Abstract
The present invention is directed to methods for determining active DPP3 in a bodily fluid sample, an assay or kit for determining active DPP3 in a bodily fluid sample, a method for diagnosing a disease or condition in a subject accompanied by or related to necrotic processes and methods of treating or preventing said disease.
Claims
1. A method for treating a disease or condition in a subject accompanied by or related to necrotic processes, wherein said disease is selected from the group consisting of heart failure, chronic heart failure, acute heart failure, myocardial infarction, infection that is microbial, infection that is viral, and sepsis, comprising administering to a subject in need thereof an effective amount of an inhibitor, wherein said inhibitor is provided by a process comprising: immunizing a mammal with peptides comprising SEQ ID No. 2, infecting or fusing a human B cell with a myeloma or hybridoma cell to produce a trioma cell, testing the immunoglobulins produced by the trioma cell for binding to DPP3, and selecting the immunoglobulins which bind to DPP3.
2. A method for treating a disease or condition in a subject accompanied by or related to necrotic processes, wherein said disease is selected from the group consisting of heart failure, chronic heart failure, acute heart failure, myocardial infarction, infection that is microbial, infection that is viral, and sepsis, comprising administering to a subject in need thereof an effective amount of an inhibitor, wherein said inhibitor is provided by a process comprising: immunizing mammal with peptides comprising SEQ ID No. 2, fusing splenocytes from the immunized mammal and cells of a myeloma or hybridoma cells, and selecting cells and testing the immunoglobulins produced by the fused cells which bind to DPP3 as the inhibitor.
3. A method for treating a disease or condition in a subject accompanied by or related to necrotic processes, wherein said disease is selected from the group consisting of heart failure, chronic heart failure, acute heart failure, myocardial infarction, infection that is microbial, infection that is viral, and sepsis, comprising administering to a subject in need thereof an effective amount of an inhibitor wherein said inhibitor is provided by a process comprising: humanizing one or more complementarity determining regions (CDRs) from a non-human mammalian immunoglobulin which is predetermined to bind to DPP3 SEQ ID No. 2, wherein the non-human immunoglobulin providing the CDRs acts as a donor and a human immunoglobulin providing framework regions (FRs) acts as an acceptor, and wherein the resulting inhibitor comprises a humanized light chain and a humanized heavy chain immunoglobulin.
4. The method of claim 3, wherein variations in the amino acid sequence of the CDRs or FRs are introduced to regain structural interactions which were abolished by the species switch for the FR sequences.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) FIG. 1A: Illustrates the Inhibition of DPP3 activity: The activity of recombinant GST-hDPP3 was measured in the presence of several different antiDPP3 antibodies. DPP3 binding antibodies, that were produced against peptides and/or full-length (FL) native DPP3, show a strong inhibitory effect of up to 70%.
(2) FIG. 1B: Illustrates the Inhibition of DPP3 activity: Inhibition curve of recombinant GST-hDPP3 with inhibitory mAbDPP3. Inhibition of DPP3 by a specific antibody is concentration dependent, with an IC.sub.50 at ˜0.2 μg/ml.
(3) FIG. 2A: Illustrates the DPP3 concentration as diagnostic marker: DPP3 concentration in EDTA plasma of healthy controls and patients with various diseases (AHF—acute heart failure, MI—myocardial infarct, sepsis, cancer, AKI—acute kidney injury, LRTI—lower respirational tract infection). Medians of patient groups differ significantly from healthy controls (Mann-Whitney test p<0.005).
(4) FIG. 2B: Illustrates the DPP3 concentration as diagnostic marker: Comparison of plasma DPP3 concentrations of patients that died shortly within admission to the emergency department and surviving patients. Surviving patients show significantly lower DPP3 levels (Mann-Whitney test p<0.05).
(5) FIG. 3: Illustrates the DPP3 activity as diagnostic marker: DPP3 activity in EDTA plasma of healthy controls and patients with various diseases (AHF—acute heart failure, sepsis, AM—acute kidney injury, LRTI—lower respirational tract infection). Medians of patient groups differ significantly from healthy controls (Mann-Whitney test p<0.0001).
(6) FIG. 4A: Illustrates the ROC plot analysis of the DPP3 activity and concentration assay: ROC analysis of healthy controls and patients suffering from AHF.
(7) FIG. 4B: Illustrates the ROC plot analysis of the DPP3 activity and concentration assay: ROC analysis of healthy controls and patients suffering from sepsis.
