DPP3 binder directed to and binding to specific DPP3-epitopes and its use in the prevention or treatment of diseases / acute conditions that are associated with oxidative stress

Abstract

The present invention provides binder directed to and binding to a DPP3 protein or functional derivative thereof and its use in a method of prevention or treatment of diseases or acute conditions in a patient, wherein said disease or acute condition is associated with oxidative stress. With this context, specifically the present invention provides a binder being directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, and wherein said DPP3 binder recognizes and binds to at least three amino acids of SEQ ID NO.: 2.

Claims

1. A dipeptidyl peptidase 3 (DPP3) binder directed to and binding to an epitope according to SEQ ID NO.: 2, and wherein said DPP3 binder recognizes and binds to at least three amino acids of SEQ ID NO.: 2, wherein said epitope is comprised in SEQ ID NO.: 1, and wherein said DPP3 binder exhibit an affinity towards DPP3 in such that the affinity constant is at least 10.sup.7 M.sup.−1, wherein said DPP3 binder is a monoclonal antibody or antigen binding fragment, and wherein the complementarity determining regions (CDR's) in the heavy chain comprises the sequences: SEQ ID NO.: 7, SEQ ID NO.:8 and SEQ ID NO.: 9 And the complementarity determining regions (CDR's) in the light chain comprises the sequences: SEQ ID.: 10, KVS and SEQ ID NO.: 11.

2. The dipeptidyl peptidase 3 binder directed to and binding to an epitope according to SEQ ID NO.: 2 of claim 1, wherein said DPP3 binder is directed to and binding to an epitope according to SEQ ID NO.: 3, and wherein said DPP3 binder recognizes and binds to at least three amino acids of SEQ ID NO.: 3.

3. The dipeptidyl peptidase 3 binder directed to and binding to an epitope according to SEQ ID NO.: 2 of claim 1, wherein said DPP3 binder is directed to and binding to an epitope according to SEQ ID NO.: 4, and wherein said DPP3 binder recognizes and binds to at least three amino acids, of SEQ ID NO.: 4.

4. The dipeptidyl peptidase 3 binder directed to and binding to an epitope according to SEQ ID NO.: 2 of claim 1, wherein said DPP3 binder is selected from a group comprising an antibody or antigen binding fragment or non-Ig scaffold.

5. The dipeptidyl peptidase 3 binder directed to and binding to an epitope according to SEQ ID NO.: 2 of claim 1, wherein said DPP3 binder is selected from a group comprising a monospecific antibody or a antigen binding fragment or a monospecific non-Ig scaffold.

6. The dipeptidyl peptidase 3 binder directed to and binding to an epitope according to SEQ ID NO.: 2 of claim 1, wherein said DPP3 binder is a humanized monoclonal antibody or antigen binding fragment, wherein the heavy chain comprises the sequence: SEQ ID NO.: 12 and wherein the light chain comprises the sequence: SEQ ID NO.: 13.

Description

EXAMPLES

1. Example 1

(1) Generation of antibodies and determination DPP3 binding ability: Several murine antibodies were produced and screened by their ability of binding human DPP3 in a specific binding assay (see table 3).

(2) 1.1. Methods:

(3) —Peptides/Conjugates for Immunization:

(4) DPP3 peptides for immunization were synthesized, see table 3, (JPT Technologies,

(5) Berlin, Germany) with an additional N-terminal cystein (if no cystein is present within the selected DPP3-sequence) residue for conjugation of the peptides to Bovine Serum Albumin (BSA). The peptides were covalently linked to BSA by using Sulfolink-coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the manual of Perbio. Recombinant GST-hDPP3 was produced by USBio (United States Biological, Salem, Mass., USA). Immunization of Mice, Immune Cell Fusion and Screening:

(6) Balb/c mice were intraperitoneally (i.p.) injected with 84 μg GST-hDPP3 or 100 μg DPP3-peptide-BSA-conjugates at day 0 (emulsified in TiterMax Gold Adjuvant), 84 μg or 100 μg at day 14 (emulsified in complete Freund's adjuvant) and 42 μg or 50 μg at day 21 and 28 (in incomplete Freund's adjuvant). At day 49 the animal received an intravenous (i.v.) injection of 42 μg GST-hDPP3 or 50 μg DPP3-peptide-BSA-conjugates dissolved in saline. Three days later the mice were sacrificed and the immune cell fusion was performed.

