ABSTRACT
In a previous study, we generated novel antithrombopoietin receptor agonist antibodies as therapeutic candidates. In this report, we investigated the in vivo effects of one of these antibodies, MA01G4344U, on primary human hematopoietic cells using xenotransplantation. NOD/Shi-scid, IL-2Rgamma(null) (NOG) mice were pretreated by total-body irradiation and received a transplant of human cord blood-derived CD34(+) cells. Weekly intraperitoneal injection of MA01G4344U (100 microg/mouse per week) or Peg-rhMGDF (5 microg/mouse per week) or phosphate-buffered saline (PBS) was performed. Human cells in peripheral blood were analyzed by flow cytometry and bone marrow cells were analyzed by flow cytometry and colony assay. MA01G4344U successfully increased the number of human CD41(+) platelets and human CD45(+) cells in peripheral blood. In the bone marrow, MA01G4344U increased the number of human CD45(+)/CD34(+) cells, which resulted in more multilineage progenitor cells. The efficacy of MA01G4344U in promoting primary human hematopoietic cells in vivo suggests its therapeutic potential for thrombocytopenic and pancytopenic disorders.
Subject(s)
Antibodies/pharmacology , Blood Cells/drug effects , Bone Marrow Cells/drug effects , Receptors, Thrombopoietin/agonists , Animals , Antigens, CD34/metabolism , Blood Cells/metabolism , Bone Marrow Cells/metabolism , Flow Cytometry , Humans , Leukocyte Common Antigens/metabolism , Male , Mice , Platelet Membrane Glycoprotein IIb/metabolism , Receptors, Thrombopoietin/immunologyABSTRACT
Tumor development is a complex and dynamic process that involves malignant, vascular, and stromal cells. Endosialin is a tumor endothelial marker (TEM) present in the microvasculature and stroma of human tumors. Cancer-associated fibroblasts (CAF) have been implicated in promoting tumor development and have been associated with mesenchymal stem cells (MSC). Since stem/progenitor cells recruited either from bone marrow or residing in nearby tissues can contribute to pathological processes we investigated endosialin in MSC using a novel monoclonal antibody. Endosialin is highly expressed by CAF and human bone marrow-derived MSC. MSC can form networks in a tube formation assay that is inhibited by an anti-endosialin antibody. Immunohistochemistry for human endosialin in xenograft tumors following co-injection of MSC and cancer cells identified MSC in tumor stroma. MSC are a potential target for anticancer therapeutic intervention and endosialin expression offers a new tool for the identification of MSC. Endosialin expression by both CAF and MSC further implies the potential contribution of MSC to tumor stroma via differentiation into tumor stromal fibroblasts.
Subject(s)
Antigens, CD/biosynthesis , Antigens, Neoplasm/biosynthesis , Biomarkers, Tumor/biosynthesis , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Endothelium, Vascular/metabolism , Neoplasms/metabolism , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Differentiation/physiology , Endothelium, Vascular/cytology , Endothelium, Vascular/pathology , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Male , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/pathology , Mice , Mice, SCID , Neoplasms/blood supply , Neoplasms/genetics , Neoplasms/pathology , Reverse Transcriptase Polymerase Chain Reaction , Stromal Cells/cytology , Stromal Cells/metabolism , Stromal Cells/pathologyABSTRACT
PURPOSE: Endosialin/CD248/tumor endothelial marker 1 is expressed in stromal cells, endothelial cells, and pericytes in various tumors; however, few studies have focused on expression in malignant cells. EXPERIMENTAL DESIGN: We studied expression of endosialin in clinical specimens, cell culture, and animal models and designed an anti-endosialin therapeutic prototype. RESULTS: Fifty human tumor cell lines and 6 normal cell types in culture were assayed by reverse transcription-PCR and/or flow cytometry for endosialin. Cell surface protein was found on 7 sarcoma lines, 1 neuroblastoma, and 4 normal cell types in culture. A fully human anti-endosialin antibody bound to human A-673 Ewing's sarcoma cells and SK-N-AS neuroblastoma cells but not HT-1080 cells. Exposure of cells to an anti-human IgG conjugated to saporin resulted in growth inhibition only of endosialin-expressing cells. Endosialin expression was assessed by immunohistochemistry in 250 clinical specimens of human cancer including 20 cancer subtypes. Endosialin is frequently found in human cancers. Endosialin expression is mainly a perivascular feature in carcinomas, with some expression in stromal cells. In sarcomas, endosialin is expressed by malignant cells, perivascular cells, and stromal cells. Development and characterization of experimental models for studying endosialin biology in sarcomas and evaluating anti-endosialin therapies is presented. CONCLUSIONS: Findings suggest that an anti-endosialin immunotoxin might be a promising therapeutic approach for endosialin-positive neoplasia, especially synovial sarcoma, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, and osteosarcoma. Thus, a diagnostic/therapeutic targeted therapeutic approach to treatment of endosialin-expressing tumors may be possible.