(8) FIG. 5: Illustrates the Safety of mAbDPP3 treatment (blood pressure): Healthy rats treated with PBS or mAbDPP3 (5.75 mg/kg). Blood pressure (BP) was measured and recorded via a catheter inserted into the Arteria carotis communis dextra. The administration and sampling catheter were inserted into the Vena jugularis sinistra. Treatment of slightly increases relative blood pressure compared to PBS treated rats (n=3 per group).
(9) FIG. 6: Illustrates the Influence of mAbDPP3 on mortality of septic mice: Septic mice (CLP model) were treated with PBS or mAbDPP3 (1.9 mg/kg) 5 minutes before and 2 h after CLP. Mortality was monitored over 7 days. The Kaplan-Meyer plot shows increased survival of septic mice after mAbDPP3 treatment.
(10) FIG. 7A: Illustrates the Influence of mAbDPP3 on heart failure of septic rats: Experimental design of heart failure study of rats in septic shock.
(11) FIG. 7B: Illustrates the Influence of mAbDPP3 on heart failure of septic rats: CLP induces heart failure in rats, as indicated by a decreased shortening fraction compared to sham animals. This shortening fraction is significantly increased by mAbDPP3 treatment (2 mg/kg; n≥7 per group; Mann-Whitney test p<0.0001).
(12) FIG. 7C: Illustrates the Influence of mAbDPP3 on heart failure of septic rats: Mean blood pressure of vehicle treated septic rats decreases with time whereas mAbDPP3 treatment leads to a significant increase in mBP (2 mg/kg; n≥7 per group; Mann-Whitney test p<0.005).
(13) FIG. 8: Illustrates the Influence of mAbDPP3 on tumor growth in vitro: Soft-Agar Assay with tumor cell lines (lung, colon and breast cancer). Addition of antiDPP3 antibody reduces tumor cell growth.
(14) FIG. 9A: Illustrates the Influence of mAbDPP3 on tumor growth in vivo: Mice with xenograft of breast tumor cells (n=10 per group) were treated with PBS or mAbDPP3. Growth curve of relative tumor volume over 24 days shows a decreased tumor growth in mAbDPP3 treated mice.
(15) FIG. 9B: Illustrates the Influence of mAbDPP3 on tumor growth in vivo: Comparison of time the breast cell tumor needs to 20-fold increase its volume with and without mAbDPP3 treatment. Growth takes significantly longer with mAbDPP3 treatment (Mann-Whitney test, p<0.05).
(16) FIG. 9C: Illustrates the Influence of mAbDPP3 on tumor growth in vivo: Mice with xenograft of colon tumor cells (n=10 per group) were treated with PBS or mAbDPP3. Growth curve of relative tumor volume over 30 days shows a decreased tumor growth in mAbDPP3 treated mice.
(17) FIG. 9D: Illustrates the Influence of mAbDPP3 on tumor growth in vivo: Comparison of time the colon cell tumor needs to 10-fold increase its volume with and without mAbDPP3 treatment. Growth takes longer with mAbDPP3 treatment.
(18) FIG. 10: Illustrates the DPP3 activity as diagnostic marker (II): DPP3 activity in EDTA plasma of healthy controls and patients with various diseases (acute myocardial infarction (AMI), cardiogenic shock, septic shock and liver failure). Medians of patient groups differ significantly from healthy controls (Mann-Whitney test p<0.05).
(19) FIG. 11: Illustrates the Effect of DPP3 on blood pressure in healthy rats: Healthy male Wistar rats were injected with 0.2 mg/kg recombinant GST-hDPP3. Blood pressure (BP) was measured and recorded via a catheter inserted into the Arteria carotis communis dextra. DPP3 was injected i.v. via the tail vain. DPP3 injection leads to decreased BP.
(20) FIG. 12A: Illustrates Peptide and small molecule inhibitors of DPP3.
(21) FIG. 12B: Illustrates Further Peptide and small molecule inhibitors of DPP3.
(22) FIG. 12 C: Illustrates Even Further Peptide and small molecule inhibitors of DPP3.
(23) FIG. 13: Illustrates Immunogen sequence, designation and characteristics of produced anti-DPP3 antibodies.
(24) FIG. 14: Illustrates Comparison of DPP3 values of diseased patients and healthy controls in a sandwich type immune assay.
(25) FIG. 15: Illustrates Comparison of DPP3 values of diseased patients and healthy controls in a sandwich type immune assay and in an enzyme capture activity assay.
(26) FIG. 16: Illustrates Data of ROC analysis (AUC—area under the curve; CI—confidence interval).
(27) FIG. 17: Illustrates Experimental groups.
(28) FIG. 18: Illustrates Overview of treatment strategy.
(29) FIG. 19: Illustrates Concentrations of native or recombinant DPP3 in samples before and after affinity chromatography with depicted anti-DPP3 antibodies.