(7) Splenocytes from the immunized mice and cells of the myeloma cell line SP2/0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37° C. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium [RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT-Supplement]. After one week, the HAT medium was replaced with HT Medium for three passages followed by returning to the normal cell culture medium.

(8) The cell culture supernatants were primarily screened for recombinant DPP3 binding IgG antibodies two weeks after fusion. Therefore, recombinant GST-tagged hDPP3 (USBiologicals, Salem, USA) was immobilized in 96-well plates (100 ng/well) and incubated with 50 μl cell culture supernatant per well for 2 hours at room temperature. After washing of the plate, 50 μl/well POD-rabbit anti mouse IgG was added and incubated for 1 h at RT. After a next washing step, 50 μl of a chromogen solution (3.7 mM o-phenylendiamin in citrate/hydrogen phosphate buffer, 0.012% H.sub.2O.sub.2) were added to each well, incubated for 15 minutes at RT and the chromogenic reaction stopped by the addition of 50 μl 4N sulfuric acid. Absorption was detected at 490 mm

(9) The positive tested microcultures were transferred into 24-well plates for propagation.

(10) After retesting the selected cultures were cloned and recloned using the limiting-dilution technique and the isotypes were determined.

(11) —Mouse Monoclonal Antibody Production

(12) Antibodies raised against GST-tagged human DPP3 or DPP3-peptides were produced via standard antibody production methods (Marx et al. 1997) and purified via Protein A. The antibody purities were ≥90% based on SDS gel electrophoresis analysis.

(13) —Characterization of Antibodies—Binding to hDPP3 and/or Immunization Peptide

(14) To analyze the capability of DPP3/immunization peptide binding by the different antibodies and antibody clones a binding assay was performed:

(15) a) Solid phase Recombinant GST-tagged hDPP3 (SEQ ID No. 1) or a DPP3 peptide (immunization peptide, SEQ ID No. 2) was immobilized onto a high binding microtiter plate surface (96-Well polystyrene microplates, Greiner Bio-One international AG, Austria, 1 μg/well in coupling buffer [50 mM Tris, 100 mM NaCl, pH7.8], 1 h at RT). After blocking with 5% bovine serum albumin, the microplates were vacuum dried.

(16) b) Labelling Procedure (Tracer)

(17) 100 μg (100 μl) of the different antiDPP3 antibodies (detection antibody, 1 mg/ml in PBS, pH 7.4) were mixed with 10 μl acridinium NHS-ester (1 mg/ml in acetonitrile, InVent GmbH, Germany; EP 0 353 971) and incubated for 30 min at room temperature. Labelled antiDPP3 antibody was purified by gel-filtration HPLC on Shodex Protein 5 μm KW-803 (Showa Denko, Japan). The purified labeled antibody was diluted in assay buffer (50 mmol/l potassium phosphate, 100 mmol/l NaCl, 10 mmol/l Nae-EDTA, 5 g/l bovine serum albumin, 1 g/l murine IgG, 1 g/l bovine IgG, 50 μmol/l amastatin, 100 μmol/l leupeptin, pH 7.4). The final concentration was approx. 5-7*10.sup.6 relative light units (RLU) of labelled compound (approx. 20 ng labeled antibody) per 200 acridinium ester chemiluminescence was measured by using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

(18) c) hDPP3 Binding Assay

(19) The plates were filled with 200 μl of labeled and diluted detection antibody (tracer) and incubated for 2-4 h at 2-8° C. Unbound tracer was removed by washing 4 times with 350 μl washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Well-bound chemiluminescence was measured by using the Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG). Characterization of Antibodies—hDPP3-inhibition Analysis