Subject(s)
Antigens, CD/biosynthesis , Antigens, Neoplasm/biosynthesis , Carcinoma/metabolism , Immunotoxins/pharmacology , Neoplasms/metabolism , Sarcoma/metabolism , Animals , Antigens, CD/genetics , Antigens, Neoplasm/genetics , Carcinoma/genetics , Cell Line, Tumor , Flow Cytometry , Humans , Immunoglobulin G/pharmacology , Immunohistochemistry , Neoplasms/genetics , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Ribosome Inactivating Proteins, Type 1/toxicity , Saporins , Sarcoma/geneticsABSTRACT
Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45-/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow-derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC.
Subject(s)
Antigens, CD/metabolism , Antigens, Neoplasm/metabolism , Endothelium, Vascular/metabolism , Antigens, CD/immunology , Antigens, Neoplasm/immunology , Base Sequence , Cells, Cultured , DNA Primers , Endothelium, Vascular/cytology , Flow Cytometry , Humans , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Two of the three components of anthrax toxin, protective antigen (PA) and lethal factor (LF), together known as lethal toxin (LeTx), reportedly show anti-tumor activity in melanoma in vitro and in vivo. The growth inhibitory activity of LeTx in culture was determined in nine human cancer cell lines, including melanoma, neuroblastoma and adenocarcinoma cells, as well as in human umbilical vein endothelial cells (HUVEC). The contribution of the two known PA receptor proteins, ANTXR1/TEM8 and ANTXR2/CMG2, to the sensitivity of the cells was assessed. The efficacy of LeTx was evaluated in vivo in the SK-N-AS neuroblastoma and SK-MEL-28 melanoma tumor xenograft models. Sensitivity to LeTx in vitro was observed in the neuroblastoma and colorectal adenocarcinoma cells and HUVEC, as well as melanoma cells. ANTXR1/TEM8 and ANTXR2/CMG2 protein expression studies suggested that a certain threshold of the PA receptor protein level must be met for sensitivity to LeTx to be observed. However, although the SK-N-AS neuroblastoma cells expressed the highest levels of receptor proteins and achieved the lowest IC50 in vitro (0.1 ng/ml), we observed no correlation between either the ANTXR1/TEM8 or ANTXR2/CMG2 protein levels and sensitivity to LeTx in vitro. In vivo, LeTx was an active anti-tumor agent when administered intravenously to mice bearing the human SK-N-AS or SK-MEL-28 tumor xenografts. The tumor growth delays were 6-8 days with a lower dose regimen and 14-16 days with a higher dose regimen for the two tumor models. These in vitro data suggest that LeTx may have broad therapeutic indications in cancer and the in vivo studies demonstrate that LeTx has systemic efficacy in neuroblastoma as well as melanoma. The therapeutic potential of LeTx needs to be further investigated in non-melanoma tumor models expressing the ANTXR1/TEM8 and/or ANTXR2/CMG2 protein.
