 (LFA-1)/ICAM-1 interaction between cytotoxic effector cells and ICAM-1⁺ target tumor cells. The cytotoxic activity of lymphokine-activated killer (LAK) cells incubated with spent media from ICAM-1⁺ tumor cells, especially interferon-γ-stimulated tumor cells, was significantly decreased as compared with that of LAK cells treated with fresh culture medium without ICAM-1 antigen. Treatment of LAK cells with spent media from ICAM-1⁻ tumor cells did not cause a significant decrease of the cytolytic activity towards ICAM-1⁺ tumor cells. These findings suggest that shedding of ICAM-1 antigen could be involved in binding of LFA-1 to LAK cells, resulting in reduced cytolytic activity.     

Expression and Functional Role of CD54/Intercellular Adhesion Molecule-1 (ICAM-1) on Human Blood Cells

CD54/Intercellular Adhesion Molecule-1 (ICAM-1) is a cell adhesion molecule largely distributed among normal and neoplastic tissues. Through the binding to its ligand(s) CD54 plays a key role in cell to cell interactions leading to the immune response. Recently, CD54 expression has been investigated on hematopoietic cells: the antigen is predominantly expressed in the early stages of normal hematopoiesis and during the activation of blood cells. As regards to hematological malignancies, CD54 is strongly expressed on neoplastic cells from “stem cell derived” neoplasms. In AML, CD54 expression is related with other differentiation-linked molecules such as CD34 and HLA-DR and is significantly correlated with FAB morphological classification. In lymphoproliferative disorders, a high CD54 expression is associated with germinal centre lymphomas. This review summarizes our current understanding of CD54 with emphasis on recent advances and reference to unresolved issues such as its prognostic role in the clinical outcome of oncohematological diseases.     

ICAM-1 (Intercellular Adhesion Molecule-1) Gene Transfection Inhibits Lymph Node Metastasis by Human Gastric Cancer Cells

Lymph node metastasis is one of the prognostic factors in gastric cancer. We have previously reported that decreased intercellular adhesion molecule-1 (ICAM-1) expression on cancer cells is associated with lymph node metastasis using a gastric cancer cell. In this study, we transfected ICAM-1 gene into a gastric cancer cell line, 2MLN, and analyzed the effect on lymph node metastasis in vitro and in vivo. A significantly greater amount of peripheral blood mononuclear cells (PBMC) adhered to ICAM-1 transfected 2MLN cells, 2MLN/ICAM cells, than to 2MLN/Vector cells. The lysis of 2MLN/ICAM cells by PBMC was significantly increased compared with that of 2MLN/Vector cells. The tumor growth rate of 2MLN/ICAM cells was significantly decreased in vivo. Lymph node metastases caused by 2MLN/ICAM cells were recognized as being fewer in number and smaller, while many lymph node metastases were caused by 2MLN cells. Histologic findings showed that leukocytes were heavily infiltrated in both the 2MLN/ICAM tumors and metastatic lesions, while only a few leukocytes were observed in the lesions associated with 2MLN cells. The above findings indicate that ICAM-1 gene transduction could prove to be an effective gene therapy for lymph node metastasis of gastric cancer.     

食管鳞癌中ICAM-1蛋白的表达及意义

目的 研究细胞间粘附分子-1(ICAM-1)在食管鳞癌组织中的表达及意义.方法 采用免疫组织化学S-P法检测49例食管鳞癌组织及38例正常食管黏膜组织中ICAM-1蛋白的表达.结果 食管鳞癌组织中ICAM-1蛋白表达率为46.9%(23/49),显著高于其在正常食管黏膜组织中的表达0.0%(0/38);ICAM-1蛋白表达率在有淋巴结转移组明显高于无淋巴结转移组;其表达率随癌组织浸润深度的加深而增加;在不同分化程度的癌组织中其阳性率也有一定差异,Ⅰ级与Ⅱ、Ⅲ级之间差异有统计学意义(P＜0.05).结论 ICAM-1在食管鳞癌的发生发展中起重要作用,与肿瘤的浸润转移密切相关.     

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.     

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.     

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.     

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.     

A Novel Human Monoclonal Antibody Directed to a Tumor-associated Antigen

Twelve human monoclonal antibodies (HuMAb) were established by the fusion of (mouse × human) heteromyeloma cells with B-lymphoblastoid cells derived from the regional lymph nodes of three patients with squamous cell carcinoma of the lung. They were tested for reactivity to two kinds of proteins (purified protein derivatives and bovine serum albumin) by ELISA, Sq-19 (squamous cell carcinoma) culture cells by indirect membrane immunofluorescence tests, and Sq-19 tumor xenograft by immunohistological study. Among them, one HuMAb 904F (IgM, λ) was selected. In indirect membrane immunofluorescence tests, this 904F antibody reacted with various kinds of cell lines, e.g. lung cancer, esophageal cancer, endometrial cancer, and stomach cancer. It did not react with malignant hematopoietic and diploid fibroblast cell lines. Immunohistologically, it stained the tumor nests of squamous cell carcinoma, adenocarcinoma, and large cell carcinoma of the lung. It also stained those of esophagus and colon, but not those of small cell carcinoma of lung, or stomach. On frozen-section specimens of normal tissues from various organs, it showed only limited areas of positive staining. Limited positive findings were observed at a reticular zone of the adrenal gland, at the esophagus as weak staining, and at islets of the pancreas as very weak staining. Western blotting analysis demonstrated that it recognized a 54 kDa trypsin-sensitive molecule which is expressed on the surface of tumor cells. These results suggest the 904F monoclonal antibody detects a novel tumor-associated antigen which is recognized by the human immune system.     