(20) To analyze the capability of DPP3 inhibition by the different antibodies and antibody clones a DPP3 activity assay with known procedure (Jones et al., 1982) was performed. Recombinant GST-tagged hDPP3 was diluted in assay buffer (25 ng/ml GST-DPP3 in 50 mM Tris-HCl, pH7.5 and 100 μM ZnCl.sub.2) and 200 μl of this solution incubated with 10 μg of the respective antibody at room temperature. After 1 hour of pre-incubation, fluorogenic substrate Arg-Arg-βNA (20 μl, 2 mM) was added to the solution and the generation of free βNA over time was monitored using the Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG) at 37° C. Fluorescence of βNA is detected by exciting at 340 nm and measuring emission at 410 nm. Slopes (in RFU/min) of increasing fluorescence of the different samples are calculated. The slope of GST-hDPP3 with buffer control is appointed as 100% activity. The inhibitory ability of a possible capture-binder is defined as the decrease of GST-hDPP3 activity by incubation with said capture-binder in percent.

(21) 1.2. Results:

(22) The following table represents a selection of obtained antibodies and their binding rate in Relative Light Units (RLU) as well as their relative inhibitory ability (%; table 3). The monoclonal antibodies raised against the below depicted DPP3 regions, were selected by their ability to bind recombinant DPP3 and/or immunization peptide, as well as by their inhibitory potential.

(23) All antibodies raised against the GST-tagged, full length form of recombinant hDPP3 show a strong binding to immobilized GST-tagged hDPP3. Also antibodies raised against the SEQ ID 2 peptide bind to GST-hDPP3. The SEQ ID 2 antibodies also strongly bind to the immunization peptide. Those antibodies were characterized in more detail (see example 2). The monoclonal antibody AK1967, with the ability of inhibiting DPP3 activity by 70%, was chosen as possible therapeutic antibody and was also used as template for chimerization and humanization.

(24) TABLE-US-00004 TABLE 3 list of antibodies raised against full-length  or sequences of hDPP3 and their ability to bind hDPP3 (SEQ ID No. 1) or immunization peptide  (SEQ ID No. 2) in RLU, as well as the maximum inhibition of recombinant GST-hDPP3.  immunization HDPP3 peptide Max. Sequence Antigen/ HDPP3 binding binding Inhibition number Immunogen region Clone [RLU] [RLU] of HDPP3 SEQ ID: 1 GST tagged   1-737 2552 3,053,621 0 65% recombinant 2553 3,777,985 0 35% FL-hDPP3 2554 1,733,815 0 30% 2555 3,805,363 0 25% SEQ ID: 2 CETVINPETGE 474-493 1963 141,822 2,163,038 60% QIQSWYRSGE 1964 100,802 2,041,928 60% 1965 99,493 1,986,794 70% 1966 118,097 1,990,702 65% 1967 113,736 1,909,954 70% 1968 105,696 2,017,731 65% 1969 82,558 2,224,025 70%

2. Example 2

(25) Antibodies raised against SEQ ID NO. 2 were characterized in more detail (epitope mapping, binding affinities, specificity, inhibitory potential). Here the results for clone 1967 of SEQ ID NO. 2 (“AK1967”) are shown as an example.

(26) 2.1. Methods:

(27) —Determination of AK1967 epitope on DPP3:

(28) For epitope mapping of AK1967 a number of N- or C-terminally biotinylated peptides were synthesized (peptides&elephants GmbH, Hennigsdorf, Germany). These peptides include the sequence of the full immunization peptide (SEQ ID No. 2) or fragments thereof, with stepwise removal of one amino acid from either C- or N-terminus (see table 5 for a complete list of peptides).

(29) a) Solid Phase

(30) High binding 96 well plates were coated with 2 μg Avidin per well (Greiner Bio-One international AG, Austria) in coupling buffer (500 mM Tris-HCl, pH 7.8, 100 mM NaCl). Afterwards plate were washed and filled with specific solutions of biotinylated peptides (10 ng/well; buffer—1×PBS with 0.5% BSA)

(31) b) Labelling Procedure (Tracer)

(32) AntiDPP3 antibody AK1967 was labelled with a chemiluminescence label according to Example 1.