Subject(s)
Antigens, Bacterial/therapeutic use , Bacterial Toxins/therapeutic use , Melanoma, Experimental/drug therapy , Neuroblastoma/drug therapy , Receptors, Peptide/analysis , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Child , Female , Humans , Male , Melanoma, Experimental/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Middle Aged , Neuroblastoma/pathology , Xenograft Model Antitumor AssaysABSTRACT
The formation of functional, mature blood vessels depends on the interaction between endothelial cells and pericytes. Commonality exists in the processes involved in vasculature development between tissues whether healthy or diseased. Endosialin/TEM 1 is a cell membrane protein that is expressed in blood vessels during embryogenesis and tumorigenesis but not in normal mature vessels. Antibodies developed to human endosialin were used to investigate endosialin expression and function in human prenatal brain pericytes and pericytes residing in tumors. Anti-endosialin was capable of preventing pericyte tube formation in culture and inhibited migration. Brain pericytes in culture had higher levels of endosialin/TEM 1 than TEMs-2, -3, -4, -5, -7, and -8. Immunocytochemistry revealed that endosialin was present in the cytoplasmic body and in the elongated extensions essential to pericyte function. Transgenic mice engineered to express human endosialin bred on an immunocompromised background allowed the growth of human tumor xenografts. In human colon carcinoma Colo205 and HT29 xenografts grown in human endosialin-transgenic mice, endosialin expression was largely confined to NG2-expressing perivascular cells and not CD31-positive endothelial cells. Similar methods applied to human ovarian and colon tumors confirmed endosialin expression by pericytes. The data indicate that endosialin is strongly expressed by pericytes during periods of active angiogenesis during embryonic and tumor development. Anti-endosialin antibodies may have value in identifying vasculature in malignant tissues. With the appropriate agent, targeting endosialin may interfere with blood vessel growth during tumor development.
Subject(s)
Antigens, CD/metabolism , Antigens, Neoplasm/metabolism , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/metabolism , Neovascularization, Pathologic , Neovascularization, Physiologic , Pericytes/cytology , Pericytes/metabolism , Animals , Antigens, CD/genetics , Antigens, Neoplasm/genetics , Base Sequence , Cell Line, Tumor , Cells, Cultured , DNA Primers/genetics , Embryonic Development , Female , Gene Expression , Humans , Male , Mice , Mice, Inbred C57BL , Mice, SCID , Mice, Transgenic , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transplantation, HeterologousABSTRACT
PURPOSE: Substantial evidence indicates that supraoligomerization of the death receptors for Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is necessary for efficient activation of the apoptotic pathway. Bivalent IgG antibodies can induce the efficient apoptosis by mimicking the natural ligands but only after these antibodies are further oligomerized by cross-linking. In this study, we generated a novel agonist antibody to TRAIL receptor 2 (TRAIL-R2) capable of inducing apoptosis without cross-linking and elucidated its mode of action and efficacy. EXPERIMENTAL DESIGN: A fully human antibody to TRAIL-R2, KMTR2, was generated from KM Mouse immunized with TRAIL-R2 ectodomain. Apoptosis-inducing activities of unfractionated or purified monomeric IgG of KMTR2 was evaluated in the presence or absence of cross-linkers, secondary antibodies or Fc receptor-expressing effector cells, against human colorectal adenocarcinoma Colo205. Oligomerization of TRAIL-R2 was analyzed by size exclusion chromatography and confocal microscopy, and in vivo efficacy was examined in Colo205 xenograft model. RESULTS: KMTR2 specifically recognized TRAIL-R2 and induced apoptosis with or without cross-linking. Size exclusion chromatography showed that the apoptosis activity coeluted with monomeric IgG and was effective independent of secondary antibody or Fc receptor-expressing effector cells. The antibody formed supracomplexes with soluble recombinant and membrane-anchored TRAIL-R2 and enhanced clustering of TRAIL-R2 on cell surface without cross-linking. KMTR2 was dramatically efficacious in reducing established human tumor. CONCLUSION: Our findings indicate that novel agonist antibody KMTR2 can direct antibody-dependent oligomerization of TRAIL-R2 and initiates efficient apoptotic signaling and tumor regression independent of host effector function. Thus, the direct agonist would be a lead candidate for cancer therapeutics.
