Anti-infliximab antibody concentrations can guide treatment intensification in patients with Crohn's disease who lose clinical response.

BACKGROUND: The presence of antibodies towards infliximab (ATI) is associated with lower infliximab (IFX) trough concentrations and loss of response. IFX treatment intensification is effective for restoring response in most, but not all patients with Crohn's disease (CD). AIM: To compare outcome, pharmacokinetics and immunogenicity of treatment intensification strategies in patients with CD who lost clinical response to IFX. METHODS: A retrospective cohort study was conducted, including 103 patients with CD who lost clinical response during IFX maintenance therapy and therefore received a double dose IFX (10 mg/kg) and/or a next infusion after a shortened interval. IFX and ATI concentrations were measured in consecutive trough samples, just before (T0) and after (T+1) treatment intensification. RESULTS: Clinical response (physicians' global assessment) and biological response and remission (CRP) were restored in 63%, 42% and 24% of patients (evaluated at T+1). Treatment intensification increased IFX trough concentrations from 1.2 µg/mL [0.3-3.6] at T0 to 3.6 µg/mL [0.5-10.2] at T+1 (P < .0001). Using a drug tolerant assay, ATI were detected in the T0 sample of 47% of patients. ATI negatively impacted the achieved IFX trough concentration (Spearman r -0.57, P < .0001) and the probability of clinical response (P = 0.034) at T+1. When ATI were quantifiable but <282 ng/mL eq. at T0, combined interval shortening and dose doubling was more effective for restoring therapeutic IFX trough concentrations (≥3 µg/mL at T+1) than dose doubling alone, which in turn was more effective than interval shortening alone (P < .001). CONCLUSION: Antibodies towards infliximab can guide clinical decision-making on treatment intensification.

Increased zymogen activity of thrombin-activatable fibrinolysis inhibitor prolongs clot lysis.

BACKGROUND AND OBJECTIVES: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a zymogen that can be activated by proteolytic cleavage into the active enzyme TAFIa. Hydrolysis of the C-terminal lysines on fibrin by TAFIa results in a down-regulation of fibrinolysis. Recent studies demonstrated that the zymogen also exerts an intrinsic enzymatic activity. Our objective was to identify and characterize zymogen-stimulatory nanobodies. METHODS AND RESULTS: The screening of 24 nanobodies against TAFI revealed that two nanobodies (i.e. Vhh-TAFI-a51 and Vhh-TAFI-i103) were able to stimulate the zymogen activity 10- to 21-fold compared with the baseline zymogen activity of TAFI. The increase in catalytic efficiency can be attributed mainly to an increased catalytic rate, as no change in the K(M) -value was observed. The stability, the susceptibility towards PTCI and GEMSA and the kinetics of the stimulated zymogen activity differ significantly from those of TAFIa activity. Epitope mapping revealed that both Asp(75) and Thr(301) are major determinants in the binding of these nanobodies to TAFI. Localization of the epitope strongly suggests that this instability is as a result of a disruption of the stabilizing interactions between the activation peptide and the dynamic flap region (residues 296-350). In TAFI-depleted plasma reconstituted with a non-activatable variant of TAFI (TAFI-R92A), clot lysis could be prolonged by nanobody-induced stimulation of its zymogen activity as well as by increasing its concentration. CONCLUSIONS: Increasing the zymogen activity of TAFI results in an antifibrinolytic effect.

TAFIa inhibiting nanobodies as profibrinolytic tools and discovery of a new TAFIa conformation.

BACKGROUND: Because activated thrombin activatable fibrinolysis inhibitor (TAFIa) has very powerful antifibrinolytic properties, co-administration of t-PA and a TAFIa inhibitor enhances t-PA treatment. OBJECTIVE: We aimed to generate nanobodies specifically inhibiting the TAFIa activity and to test their effect on t-PA induced clot lysis. RESULTS: Five nanobodies, raised towards an activated more stable TAFIa mutant (TAFIa A(147) -C(305) -I(325) -I(329) -Y(333) -Q(335) ), are described. These nanobodies inhibit specifically TAFIa activity, resulting in an inhibition of up to 99% at a 16-fold molar excess of nanobody over TAFIa, IC(50) 's range between 0.38- and > 16-fold molar excess. In vitro clot lysis experiments in the absence of thrombomodulin (TM) demonstrate that the nanobodies exhibit profibrinolytic effects. However, in the presence of TM, one nanobody exhibits an antifibrinolytic effect whereas the other nanobodies show a slight antifibrinolytic effect at low concentrations and a pronounced profibrinolytic effect at higher concentrations. This biphasic pattern was highly dependent on TM and t-PA concentration. The nanobodies were found to bind in the active-site region of TAFIa and their time-dependent differential binding behavior during TAFIa inactivation revealed the occurrence of a yet unknown intermediate conformational transition. CONCLUSION: These nanobodies are very potent TAFIa inhibitors and constitute useful tools to accelerate fibrinolysis. Our data also demonstrate that the profibrinolytic effect of TAFIa inhibition may be reversed by the presence of TM. The identification of a new conformational transition provides new insights into the conformational inactivation of the unstable TAFIa.