(33) c) Peptide Binding Assay

(34) The plates were filled with 200 μl of labeled and diluted detection antibody (tracer) and incubated for 4 h at room temperature. Unbound tracer was removed by washing 4 times with 350 μl washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Well-bound chemiluminescence was measured by using the Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG). Binding of AK1967 to the respective peptides is determined by evaluation of the relative light units (RLU). Any peptide that shows a significantly higher RLU signal than the unspecific binding of AK1967 is defined as AK1967 binder. The combinatorial analysis of binding and non-binding peptides reveals the specific DPP3 epitope of AK1967.

(35) —Determination of Binding Affinities using Octet:

(36) The experiment was performed using Octet Red96 (ForteBio). AK1967 was captured on kinetic grade anti-humanFc (AHC) biosensors. The loaded biosensors were then dipped into a dilution series of recombinant GST-tagged human DPP3 (100, 33.3, 11.1, 3.7 nM). Association was observed for 120 seconds followed by 180 seconds of dissociation. The buffers used for the experiment are depicted in table 4. Kinetic analysis was performed using a 1:1 binding model and global fitting.

(37) TABLE-US-00005 TABLE 4 Buffers used for Octet measurements Buffer Composition Assay Buffer PBS with 0.1% BSA, 0.02% Tween-21 Regeneration Buffer 10 mM Glycine buffer (pH 1.7) Neutralization Buffer PBS with 0.1% BSA, 0.02% Tween-21

(38) —Western Blot Analysis of Binding Specificity of AK1967:

(39) Blood cells from human EDTA-blood were washed (3× in PBS), diluted in PBS and lysed by repeated freeze-thaw-cycles. The blood cell lysate had a total protein concentration of 250 μg/ml, and a DPP3 concentration of 10 μg/ml. Dilutions of blood cell lysate (1:40, 1:80, 1:160 and 1:320) and of purified recombinant human His-DPP3 (31.25-500 ng/ml) were subjected to SDS-PAGE and Western Blot. The blots were incubated in 1.) blocking buffer (1×PBS-T with 5% skim milk powder), 2.) primary antibody solution (AK1967 1:2.000 in blocking buffer) and 3.) HRP labelled secondary antibody (goat anti mouse IgG, 1:1.000 in blocking buffer). Bound secondary antibody was detected using the Amersham ECL Western Blotting Detection Reagent and the Amersham Imager 600 UV (both from GE Healthcare).

(40) —DPP3 Inhibition Assay:

(41) To analyze the capability of DPP3 inhibition by AK1967 a DPP3 activity assay with known procedure (Jones et al., 1982) was performed. Recombinant GST-tagged hDPP3 was diluted in assay buffer (25 ng/ml GST-DPP3 in 50 mM Tris-HCl, pH7,5) and increasing concentrations of AK1967 were added. Fluorogenic substrate Arg-Arg-βNA was added to the solution and the generation of free βNA over time was monitored using the Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG) at 37° C. Fluorescence of βNA is detected by exciting at 340 nm and measuring emission at 410 nm. Slopes (in RFU/min) of increasing fluorescence of the different samples are calculated. The slope of GST-hDPP3 with buffer control is appointed as 100% activity. The inhibitory ability AK1967 is defined as the decrease of GST-hDPP3 activity by incubation with said antibody in percent. The resulting lowered DPP3 activities are shown in an inhibition curve in FIG. 1C.

(42) 2.2. Results:

(43) —Epitope Mapping:

(44) The analysis of peptides that AK1967 binds to and does not bind to revealed the DPP3 sequence INPETG (SEQ ID No. 3) as necessary epitope for AK1967 binding (see table 5).

(45) TABLE-US-00006 TABLE 5 Peptides used for Epitope mapping of AK1967

(46) —Binding Affinity:

(47) AK1967 binds with an affinity of 2.2*10.sup.−9 M to recombinant GST-hDPP3 (for more details see table 6 and for kinetic curves see FIG. 1A).

