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1.
Nat Commun ; 11(1): 2992, 2020 06 12.
Article in English | MEDLINE | ID: mdl-32532974

ABSTRACT

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC's anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC's cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC's anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.


Subject(s)
Antibodies, Monoclonal/pharmacology , Hemophilia A/prevention & control , Immunoglobulin Fab Fragments/immunology , Protein C Inhibitor/pharmacology , Protein C/antagonists & inhibitors , Animals , Antibodies, Monoclonal/classification , Antibodies, Monoclonal/immunology , Bleeding Time , Cell Membrane Permeability/drug effects , Cells, Cultured , Crystallography, X-Ray , Hemophilia A/blood , Hemorrhage/prevention & control , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/physiology , Humans , Immunoglobulin Fab Fragments/metabolism , Macaca fascicularis , Male , Protein C/chemistry , Protein C/immunology , Protein C/metabolism , Protein C Inhibitor/blood , Protein C Inhibitor/pharmacokinetics
2.
Mol Cancer Ther ; 16(5): 893-904, 2017 05.
Article in English | MEDLINE | ID: mdl-28292941

ABSTRACT

C4.4A (LYPD3) has been identified as a cancer- and metastasis-associated internalizing cell surface protein that is expressed in non-small cell lung cancer (NSCLC), with particularly high prevalence in the squamous cell carcinoma (SCC) subtype. With the exception of skin keratinocytes and esophageal endothelial cells, C4.4A expression is scarce in normal tissues, presenting an opportunity to selectively treat cancers with a C4.4A-directed antibody-drug conjugate (ADC). We have generated BAY 1129980 (C4.4A-ADC), an ADC consisting of a fully human C4.4A-targeting mAb conjugated to a novel, highly potent derivative of the microtubule-disrupting cytotoxic drug auristatin via a noncleavable alkyl hydrazide linker. In vitro, C4.4A-ADC demonstrated potent antiproliferative efficacy in cell lines endogenously expressing C4.4A and inhibited proliferation of C4.4A-transfected A549 lung cancer cells showing selectivity compared with a nontargeted control ADC. In vivo, C4.4A-ADC was efficacious in human NSCLC cell line (NCI-H292 and NCI-H322) and patient-derived xenograft (PDX) models (Lu7064, Lu7126, Lu7433, and Lu7466). C4.4A expression level correlated with in vivo efficacy, the most responsive being the models with C4.4A expression in over 50% of the cells. In the NCI-H292 NSCLC model, C4.4A-ADC demonstrated equal or superior efficacy compared to cisplatin, paclitaxel, and vinorelbine. Furthermore, an additive antitumor efficacy in combination with cisplatin was observed. Finally, a repeated dosing with C4.4A-ADC was well tolerated without changing the sensitivity to the treatment. Taken together, C4.4A-ADC is a promising therapeutic candidate for the treatment of NSCLC and other cancers expressing C4.4A. A phase I study (NCT02134197) with the C4.4A-ADC BAY 1129980 is currently ongoing. Mol Cancer Ther; 16(5); 893-904. ©2017 AACR.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Adhesion Molecules/immunology , Immunoconjugates/administration & dosage , Aminobenzoates/chemistry , Aminobenzoates/immunology , Animals , Antibodies, Monoclonal/immunology , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/immunology , Cell Adhesion Molecules/antagonists & inhibitors , Cell Line, Tumor , Cisplatin/administration & dosage , Cisplatin/immunology , GPI-Linked Proteins/antagonists & inhibitors , GPI-Linked Proteins/immunology , Humans , Immunoconjugates/chemistry , Immunoconjugates/immunology , Mice , Oligopeptides/chemistry , Oligopeptides/immunology , Paclitaxel/administration & dosage , Paclitaxel/immunology , Vinblastine/administration & dosage , Vinblastine/analogs & derivatives , Vinblastine/immunology , Vinorelbine , Xenograft Model Antitumor Assays
3.
J Nucl Med ; 50(3): 435-43, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19223400