Lysozyme inhibitor conferring bacterial tolerance to invertebrate type lysozyme.

Invertebrate (I-) type lysozymes, like all other known lysozymes, are dedicated to the hydrolysis of peptidoglycan, the major bacterial cell wall polymer, thereby contributing to the innate immune system and/or digestive system of invertebrate organisms. Bacteria on the other hand have developed several protective strategies against lysozymes, including the production of periplasmic and/or membrane-bound lysozyme inhibitors. The latter have until now only been described for chicken (C-) type lysozymes. We here report the discovery, purification, identification and characterization of the first bacterial specific I-type lysozyme inhibitor from Aeromonas hydrophila, which we designate PliI (periplasmic lysozyme inhibitor of the I-type lysozyme). PliI has homologs in several proteobacterial genera and contributes to I-type lysozyme tolerance in A. hydrophila in the presence of an outer membrane permeabilizer. These and previous findings on C-type lysozyme inhibitors suggest that bacterial lysozyme inhibitors may have an important function, for example, in bacteria-host interactions.

Discovery of novel mechanisms and molecular targets for the inhibition of activated thrombin activatable fibrinolysis inhibitor.

BACKGROUND: Thrombin activatable fibrinolysis inhibitor (TAFI) is an important regulator of fibrinolysis and an attractive target to develop profibrinolytic drugs. OBJECTIVE: To analyze the (inhibitory) properties of five monoclonal antibodies (mAbs) directed towards rat TAFI (i.e. MA-RT13B2, MA-RT30D8, MA-RT36A3F5, MA-RT36B2 and MA-RT82F12). METHODS AND RESULTS: Direct interference of the mAb with rat activated TAFI (TAFIa) activity was assayed using a chromogenic activity assay. This revealed reductions of 79% +/- 1%, 54% +/- 4%, and 19% +/- 2% in activity in the presence of a 16-fold molar excess of MA-RT13B2, MA-RT36A3F5, and MA-RT82F12, respectively whereas MA-RT30D8 and MA-RT36B2 had no direct inhibitory effect. Additionally, MA-RT13B2 and MA-RT36A3F5 reduced rat TAFIa half-life by 56% +/- 2% and 61% +/- 3%. Tissue-type plasminogen activator mediated in vitro clot lysis was determined using rat plasma. Compared to potato tuber carboxypeptidase inhibitor, MA-RT13B2, MA-RT30D8, MA-RT36A3F5, and MA-RT82F12 reduced clot lysis times by 86% +/- 14%, 100% +/- 5%, 100% +/- 10%, and 100% +/- 11%, respectively. During epitope mapping, Arg(227) and Ser(251) were identified as major residues interacting with MA-RT13B2. Arg(188) and His(192) contribute to the interaction with MA-RT36A3F5. Arg(227), Ser(249), Ser(251), and Tyr(260) are involved in the binding of MA-RT30D8 and MA-RT82F12 with rat TAFI(a). The following mechanisms of inhibition have been deduced: MA-RT13B2 and MA-RT36A3F5 have a destabilizing effect on rat TAFIa whereas MA-RT30D8 and MA-RT82F12 partially block the access to the active site of TAFIa or interact with the binding of TAFIa to the blood clot. CONCLUSIONS: The described inhibitory mAb towards rat TAFIa will facilitate TAFI research in murine models. Additionally, we reveal novel molecular targets for the direct inhibition of TAFIa through different mechanisms.

Characterization of rat thrombin-activatable fibrinolysis inhibitor (TAFI)--a comparative study assessing the biological equivalence of rat, murine and human TAFI.