Subject(s)
Antibodies, Monoclonal/pharmacology , Apoptosis/drug effects , Receptors, Tumor Necrosis Factor/immunology , Xenograft Model Antitumor Assays/methods , Animals , Antibody Specificity , Cell Line, Tumor , Cell Survival/drug effects , Dimerization , Dose-Response Relationship, Drug , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor/chemistry , Remission InductionABSTRACT
The fully human monoclonal antibody KMTR2 acts as a strong direct agonist for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 (TRAIL-R2), which is capable of inducing apoptotic cell death without cross-linking. To investigate the mechanism of direct agonistic activity induced by KMTR2, the crystal structure of the extracellular region of TRAIL-R2 and a Fab fragment derived from KMTR2 (KMTR2-Fab) was determined to 2.1 Å resolution. Two KMTR2-Fabs assembled with the complementarity-determining region 2 of the light chain via two-fold crystallographic symmetry, suggesting that the KMTR2-Fab assembly tended to enhance TRAIL-R2 oligomerization. A single mutation at Asn53 to Arg located at the two-fold interface in the KMTR2 resulted in a loss of its apoptotic activity, although it retained its antigen-binding activity. These results indicate that the strong agonistic activity, such as apoptotic signaling and tumor regression, induced by KMTR2 is attributed to TRAIL-R2 superoligomerization induced by the interdimerization of KMTR2.
Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/pharmacology , Protein Multimerization/drug effects , Receptors, TNF-Related Apoptosis-Inducing Ligand/agonists , Receptors, TNF-Related Apoptosis-Inducing Ligand/chemistry , Animals , Antibodies, Monoclonal/genetics , Apoptosis/drug effects , Binding Sites/genetics , CHO Cells , Cell Line, Tumor , Cell Survival/drug effects , Cricetinae , Cricetulus , Crystallography, X-Ray , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin Fab Fragments/pharmacology , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Mutation, Missense , Protein Binding , Protein Structure, Tertiary , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , StereoisomerismABSTRACT
BACKGROUND: CD44 is a major cellular receptor for hyaluronic acids. The stem structure of CD44 encoded by ten normal exons can be enlarged by ten variant exons (v1-v10) by alternative splicing. We have succeeded in preparing MV5 fully human IgM and its class-switched GV5 IgG monoclonal antibody (mAb) recognizing the extracellular domain of a CD44R1 isoform that contains the inserted region coded by variant (v8, v9 and v10) exons and is expressed on the surface of various human epithelial cancer cells. METHODS AND PRINCIPAL FINDINGS: We demonstrated the growth inhibition of human cancer xenografts by a GV5 IgG mAb reshaped from an MV5 IgM. The epitope recognized by MV5 and GV5 was identified to a v8-coding region by the analysis of mAb binding to various recombinant CD44 proteins by enzyme-linked immunosorbent assay. GV5 showed preferential reactivity against various malignant human cells versus normal human cells assessed by flow cytometry and immunohistological analysis. When ME180 human uterine cervix carcinoma cells were subcutaneously inoculated to athymic mice with GV5, significant inhibition of tumor formation was observed. Furthermore, intraperitoneal injections of GV5markedly inhibited the growth of visible established tumors from HSC-3 human larynx carcinoma cells that had been subcutaneously transplanted one week before the first treatment with GV5. From in vitro experiments, antibody-dependent cellular cytotoxicity and internalization of CD44R1 seemed to be possible mechanisms for in vivo anti-tumor activity by GV5. CONCLUSIONS: CD44R1 is an excellent molecular target for mAb therapy of cancer, possibly superior to molecules targeted by existing therapeutic mAb, such as Trastuzumab and Cetuximab recognizing human epidermal growth factor receptor family.