Biomedical subjects

Brent Larsen

Publications and source records attributed to Brent Larsen.

8 recordsLinked to original sources

PubMed2006

Obligate multivalent recognition of cell surface tomoregulin following selection from a multivalent phage antibody library.

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.

Animals↗

PubMed2006

Tab2, a novel recombinant polypeptide tag offering sensitive and specific protein detection and reliable affinity purification.

The detection and purification of proteins are often time-consuming and frequently involve complicated protocols. The addition of a peptide tag to recombinant proteins can make this process more efficient. Many of the commonly used tags, such as Flagtrade mark, Myc, HA and V5 are recognized by specific monoclonal antibodies and therefore, allow immunoaffinity-based purification. Enhancing the current scope of flexibility in using diverse peptide tags, we report here the development of a novel, short polypeptide tag (Tab2) for detection and purification of recombinant proteins. The Tab2 epitope corresponds to the NH2-terminal seven amino acid residues of human TGFalpha. A monoclonal anti-Tab2 antibody was raised and characterized. To investigate the potential of this peptide sequence as a novel tag for recombinant proteins, we expressed several different recombinant proteins containing this tag in E. coli, baculovirus, and mammalian cells. The data presented demonstrates the Tab2 tag-anti-Tab2 antibody combination is a reliable tool enabling specific Western blot detection, FACS analysis, and immunoprecipitation as well as non-denaturing protein affinity purification.

Amino Acid Motifs↗

PubMed2006

Amplified anticoagulant activity of tissue factor-targeted thrombomodulin: in-vivo validation of a tissue factor-neutralizing antibody fused to soluble thrombomodulin.

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.

Animals↗

PubMed2006

Anti-EphA2 antibodies decrease EphA2 protein levels in murine CT26 colorectal and human MDA-231 breast tumors but do not inhibit tumor growth.

The EphA2 receptor tyrosine kinase has been shown to be over-expressed in cancer and a monoclonal antibody (mAb) that activates and down-modulates EphA2 was reported to inhibit the growth of human breast and lung tumor xenografts in nude mice. Reduction of EphA2 levels by treatment with anti-EphA2 siRNA also inhibited tumor growth, suggesting that the anti-tumor effects of these agents are mediated by decreasing the levels of EphA2. As these studies employed human tumor xenograft models in nude mice with reagents whose cross reactivity with murine EphA2 is unknown, we generated a mAb (Ab20) that preferentially binds, activates, and induces the degradation of murine EphA2. Treatment of established murine CT26 colorectal tumors with Ab20 reduced EphA2 protein levels to approximately 12% of control tumor levels, yet had no effect on tumor growth. CT26 tumor cell colonization of the lung was also not affected by Ab20 administration despite having barely detectable levels of EphA2. We also generated and tested a potent agonistic mAb against human EphA2 (1G9-H7). No inhibition of humanMDA-231 breast tumor xenograft growth was observed despite evidence for >85% reduction of EphA2 protein levels in the tumors. These results suggest that molecular characteristics of the tumors in addition to EphA2 over-expression may be important for predicting responsiveness to EphA2-directed therapies.

Animals↗

PubMed2005

Identification of a novel prostate tumor target, mindin/RG-1, for antibody-based radiotherapy of prostate cancer.

Gene expression analysis showed that a human mindin homologue, mindin/RG-1, is expressed selectively in prostate tissues and that its expression level is elevated in some prostate tumors. Mindin/RG-1 protein expression is maintained in >80% of prostate cancers metastatic to bone or lymph nodes as well as in locally recurrent tumors in androgen-unresponsive patients. In contrast, mindin/RG-1 expression in other normal tissues is significantly lower than that seen in the prostate. A fully human antibody, 19G9, was generated against mindin/RG-1 protein and was shown to accumulate at high abundance in LNCaP tumor xenografts. Conjugates of this antibody with the chelator CHX-A''-DTPA were generated and radiolabeled with either 111In, 90Y, or 86Y. Small animal positron emission tomography imaging with the 86Y-radiolabeled conjugate showed very specific accumulation of the antibody in LNCaP tumor xenografts with clear tumor delineation apparent at 4 hours. The therapeutic efficacy of [90Y]-CHX-A''-DTPA-19G9 was evaluated in mice bearing LNCaP xenografts. A dose-finding study identified a nontoxic therapeutic dose to be approximately 75 microCi. Significant antitumor effects were seen with a single administration of radiolabeled antibody to animals bearing 200 to 400 mm3 tumors. Inhibition of tumor growth was observed in all treated animals over a 49-day period. At 49 days posttreatment, slow tumor growth recurred but this could be prevented for an additional 40-day period by a second administration of a 75 microCi dose at day 49. We conclude that [90Y]-CHX-A''-DTPA-19G9 is a novel antibody conjugate that has considerable promise for therapy of metastatic prostate cancer in androgen-unresponsive patients.