ABSTRACT

UNLABELLED: To optimize in vivo tissue uptake kinetics and clearance of engineered monoclonal antibody (mAb) fragments for radiotherapeutic and radiodiagnostic applications, we compared the biodistribution and tumor localization of four (111)In- and (86)Y-labeled antibody formats, derived from a single antimindin/RG-1 mAb, in a prostate tumor model. The IgG, diabody, single-chain variable domain (scFv), and novel miniantibody formats, composed of the human IgE-C(H)4 and a modified IgG1 hinge linked to scFv domains, were compared. METHODS: Antibodies were first derivatized with the bifunctional chelator CHX-A''-diethylenetriamine pentaacetic acid and then bound to the radiometal to create radiolabeled immunoconjugates. Human LNCaP xenografts were grown in nude mice, and (111)In- or (86)Y-labeled antibodies were administered intravenously. Tissues were harvested at different times, and the level of antibody deposition was determined by measuring radioactivity. Whole-body small-animal PET of mice receiving (86)Y-labeled antibodies was performed at 6 time points and colocalized with simultaneous micro-CT imaging. RESULTS: The biodistributions of (111)In and (86)Y antibodies were quite similar. The blood, tumor, kidney, and liver tissues contained varying levels of radioactivity. The antibody accumulation in the tumor correlated with molecular size. The IgG steadily increased with time to 24.1 percentage injected dose per gram (%ID/g) at 48 h. The miniantibody accumulated at a similar rate to reach a lower level (14.2 %ID/g) at 48 h but with a higher tumor-to-blood ratio than the IgG. Tumor accumulation of the diabody peaked at 3 h, reaching a much lower level (3.7 %ID/g). A combination of rapid clearance and lower relative affinity of the scFv precluded deposition in the tumor. Small-animal PET results correlated well with the biodistribution results, with similar tumor localization patterns. CONCLUSION: The larger antibody formats (IgG and miniantibody) gave higher tumor uptake levels than did the smaller formats (diabody and scFv). These larger formats may be more suitable for radioimmunotherapy applications, evidenced by the preclinical efficacy previously shown by a report on the IgG format. The smaller formats were rapidly cleared from circulation, and the diabody, which accumulated in the tumor, may be more suitable for radiodiagnostic applications.


Subject(s)
Antibodies, Monoclonal/pharmacokinetics , Prostatic Neoplasms/metabolism , Radiopharmaceuticals/pharmacokinetics , Animals , Antibodies, Monoclonal/chemistry , Cell Line, Tumor , Chelating Agents/chemistry , Humans , Indium Radioisotopes , Isothiocyanates/chemistry , Male , Mice , Mice, Nude , Neoplasm Transplantation , Pentetic Acid/analogs & derivatives , Pentetic Acid/chemistry , Positron-Emission Tomography , Radiopharmaceuticals/chemistry , Tissue Distribution , Tomography, X-Ray Computed , Transplantation, Heterologous , Yttrium Radioisotopes
4.
J Biomol Screen ; 11(8): 985-95, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17092910

ABSTRACT

A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.


Subject(s)
Antigens, Surface/immunology , Bacteriophages/immunology , Immunoglobulin Variable Region/immunology , Membrane Proteins/pharmacology , Neoplasm Proteins/pharmacology , Peptide Library , Animals , Antibodies , Bacteriophages/genetics , CHO Cells , Cell Line , Cricetinae , Cricetulus , Enzyme-Linked Immunosorbent Assay/methods , Humans , Immunoglobulin G/metabolism , Sequence Analysis, Protein , Transfection , Viral Load
5.
Thromb Haemost ; 96(3): 317-24, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16953273