BACKGROUND AND OBJECTIVES: Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis. Although rat models to study the role of TAFI are available, the biochemical properties of rat TAFI are not well investigated and immunologic tools are lacking. Therefore, we have characterized recombinant rat TAFI-6His and compared its properties with those of human TAFI as well as of murine TAFI-V5-6His. METHODS AND RESULTS: TAFI from all three species is activatable by the thrombin-thrombomodulin complex, generating a highly unstable protein (TAFIa). Half-lives at 37 degrees C are 8.5+/-0.6 min, 3.4+/-0.4 min and 2.2+/-0.2 min for human, rat and murine TAFIa, respectively. The 50% clot lysis times are 6+/-1 min for TAFI-depleted rat plasma and 137+/-34 min, 62+/-9 min and 50+/-8 min when TAFI-depleted rat plasma is supplemented with 0.02 U of human, rat or murine TAFIa, respectively, which correlates with their half-lives. Upon incubation with the thrombin-thrombomodulin complex, the 36-kDa fragment of rat and murine TAFI is not cleaved into 25-kDa and 11-kDa fragments. Upon incubation of rat TAFI and murine TAFI with plasmin, a 32-kDa fragment is formed due to cleavage at Arg147, in contrast to the formation of a 36-kDa fragment for human TAFI. Concomitantly, activity levels upon plasmin incubation are drastically reduced for rat and murine TAFI. CONCLUSIONS: Recombinant human, rat and murine TAFI have similar but not identical biochemical characteristics, suggesting a similar role during fibrinolysis in vivo.

Modulation of TAFI function through different pathways--implications for the development of TAFI inhibitors.

OBJECTIVE: To elucidate the mechanism and the binding regions of monoclonal antibodies (MA) that interfere with thrombin-activatable fibrinolysis inhibitor (TAFI)/activated thrombin-activatable fibrinolysis inhibitor (TAFIa) activity. RESULTS: Of 42 MA, 19 interfere with the TAFI activation/TAFIa activity resulting in an inhibition of up to 92%. Characterization of the mechanism of inhibition revealed that 14 MA blocked the activation of TAFI by thrombin/thrombomodulin completely whereas five MA interfered directly with the enzymatic activity of TAFIa. Surprisingly, the former, except one, induced a significant reduction of clot lysis time whereas the latter did not. Affinity studies using a human/murine TAFI chimer revealed that the binding region of the 14 activation blocking MA is located between AA1 and AA67. MA that inhibit exclusively the activation of TAFI by thrombin/thrombomodulin bind to Gly66. A MA that inhibits the activation of TAFI by both thrombin/thrombomodulin and plasmin binds to Val41. The MA that interfere with the enzymatic activity bind to the TAFIa moiety. CONCLUSIONS: The current study reveals at least three different putative molecular targets in the search for pharmacologically active compounds to modulate TAFIa activity.

Structural determinants in the stability of the serpin/proteinase complex.

Serpins inhibit serine proteinases through formation of stable 1:1 complexes. In this study we have evaluated the effects of PAI-1 neutralizing antibodies (MA) on the stability of PAI-1/proteinase complexes, partially destabilized through prolongation of the reactive center loop. MA-8H9D4, reacting with residues Arg(300), Gln(303), and Asp(305), had no effect on the stability. In contrast, MA-33H1F7 and MA-55F4C12, reacting with alpha-helix F and the turn connecting hF with s3A, affected significantly and proteinase-dependently formed PAI-1/proteinase complexes. That is, MA-33H1F7 increased the stability of both PAI-1/t-PA and u-PA complexes (7- and 3-fold, respectively) whereas MA-55F4C12 stabilized PAI-1/t-PA complexes (3-fold) but destabilized PAI-1/u-PA complexes (2-fold). It is concluded that interference with the docking site of the cognate proteinase in the preformed serpin/proteinase complex may affect the intrinsic stability. We hypothesize that this is the consequence of a decreased or increased torsion of the RCL on the catalytic triad in the proteinase.

Elucidation of a novel epitope of a substrate-inducing monoclonal antibody against the serpin PAI-1.

Plasminogen activator inhibitor-1 (PAI-1) is the most important physiological inhibitor of plasminogen activators. Inhibition of PAI-1 constitutes a putative strategy for the prevention of cardiovascular disease. The monoclonal antibody MA-8H9D4 inhibits PAI-1 activity by inducing a substrate behavior in PAI-1. To identify the epitope, a rational approach was used to design various PAI-1 alanine mutants (n = 16) for evaluation of their affinity. PAI-1-R300A, PAI-1-Q303A and PAI-1-D305A had affinities for MA-8H9D4 of < 10(5) M(-1), 2.0 x 10(8) M(-1) and 2.5 x 10(8) M(-1), respectively, whereas the affinity of wtPAI-1 is 3.3 x 10(9) M(-1). The epitope on the axis of arginine 300, glutamine 303 and aspartic acid 305, located on the loop between alpha-helix I and beta-strand 5A, demonstrates that MA-8H9D4 interferes with the final locking step in the serpin/proteinase interaction, thereby explaining its substrate inducing properties. The location of the epitope as well as the proposed mechanism of action is clearly different from that of other substrate inducing monoclonal antibodies against PAI-1. Elucidation of this novel epitope and the previously unidentified molecular mechanism opens new perspectives for the rational development of PAI-1-neutralizing compounds, as well as for the further exploration of synergistic effects between different PAI-1-inhibiting compounds.