(48) TABLE-US-00007 TABLE 6 Kinetic Constants of AK1967 affinity measurements KD (M) kon(1/Ms) kdis(1/s) Full X{circumflex over ( )}2 Full R{circumflex over ( )}2 2.2E−09 1.6E+05 3.5E−04 0.0413 0.9987

(49) —Specificity:

(50) The only protein detected with AK1967 as primary antibody in lysate of blood cells was DPP3 at 80 kDa (FIG. 1B). The total protein concentration of the lysate was 250 μg/ml whereas the estimated DPP3 concentration is about 10 μg/ml. Even though there is 25 times more unspecific protein in the lysate, AK1967 binds and detects specifically DPP3 and no other unspecific binding takes place.

(51) —Inhibitory Potential:

(52) AK1967 inhibits 15 ng/ml DPP3 in a specific DPP3 activity assay with an IC50 of about 15 ng/ml (FIG. 1C).

3. Example 3

(53) A septic shock model was used to induce heart failure in rats and then to characterize AK1967's influence on oxidative stress in myocardium.

(54) 3.1. Methods:

(55) —Study Design

(56) The study flow is depicted in FIG. 2 A below. After CLP or sham surgery the animals were allowed to rest for 20 hours with free access to water and food. Afterwards they were anesthetized, tracheotomy done and arterial and venous line laid. At 24 hours after CLP surgery either AK1967 or vehicle (saline) were administered with 2 mg/kg. As a safety measure hemodynamics were monitored invasively and continuously from t=0 till 3 h.

(57) —CLP Model of Septic Shock

(58) Male Wistar rats (2-3 months, 300 to 400 g, group size refer to table 7) from the Centre d'élevage Janvier (France) were allocated randomly to one of three groups. All the animals were anesthetized using ketamine hydrochloride (90 mg/kg) and xylazine (9 mg/kg) intraperitoneally (i.p.). For induction of polymicrobial sepsis, cecal ligation and puncture (CLP) was performed using Rittirsch's protocol with minor modification. A ventral midline incision (1.5 cm) was made to allow exteriorization of the cecum. The cecum is then ligated just below the ileocecal valve and punctured once with an 18-gauge needle. The abdominal cavity is then closed in two layers, followed by fluid resuscitation (3 ml/100 g body of weight of saline injected subcutaneously) and returning the animal to its cage. Sham animals were subjected to surgery, without getting their cecum punctured.

(59) —Experimentation Time Points and Animal Groups

(60) At t=0 (baseline) all CLP animals are in septic shock and developed a decrease in heart function (low blood pressure, low shortening fraction). At this time point AK1967 (2 mg/kg) or vehicle (saline) were injected (i.v.) and saline infusion was started. There were 1 control group and 2 CLP groups which are summarized in the table below (table 7). At the end of the experiment, the animals were euthanized, and organs (e.g. heart) harvested for subsequent analysis.

(61) TABLE-US-00008 TABLE 7 list of experimental groups Group Group size CLP treatment 1 - sham 4 no saline 2 - CLP-saline 5 yes saline 3 - CLP-AK1967 5 yes AK1967

(62) —DHE Labeling of ROS in Myocardium

(63) Dihydroethidium (DHE; Sigma-Aldrich) staining was used to evaluate the in situ levels of superoxide anion in the myocardium. Cardiac cryostat sections (7 μm) of the ventricles were incubated with DHE (37 μM) for 30 min in a dark humidified chamber. Acquisition of fluorescent images of ethidium bromide with Leica fluorescence microscope was performed under identical setting whatever the block tissue. The stained area was measured with IPLab software and expressed as a percentage of area of interest (% of ROI).

(64) 3.2. Results:

(65) Rats with septic shock induced heart failure after CLP surgery develop high amounts of reactive oxygen species (ROS) in their myocardium, whereas sham operated animals show almost no oxidative stress (FIGS. 2B and C). Treatment of the sick (CLP) animals with AK1967 reduces the oxidative stress levels in the myocardium to levels of healthy (sham-operated) animals. This strong ROS decrease is achieved within only 3 hours of treatment (FIGS. 2B and C).