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Hyaluronan Receptors/metabolism , Neoplasms/drug therapy , Neoplastic Stem Cells/drug effects , Animals , Antibodies, Monoclonal/immunology , Antibody Specificity/immunology , Antibody-Dependent Cell Cytotoxicity/drug effects , Antibody-Dependent Cell Cytotoxicity/immunology , Antineoplastic Agents/immunology , Cell Line, Tumor , Epitopes/genetics , Epitopes/immunology , Epitopes/metabolism , Female , Flow Cytometry , Genetic Variation , HCT116 Cells , HEK293 Cells , HeLa Cells , Humans , Hyaluronan Receptors/genetics , Hyaluronan Receptors/immunology , Immunohistochemistry , Lymphocytes/drug effects , Lymphocytes/immunology , Mice , Mice, Nude , Neoplasms/immunology , Neoplasms/metabolism , Neoplastic Stem Cells/immunology , Neoplastic Stem Cells/metabolism , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Isoforms/metabolism , Rats , Rats, Inbred F344 , Xenograft Model Antitumor AssaysABSTRACT
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2 L) preferentially induces apoptosis in human tumor cells through its cognate death receptors DR4 or DR5, thereby being investigated as a potential agent for cancer therapy. Here, we applied fully human anti-human TRAIL receptor monoclonal antibodies (mAbs) to specifically target one of death receptors for TRAIL in human glioma cells, which could also reduce potential TRAIL-induced toxicity in humans. Twelve human glioma cell lines treated with several fully human anti-human TRAIL receptor mAbs were sensitive to only anti-DR5 mAbs, whereas they were totally insensitive to anti-DR4 mAb. Treatment with anti-DR5 mAbs exerted rapid cytotoxicity and lead to apoptosis induction. The cellular sensitivity was closely associated with cell-surface expression of DR5. Expression of c-FLIP(L), Akt, and Cyclin D1 significantly correlated with sensitivity to anti-DR5 mAbs. Primary cultures of glioma cells were also relatively resistant to anti-DR5 mAbs, exhibiting both lower DR5 and higher c-FLIP(L) expression. Downregulation of c-FLIP(L) expression resulted in the sensitization of human glioma cells to anti-DR5 mAbs, whereas overexpression of c-FLIP(L) conferred resistance to anti-DR5 mAb. Treatment of tumor-burden nude mice with the direct agonist anti-DR5 mAb KMTR2 significantly suppressed growth of subcutaneous glioma xenografts leading to complete regression. Similarly, treatment of nude mice bearing intracerebral glioma xenografts with KMTR2 significantly elongated lifespan without tumor recurrence. These results suggest that DR5 is the predominant TRAIL receptor mediating apoptotic signals in human glioma cells, and sensitivity to anti-DR5 mAbs was determined at least in part by the expression level of c-FLIP(L) and Akt. Specific targeting of death receptor pathway through DR5 using fully human mAbs might provide a novel therapeutic strategy for intractable malignant gliomas.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/pharmacology , Glioma/drug therapy , Glioma/pathology , Receptors, TNF-Related Apoptosis-Inducing Ligand/antagonists & inhibitors , Receptors, TNF-Related Apoptosis-Inducing Ligand/immunology , Animals , Cell Line, Tumor , Female , Glioma/immunology , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Xenograft Model Antitumor AssaysABSTRACT
We enhanced the activities of two agonist antibodies specific for the thrombopoietin receptor (c-MPL) by switching domains within their constant regions to those of different antibody isotypes. Our results suggest the importance of the hinge region in modulating agonist activity. The antibodies' thrombopoietin-like activity in vitro and in vivo, as well as the desirable pharmacokinetic profile conferred by retaining the whole-IgG structure, suggests that they provide a valuable option for treating thrombocytopenia.