ABSTRACT

Tissue factor (TF) exposure is a potent pro-thrombotic trigger that initiates activation of the coagulation cascade, while thrombomodulin (TM) is a potent anticoagulant protein that limits the extent of activation. Both TF neutralizing antibodies and soluble TM (sTM) are effective anticoagulants. We have developed a novel anticoagulant fusion protein, Ab(TF)-TM, by fusing a TFneutralizing single-chain antibody, Ab(TF), to an active fragment of TM. Ab(TF)-TM is a novel anticoagulant targeting to sites of TF exposure with a dual mechanism of action. The Ab(TF) portion of the molecule inhibitsTF/factorVIIa mediated activation of FIX and FX, and the TM portion of the molecule acts as a cofactor for activation of protein C. In-vitro coagulation assays show that Ab(TF)-TM more potently inhibits TF-initiated coagulation (prothrombin time) than can its individual components, Ab(TF) (20-fold) and sTM (80-fold) alone, or in combination (10-fold). In contrast, the potency of Ab(TF)-TM in the activated partial thromboplastin and thrombin clotting time assays was similar to sTM alone. In a rat model of disseminated intravascular coagulation (DIC), intravenous injection of a human TF-containing thromboplastin reagent (0.5 ml/kg) resulted in an immediate death in approximately 60% of the animals and a clinical score of approximately 2.5. Pre-injection of Ab(TF)-TM or Ab(TF) and sTM, given alone or in combination, showed dose-dependent efficacy. At a dose of 0.7 nmol/kg, Ab(TF)-TM completely prevented death and reduced clinical scores by 79%, while neitherAb(TF) nor sTM, given alone or in combination, showed significant therapeutic effects. Calculated effective doses that reduced mortality by 50% relative to that in the control group (ED(50), nmol/kg) were 0.21 for Ab(TF)-TM, 3.2 for an equimolar mixture of Ab(TF) and sTM, 4.3 for sTM and 20 for Ab(TF). Thus, Ab(TF)-TM presented 10- to 100-fold enhancement of the anticoagulant potency, relative to the ED(50) in Ab(TF) and sTM given either alone or in combination, in a rat DIC model.


Subject(s)
Anticoagulants/pharmacology , Thrombomodulin/blood , Thromboplastin/metabolism , Animals , Anticoagulants/chemistry , Blood Coagulation , Dimerization , Dose-Response Relationship, Drug , Humans , Male , Rats , Rats, Sprague-Dawley , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Thromboembolism , Thrombomodulin/chemistry , Thromboplastin/immunology , Time Factors
6.
Protein Expr Purif ; 45(1): 115-24, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16289982

ABSTRACT

With the recent completion of the human genome sequencing project, scientists are faced with the daunting challenge of deciphering the function of these newly found genes quickly and efficiently. Equally as important is to produce milligram quantities of the therapeutically relevant gene products as quickly as possible. Mammalian expression systems provide many advantages to aid in this task. Mammalian cell lines have the capacity for proper post-translational modifications including proper protein folding and glycosylation. In response to the needs described above, we investigated the protein expression levels driven by the human CMV in the presence or absence of intron A, the mouse and rat CMV promoters with intron A, and the MPSV promoter in plasmid expression vectors. We evaluated the different promoters using an in-house plasmid vector backbone. The protein expression levels of four genes of interest driven by these promoters were evaluated in HEK293EBNA and CHO-K1 cells. Stable and transient transfected cells were utilized. In general, the full-length human CMV, in the presence of intron A, gave the highest levels of protein expression in transient transfections in both cell lines. However, the MPSV promoter resulted in the highest levels of stable protein expression in CHO-K1 cells. Using the CMV driven constitutive promoters in the presence of intron A, we have been able to generate >10 microg/ml of recombinant protein using transient transfections.


Subject(s)
Cytomegalovirus/genetics , Gene Expression Regulation , Genetic Vectors/genetics , Promoter Regions, Genetic , Animals , CHO Cells , Cell Line , Cricetinae , Humans , Mice , RNA, Messenger/genetics , Rats , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Sarcoma Viruses, Murine/genetics , Transcription, Genetic/genetics , Transfection
7.
Bioorg Med Chem Lett ; 15(21): 4752-6, 2005 Nov 01.
Article in English | MEDLINE | ID: mdl-16125385

ABSTRACT

The activated Factor VII/tissue factor complex (FVIIa/TF) plays a key role in the formation of blood clots. Inhibition of this complex may lead to new antithrombotic drugs. An X-ray crystal structure of a fluoropyridine-based FVIIa/TF inhibitor bound in the active site of the enzyme complex suggested that incorporation of substitution at the 5-position of the hydroxybenzoic acid side chain could lead to the formation of more potent inhibitors through interactions with the S1'/S2' pocket.


Subject(s)
Enzyme Inhibitors/chemical synthesis , Factor VIIa/chemistry , Fibrinolytic Agents/chemical synthesis , Pyridines/chemical synthesis , Thromboplastin/chemistry , Binding Sites , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Factor VIIa/antagonists & inhibitors , Factor Xa Inhibitors , Fibrinolytic Agents/chemistry , Fibrinolytic Agents/pharmacology , Humans , Inhibitory Concentration 50 , Protein Binding , Prothrombin Time , Pyridines/chemistry , Structure-Activity Relationship , Thromboplastin/antagonists & inhibitors
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