Anti-murine Antibody Response to Mouse Monoclonal Antibodies in Cancer Patients

Although development of human anti-murine inununoglobulin antibody (HAMA) is often seen in patients receiving murine antibodies, the variety of methods used for detecting HAMA makes it difficult to compare directly the HAMA responses measured by different assays. In the present study, several parameters of the HAMA response to two murine monoclonal antibodies were evaluated. The anti-sialosyl Tn antibody MLS102 and anti-CA125 antibody 145-9, which were labeled with ¹¹¹ln, were injected intravenously into 17 colorectal cancer patients and 11 ovarian cancer patients for immnnoscintigraphy, respectively. HAMA was measured by enzyme-linked immunosorbent assay. There was no difference in baseline HAMA levels before antibody injection between the two groups. HAMA developed more frequently in ovarian cancer patients receiving the 145-9 antibody than in colorectal cancer patients receiving the MLS102 antibody (9/11 vs. 6/17, P <0.05). No significant difference was observed in maximal HAMA levels between the two groups of patients. However, time to reach the maximal levels was delayed and the duration of the response seemed longer in ovarian cancer patients. Among 11 patients receiving the 145-9 antibody three patients became positive for HAMA more than 2 months after antibody injection and the other two had HAMA activity in their sera for more than 17 months. HAMA response was different between the two antibodies, and late onset or long duration of HAMA response against the 145-9 antibody suggests the importance of HAMA measurement in patients who receive a second injection of murine antibodies even after a long interval.     

Molecular Identification of a Human Carcinoma-associated Glycoprotein Antigen Recognized by Mouse Monoclonal Antibody FU-MK-1

Mouse monoclonal antibody FU-MK-1, raised against a human gastric adenocarcinoma, recognizes an antigen (termed MK-1 antigen) present on the majority of carcinomas. The present study aimed to identify the MK-1 molecule and to establish its relationship to other carcinoma antigens. Immunoprecipitation studies of human tumor cell lines revealed that FU-MK-1 recognizes a monomeric membrane glycoprotein with two forms, 40 kDa (major form) and 42 kDa (minor form), and with a molecular mass of 35 kDa following treatment with the N-glycosylation inhibitor tunicamycin. The partial amino acid sequence of a main fragment of the MK-1 molecule obtained by spontaneous cleavage under hypotonic conditions was examined, and the 17 contiguous NH₂-terminal amino acids were found to be identical with residues 81–97 of the 314-residue GA733–2 protein [Szala et al.; Proc. Natl. Acad. Sci. USA , 87, 3542–3546 (1990)]. Hence, the GA733–2 cDNA was cloned and the specificity of FU-MK-1 was confirmed using four recombinant forms of the GA733–2 antigen expressed in COS-1 cells. Immunoprecipitation with FU-MK-1 of the cell lysate transfected with the full-length GA733–2 cDNA revealed two bands corresponding to those obtained from the tumor cell lines. FU-MK-1 also precipitated three other recombinant proteins consisting of amino acids 1–265, 1–201, and 1–139 of the GA733–2 protein, respectively. Furthermore, immunoblotting analysis indicated that FU-MK-1 binds to a small fragment (6 kDa) generated from a tumor cell line under hypotonic conditions, suggesting that the FU-MK-1 epitope exists on the distal 6-kDa peptide of the extracellular domain of the GA733–2 molecule. We thus conclude that the MK-1 antigen is the GA-733–2 antigen, which is currently being used as a target in clinical trials with monoclonal antibodies.     

Transfection-induced Tumor Necrosis Factor-α Increases the Susceptibility of Human Glioma Cells to Lysis by Lymphokine-activated Killer Cells: Continuous Expression of Intercellular Adhesion Molecule-1 on the Glioma Cells

To develop more effective adoptive immunotherapy, we transfected the human tumor necrosis factor-α (TNF-α) gene into human glioma cells (U251-SP), which were used as target cells. TNF-α is known to increase both the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of glioma cells and the susceptibility of glioma cells to lymphokine-activated killer (LAK) cell cytolysis. We compared the expression of ICAM-1 induced by TNF-α generated by the TNF-α gene-transfected cells with that induced by exogenously added TNF-α. When the TNF-α gene was transfected into U251-SP cells, the expression of ICAM-1 was detected on the cell surface from 3 days after the transfection and continued until at least 9 days. In contrast, it was expressed only transiently in the case of exogenously added TNF-α. Also, the cytolytic activity of LAK cells induced by transfection-induced TNF-α was significantly stronger than that induced by exogenously added TNF-α. The increased susceptibility was quenched by anti-ICAM-1 monoclonal antibody. These data indicated that continuous expression of ICAM-1 induced by TNF-α gene transfection of glioma cells resulted in higher cytolytic activity of LAK cells.