Subject(s)
Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Genetic Enhancement/methods , Protein Engineering/methods , Receptors, Thrombopoietin/genetics , Receptors, Thrombopoietin/immunology , Antibodies, Monoclonal/chemistry , Protein Structure, Tertiary , Receptors, Thrombopoietin/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Structure-Activity RelationshipABSTRACT
Infusion reactions are a major side effect of the administration of therapeutic Abs and are the result of a complex immune reaction. In this study, we report that substitutions of Fc amino acids in the anti-HLA-DR Ab HD8 reduce its ability to induce infusion reactions in rats and monkeys. We first showed that i.v. administration of IgG1- and IgG2-subclass HD8 Abs induces severe infusion reactions in monkeys. These Abs express strong complement-dependent cytotoxicity (CDC), and in vivo depletion of complement in rats by pretreatment with cobra venom factor abrogated the lethal infusion reactions generated by HD8-IgG1. Thus, the infusion reactions appear to be largely driven by the complement system. To reduce the CDC function of HD8-IgG1, its Fc region was modified by two amino acid substitutions at Pro(331)Ser and Lys(322)Ala. The modified Ab was incapable of expressing CDC in vitro and did not induce severe infusion reactions in rats and monkeys, even at extremely high doses. The modified Ab retained its Ab-dependent cellular cytotoxicity function as well as its antitumor activity in a tumor-bearing mouse model. In summary, complement appears to drive infusion reactions, and modifications that eliminate the CDC activity of an Ab also reduce its ability to induce infusion reactions.
Subject(s)
Antibody-Dependent Cell Cytotoxicity/immunology , Complement Activation/immunology , Isoantibodies/toxicity , Animals , Animals, Congenic , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/therapeutic use , CHO Cells , Cell Line , Cell Line, Tumor , Complement System Proteins/toxicity , Cricetinae , Cricetulus , HLA-DR Antigens/immunology , HLA-DR Antigens/metabolism , Humans , Immunoglobulin Fc Fragments/administration & dosage , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/metabolism , Infusions, Intravenous/adverse effects , Isoantibodies/administration & dosage , Isoantibodies/therapeutic use , Lymphoma/immunology , Lymphoma/mortality , Lymphoma/therapy , Macaca fascicularis , Male , Mice , Mice, SCID , Rats , Transplantation, HeterologousABSTRACT
HD8, a fully human monoclonal antibody specific for human leukocyte antigen-DR (HLA-DR), was generated by using the transchromosome mouse that bears the human immunoglobulin genes. HD8 could bind to all 13 tested HLA-DR-positive cell lines and 35 B-cells from healthy donors. Epitope mapping revealed that while the antibody recognizes the most polymorphic region of the HLA-DRB chain, its critical epitope residues are conserved in the major alleles. Indeed, HD8 could recognize 99.2% of HLA-DRB alleles. Since its essential epitope residues are also largely conserved in HLA-DP and HLA-DQ, HD8 could recognize 100% and 66% of the HLA-DP and HLA-DQ alleles tested, respectively. HD8 exerted strong antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in vitro, and significantly extended the life span of immunocompromised mice inoculated with non-Hodgkin lymphoma cell lines. The HD8 antibody may be highly useful in HLA-DR-targeted immunotherapy as it is likely to evoke similarly strong responses in individuals carrying different HLA-DR alleles.
Subject(s)
Antibodies, Monoclonal/pharmacology , HLA-DR Antigens/immunology , Lymphoma/therapy , Adenosine Triphosphatases , Animals , Antibodies, Monoclonal/therapeutic use , B-Lymphocytes/immunology , Cell Line , Cytotoxicity, Immunologic , Epitope Mapping , Escherichia coli Proteins , Humans , Lymphoma/genetics , Mice , Mice, TransgenicABSTRACT
When human antibody genes were expressed in the methylotrophic yeast Ogataea minuta, the secreted antibody became partially degraded. To suppress the degradation, a vacuolar protease-deficient strain was constructed and its antibody production was evaluated. Although antibody productivity was improved in the vacuolar protease-deficient strain, the secreted antibody still became partially degraded. Peptide sequencing revealed that the cleavage occurred in the CH1 region of the heavy chain, implying that the cleavage was caused by an aspartic protease, Yps1p. To inhibit this cleavage, Yps1p-deficient strains were constructed and their antibody production was evaluated. As a result, the partial degradation of the antibody was suppressed in the O. minuta multiple-protease-deficient strains.