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(70) TABLE-US-00009 SEQUENCE LISTING SEQ ID No. 1-hDPP3 aa 1-737  MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAAWYGGLAVLLQTSPEAP  YIYALLSRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNYKSFGDTK  FVPNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLRHLGLGKEGITTY  FSGNCTMEDAKLAQDFLDSQNLSAYNTRLFKEVDGEGKPYYEVRLASVLGSEPSLDS  EVTSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKAKAYAANSHQGQMLAQYIESF  TQGSIEAHKRGSRFWIQDKGPIVESYIGFIESYRDPFGSRGEFEGFVAVVNKAMSAKFE  RLVASAEQLLKELPWPPTFEKDKFLTPDFTSLDVLTFAGSGIPAGINIPNYDDLRQTEG  FKNVSLGNVLAVAYATQREKLTFLEEDDKDLYILWKGPSFDVQVGLHELLGHGSGK  LFVQDEKGAFNFDQETVINPETGEQIQSWYRSGETWDSKFSTIASSYEECRAESVGLY  LCLHPQVLEIFGFEGADAEDVIYVNWLNMVRAGLLALEFYTPEAFNWRQAHMQARF  VILRVLLEAGEGLVTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQVLKSTG  DVAGGRALYEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLEY  EASAAGLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA  SEQ ID No. 2-hDPP3 aa 474-493 (N-Cys)-immunization peptide with  additional N-terminal Cystein  CETVINPETGEQIQSWYRSGE  SEQ ID No. 3-hDPP3 aa 477-482-epitope of AK1967  INPETG  SEQ ID No. 4-hDPP3 aa 480-483  ETGE  SEQ ID No. 5-variable region of murine AK1967 in heavy chain  QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMSVGWIRQPSGKGLEWLAHIWWNDN  KSYNPALKSRLTISRDTSNNQVFLKIASVVTADTGTYFCARNYSYDYWGQGTTLTVSS  SEQ ID No. 6-variable region of murine AK1967 in light chain  DVVVTQTPLSLSVSLGDPASISCRSSRSLVHSIGSTYLHWYLQKPGQSPKLLIYKVSNR  FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK  SEQ ID No. 7-CDR1 of murine AK1967 in heavy chain  GFSLSTSGMS  SEQ ID No. 8-CDR2 of murine AK1967 in heavy chain  IWWNDNK  SEQ ID No. 9-CDR 3 of murine AK1967 in heavy chain  ARNYSYDY  SEQ ID No. 10-CDR1 of murine AK1967 in light chain  RSLVHSIGSTY  CDR2 of murine AK1967 in light chain  KVS  SEQ ID No. 11-CDR3 of murine AK1967 in light chain  SQSTHVPWT  SEQ ID No. 12-humanized AK1967-heavy chain sequence (IgG1κ backbone)  MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMS  VGWIRQPPGKALEWLAHIWWNDNKSYNPALKSRLTITRDTSKNQVVLTMTNMDPV  DTGTYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK  DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK  PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV  VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE  YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI  AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH  NHYTQKSLSLSPG  SEQ ID No. 13-humanized AK1967-light chain sequence (IgG1κ backbone)  METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLY  WYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQST  HVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK  VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT  KSFNRGEC 

FIGURE DESCRIPTION

(71) FIG. 1: AK1967 characterization (A) Association- and dissociation curve of the AK1967-DPP3 binding analysis using Octet. AK1967 loaded biosensors were dipped into a dilution series of recombinant GST-tagged human DPP3 (100, 33.3, 11.1, 3.7 nM) and association and dissociation monitored. (B) Western Blot of dilutions of blood cell lysate and detection of DPP3 with AK1967 as primary antibody. (C) Inhibition curve of native DPP3 from blood cells with inhibitory antibody AK1967. Inhibition of DPP3 by a specific antibody is concentration dependent, with an IC.sub.50 at ˜15 ng/ml when analyzed against 15 ng/ml DPP3.

(72) FIG. 2: Influence of AK1967 on oxidative stress in rats with septic shock induced heart failure

(73) (A) Experimental design of heart failure study of rats in septic shock.

(74) (B) Fluorescence images of DHE labelled myocardium of sham, CLP and CLP AK1967 animals.

(75) (C) Quantification of DHE stained areas and expression as percentage of area of interest (% of ROI).