Amino Acid Sequence↗

PubMed2005

Crystal structures of potent thiol-based inhibitors bound to carboxypeptidase B.

This paper presents the crystal structure of porcine pancreatic carboxypeptidase B (pp-CpB) in complex with a variety of thiol-based inhibitors that were developed as antagonists of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Recent studies have indicated that a selective inhibitor of TAFIa could enhance the efficacy of existing thrombolytic agents for the treatment of acute myocardial infarction, one of the most prevalent forms of heart attacks. Unfortunately, activated TAFIa rapidly degrades in solution and cannot be used for crystallographic studies. In contrast, porcine pancreatic CpB is stable at room temperature and is available from commercial sources. Both pancreatic CpB and TAFIa are zinc-based exopeptidases, and the proteins share a 47% sequence identity. The homology improves considerably in the active site where nearly all of the residues are conserved. The inhibitors used in this study were designed to mimic a C-terminal arginine residue, one of the natural substrates of TAFIa. The X-ray structures show that the thiol group chelates the active site zinc, the carboxylic acid forms a salt bridge to Arg145, and the guanidine group forms two hydrogen bonds to Asp255. A meta-substituted phenyl was introduced into our inhibitors to reduce conformational freedom. This modification vastly improved the selectivity of compounds against other exopeptidases that cleave basic residues. Comparisons between structures indicate that selectivity derives from the interaction between the guanidine group in the inhibitors and an acidic active site residue. The location of this acidic residue is not conserved in the various carboxypeptidases.

Animals↗

PubMed2005

Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.

Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.

Amino Acid Sequence↗

PubMed2004

P-selectin-targeting of the fibrin selective thrombolytic Desmodus rotundus salivary plasminogen activator alpha1.

During thrombosis, P-selectin is expressed on the surface of activated endothelial cells and platelets. We hypothesized that targeting a plasminogen activator (PA) to P-selectin would enhance local thrombolysis and reduce bleeding risk. Previously, a urokinase (uPA)/anti-P-selectin antibody (HuSZ51) fusion protein was shown to increase fibrinolysis in a hamster pulmonary embolism model. To explore the therapeutic potential of this targeting strategy, we fused the fibrin-selective Desmodus rotundus salivary PA alpha1 (dsPA alpha 1) to HuSZ51 and compared the fibrinolytic activity of P-selectin-targeted dsPA alpha 1 (HuSZ51-dsPA alpha 1) to unmodified dsPA alpha 1 in vitro and in vivo. HuSZ51-dsPA alpha 1 and dsPA alpha 1 were expressed in CHO cells and purified to homogeneity by affinity chromatography. HuSZ51-dsPA alpha 1 bound to thrombin-activated human and dog platelets with comparable affinities to that of parental antibody SZ51. The fusion protein retained the catalytic activities of dsPA alpha 1 in chromogenic and clot lysis assays, indicating that dsPA alpha 1 is fully functional when fused to HuSZ51. Compared to dsPA alpha 1, HuSZ51-dsPA alpha 1 had similar thrombolytic efficacy in a rat pulmonary embolism model and anti-thrombotic potency in a dog model of femoral artery thrombosis. However, HuSZ51-dsPA alpha 1 was less effective in lysis of preexisting arterial thrombi in the dog model. The reduced arterial thrombolysis was not due to the pharmacokinetic properties of HuSZ51-dsPA alpha 1 because antigen level and amidolytic activity were higher in plasma from HuSZ51-dsPA alpha 1-treated groups than corresponding dsPA alpha 1-treated groups. These data indicate that the thrombolytic efficacy of HuSZ51-dsPA alpha 1 varied dependent on the physical composition of thrombi. The lack of stimulation by fibrin in arterial thrombi may contribute to the attenuated thrombolytic efficacy of HuSZ51-dsPA alpha 1 in the dog model.

Animals↗